# Patent

## Title
Methods and compositions for inhibiting neddylation of proteins

## Abstract

Provided herein is a novel binding pocket within NEDD8 co-E3 proteins that binds NEDD8 E2 enzymes. Particularly at its M-Terminus. Methods are provided for screening for compounds that bind to the disclosed E2-binding pocket in NEDD8 co-E3 proteins. Compounds that bind to the E2-binding pocket and optionally inhibit the activity of NEDD8 co-E3 proteins and pharmaceutical compositions comprising the same are further provided. The NEDD8 co-E3 inhibitors find use, as agents preventing the NEDDylation of a target protein, in inhibiting cell growth and methods for treating cancers, inflammatory disorders, and pathogenic infections. The preferred inhibitors are peptides corresponding to a M-terminal fragment of Dnc1, e.g. MTLASKLKRDD, MLKLRQLQKKKQ, and MIKLFSLKQQKK, which are substituted at the M-Terminus with an uncharged group (e.g. acyl).

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is the U.S. National Stage of International Application No. PCT/US2012/038127, filed May 16, 2012, which claims the benefit of U.S. Provisional Patent Application No. 61/486,972, filed May 17, 2011, both of which are hereby incorporated by reference in their entirety.


FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with United States Government support under grant number R01GM069530 awarded by the National Institutes of Health. The United States Government has certain rights in the invention.


REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB
The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named amended439490SEQLIST.TXT, created on Feb. 1, 2014, and having a size of 246 kilobytes and is filed with the Preliminary Amendment filed on Feb. 4, 2014. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
The present invention relates to the inhibition of covalent modifications of proteins through conjugation with other proteins; particularly, neuronal precursor cell expressed developmentally downregulated protein 8 (NEDD8).
BACKGROUND OF THE INVENTION
Post-translational covalent modifications of proteins through conjugation with other proteins is an important biological mechanism for regulating protein metabolism and biological activity. The most well understood post-translational modifier is ubiquitin, an 8.5 kDa protein, that is covalently attached to lysines in a target protein. Polyubiquitination of a protein targets the protein for degradation.
Ubiquitin is conjugated to its target proteins through an enzymatic cascade involving a specific E1 activating enzyme, Uba1 or Uba6, a conjugating enzyme from the family of E2s, and a ubiquitin ligase that is typically from either the RING or HECT classes of E3s (Huang et al. (2004) Oncogene 23:1958-71). Target specificity is controlled by the particular combination of E2 and E3 proteins. For example, the multi-protein ubiquitin E3 ligase Skp, cullin, F-box containing complexes (SCFs) ubiquitinate targets involved in cell-cycle progression, transcription, metabolism, and inflammation, such as the cyclin-dependent kinase (CDK) inhibitor p27Kip1 and NFκB inhibitor. IkappaB (IκB).
Other proteins that are structurally similar to ubiquitin and are referred to as ubiquitin-like proteins (UBLs) have been identified that covalently modify cellular targets using their own pathways that are parallel to that of ubiquitin. Examples of UBLs include small ubiquitin-like modifier (SUMO) and neuronal precursor cell expressed developmentally downregulated protein 8 (NEDD8). Similar to ubiquitin, UBLs are covalently attached to a lysine on a target protein via an isopeptide linkage with a C-terminal glycine of the UBL, a process that is mediated via an E1 activating enzyme, E2 conjugating enzyme, and an E3 ligase.
The covalent modification of a target protein with the UBL NEDD8 is referred to herein as neddylation. The neddylation of cullin proteins, the best understood targets of NEDD8, is necessary for SCF-mediated ubiquitination and subsequent degradation of SCF target proteins (Podust et al. (2000) Proc Natl Acad Sci USA 97:4579-4584; Read et al. (2000) Mol Cell Biol 20:2326-2333). SCFs form a subset of cullin-containing ubiquitin E3s. Members of the large family of cullin-containing E3s are called cullin-RING E3s, and contain CUL1, CUL2, Cul3, CUL4A, CUL5, CUL7, or Parc, which are regulated by covalent ligation of NEDD8. Not surprisingly, the NEDD8 pathway enzymes play an essential role in cell proliferation in organisms ranging from fission yeast to mammals (Osaka et al. (2000) EMBO J. 19:3475-3484; Tateishi et al. (2001) J Cell Biol 155:571-579). Given the importance of NEDD8 conjugation in cell growth and inflammation, further characterization of NEDD8 pathway enzymes and the domains with which they use to interact with one another is needed in order to develop therapeutics that target the neddylation pathway for the treatment of disorders such as cancer and inflammatory diseases.
BRIEF SUMMARY OF THE INVENTION
A novel binding pocket within NEDD8 co-E3 proteins that binds NEDD8 E2 enzymes is provided. Methods are provided for screening for compounds that bind to the disclosed E2-binding pocket in NEDD8 co-E3 proteins, including computational methods and binding assays. In some embodiments, compounds that bind to the pocket inhibit the activity of NEDD8 co-E3 proteins and the subsequent neddylation of protein targets. Compounds (e.g., peptides and small molecules) that bind to the E2-binding pocket and optionally inhibit the activity of NEDD8 co-E3 proteins and pharmaceutical compositions comprising the same are provided. In some embodiments, the inhibitory peptides are amino-terminally acetylated. Methods for inhibiting NEDD8 co-E3 activity and inhibiting cell growth with the NEDD8 co-E3 inhibitors are provided, as well as methods for treating cancer, inflammatory disorders, or pathogenic infections by administering the disclosed NEDD8 co-E3 inhibitors to a subject in need thereof.
These and other aspects of the invention are disclosed in more detail in the description of the invention given below.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1A provides MaxEnt liquid chromatography-time of flight (LC-TOF) spectra of S. cerevisiae yUbc12-His6 purified from WT yeast (left), Δmak3 yeast (middle), or from coexpression with yeast NatC subunits in E. coli (right).
FIG. 1B provides a Western blot of yCul1 (aka Cdc53p) from WT yeast and the indicated deletion strains.
FIGS. 2A and 2B provide results of a pulse-chase assay monitoring [32P] ̃yNEDD8 transfer from the indicated variants of yUbc12 to yCul1C+-yRbx1 (FIG. 2A) or hUbc12 to hCul1C+-hRbx1 (FIG. 2B) in the absence (left panel) or presence (right panel) of yDcn1P (FIG. 2A) or hDcn1P (FIG. 2B). Note the differences in times between left and right, with longer times required to observe activity without yDcn1P.
FIG. 3A provides raw isothermal titration calorimetry (ITC) data for titration of hDcn1P binding to hCul1ctd-hRbx1 (left panel) and thermodynamic parameters for hDcn1P binding to hCul1ctd-hRbx1 (right panel). The measured binding Kd of 1 μM for human proteins is approximately 30-fold worse than for the yeast counterparts (yCul1C+-yRbx1:yDcn1P, Kd=28 nM3).
FIG. 3B provides an autoradiograph of pulse-chase assays monitoring [32] ̃hNEDD8 transfer from hUbc12AcMet to 125 nM hCul1ctd-hRbx1 in the absence or presence of the indicated fold molar excess of hDcn1P. hDcn1P concentration-dependence in pulse-chase assays demonstrates activation while maintaining the absolute requirement for N-terminal acetylation of hUbc12. At saturating hDcn1P concentrations based on Kd measured above, hDcn1P demonstrates a striking co-E3 activity for hNEDD8 only with hUbc12AcMet.
FIG. 3C demonstrates that Cul1 neddylation requires Rbx1 RING domain, even in the presence of Dcn1P and N-terminally acetylated Ubc12AcMet and provides phosphorimager data for a time course of pulse-chase assays monitoring [32P] ̃yNEDD8 transfer from yUbc12AcMet to yCul1C+-yRbx1 and yCul1C+-yRbx1R in the absence or presence of yDcn1P (left panel) or [32P] ̃-hNEDD8 transfer from hUbc12AcMet to hCul1C+-hRbx1 and hCul1C+-hRbx1R in the absence or presence of hDcn1P (right panel). Assays were carried out at a final concentration of 500 nM y or hCul1 or a 500 nM mix of y or hCul1 with y or hDcn1P. Experiments with yeast proteins were performed at 18° C. instead of 4° C. as in other experiments. These differences in experimental design were implemented to highlight the necessity of the RING domain for cullin neddylation. Note differences in time-scales as some chase timepoints are on the order of seconds, and others minutes.
FIG. 4 provides a sequence alignment of Uba3, the Ubc12-binding subunit of NEDD8's E1 enzyme. The ClustalW sequence alignment was generated in MegAlign of Uba3, the Ubc12-binding subunit of NEDD8's heterodimeric E1 enzyme, from human (SEQ ID NO: 59), mouse (SEQ ID NO: 61), rat (SEQ ID NO: 74, Xenopus tropicalis (SEQ ID NO: 75), zebrafish (SEQ ID NO: 76), S. pombe (SEQ ID NO: 77), and S. cerevisiae (SEQ ID NO: 78). Boxes indicate Ubc12-interacting regions identified in crystal structures of human Uba3 complexes with Ubc12 (Huang et al. (2004) Nat Struct Mol Biol 11(10):927-935; Huang et al. (2007) Nature 445(7126):394-398), which are notably absent from S. cerevisiae Uba3p. Black boxes encircling the alignment indicate regions of hUba3 interacting with hUbc12's N-terminal extension. Gray boxes encircling the alignment correspond to Uba3's ubiquitin-fold domain, known to bind the catalytic domain of Ubc12.
FIG. 5A provides a depiction of the structure of hCul1WHB-hDcn1P-AcetylhUbc121-15. hDcn1P is displayed in surface-mode, colored according to conservation between 5 human and yeast Dcn1P orthologs (white—not conserved; black—100% conserved). Acetyl-hUbc121-15 peptide is shown as a helix and stick figure. hCul1WHB is not shown for simplification.
FIG. 5B provides a close-up depiction of Acetyl-hUbc121-15 peptide N-terminus binding to hDcn1P. Left—interacting residues are shown as sticks with electrostatic interactions as dashes. Right—hUbc12's N-acetyl-methionine and residues 2 and 4 as spheres in a mesh view of the hDcn1P crevasse.
FIG. 5C provides a close-up depiction of Acetyl-yUbc121-24 peptide N-terminus binding to yDcn1P. Left—interacting residues are shown as sticks with electrostatic interactions as dashes. Right—yUbc12's N-acetyl-methionine and residues 2 and 4 as spheres in a mesh view of the yDcn1P crevasse.
FIG. 6 provides a sequence alignment of Dcn1P family members. Structure-based sequence alignment of Dcn1P (also called “PONY” (Kurz et al. (2007) Nature 445(7126):394-398)) domains from yeast Dcn1 (SEQ ID NO: 49) and human Dcn1 family members 1-5 (also called DCUND1-DCUND5; SEQ ID NOs: 34, 37, 40, 43, and 46, respectively). Secondary structural elements are indicated above (Kurz et al. (2007) Nature 445(7126):394-398). Black asterisks above the alignment indicate contact sites between yDcn1P and the N-terminally acetylated yUbc12 peptide. Black asterisks below the alignment indicate contact sites between hDcn1P and the N-terminally acetylated hUbc12 peptide. Gray asterisks below the alignment indicate contact sites between hDcn1P and the hCul1WHB sub-domain.
FIG. 7A provides an autoradiograph of pulse-chase assays monitoring [32P]-yNEDD8 transfer from yUbc12Met (top) or yUbc12AcMet (bottom) to yCul1C+-yRbx1 in the absence or presence of the indicated yDcn1P variants (note differences in times between top and bottom panels).
FIG. 7B provides Western blots for yCul1 (aka Cdc53p, top) or HA-tag (bottom) from mid-log whole cell extracts from Δdcn1 or Δdcn1/Δmak10 yeast harboring empty or WT Dcn1-HA, or Y190A mutant Dcn1-HA expression vectors.
FIG. 8A provides an autoradiograph of pulse-chase assays in the absence or presence (500 nM) of the indicated DCUN1D family members monitoring tranfer of NEDD8 from 40 nM Ubc12AcMet ̃NEDD8 (upper panel) or 40 nM Ube2fAcMet ̃NEDD8 (lower panel) to 500 nM Cul1cTD-Rbx1, demonstrating that DCUN1D(1-5) family members stimulate Ubc12AcMet and Ube2fAcMetNEDD8 transfer to Cul1CTD-Rbx1.
FIG. 8B provides an autoradiograph of pulse-chase assays in the absence or presence of 500 nM DCUN1D1 monitoring transfer of NEDD8 from 40 nM Ubc12AcMet ̃NEDD8 to 500 nM Cul(1-5)CTD-Rbx1, demonstrating that DCUN1D1 potentiates NEDD8 transfer from Ubc12AcMet to Cul(1-5) family members.
FIGS. 9A and 9B demonstrate the inhibition of DCUN1D1 potentiated NEDD8 modification of Cul1CTD-Rbx1.
FIG. 9A presents phosphoimager data of pulse-chase assays monitoring the transfer of NEDD8 from 40 nM Ubc12AcMet ̃NEDD8 to 125 nM Cul1CTD-Rbx1 in the absence or presence of 125 nM DCUN1D1P and the indicated amounts of the peptide inhibitor Acetyl-yUbc121-12(6:10 Staple).
FIG. 9B provides quantification of FIG. 9A. The amount of Cul1CTD ̃NEDD8 formed at each time point was quantified and normalized with the amount of Cul1CTD ̃NEDD8 formed at two minutes in the presence of DCUN1D1P and absence of inhibitor set to one.



DETAILED DESCRIPTION OF THE INVENTION
A novel binding pocket in NEDD8 co-E3 proteins that interacts with the amino-terminal (N-terminal) extension of N-terminally acetylated NEDD8 E2 conjugating enzymes is provided. The disclosed E2-binding pocket of NEDD8 co-E3 proteins finds use in methods for identifying inhibitors of the neddylation cascade. Compounds (e.g., peptides, small molecules) that bind to the disclosed binding pocket and inhibit co-E3 activity are provided herein. These co-E3 inhibitors find use in treating diseases such as cancer, inflammatory disorders, and pathogenic infections.
As used herein, the terms “NEDD8” and “neuronal precursor cell expressed developmentally downregulated protein 8” refer to a member of the family of ubiquitin-like proteins (UBLs) that is covalently attached to target proteins. The human, mouse, and rat NEDD8 sequences are each 81 amino acids in length and are about 6 kDa. The terms “NEDD8” and “neuronal precursor cell expressed developmentally down-regulated protein 8” also refer to the Saccharomyces cerevisiae Rub1 protein. Nucleotide and amino acid sequences of NEDD8 proteins are known in the art. Non-limiting examples of NEDD8 sequences include Homo sapiens NEDD8, the nucleotide and amino acid sequences of which are set forth in GenBank Ace. Nos. NM_006156 and NP_006147, respectively (and in SEQ ID NOs: 50 and 51, respectively); Mus musculus NEDD8, the nucleotide and amino acid sequences of which are set forth in GenBank Acc. Nos. NM_008683 and NP_032709, respectively (and in SEQ ID NOs: 52 and 53, respectively) (Kamitani et al. (1997) J Biol Chem 272:28557-28562; Kumar et al. (1992) Biochem Biophys Res Comm 185:1155-1161, each of which are herein incorporated by reference in its entirety); and Saccharomyces cerevisiae Rub1, the nucleotide and amino acid sequences of which are set forth in GenBank Acc. Nos. Y16890 and CAA76516, respectively (and in SEQ ID NOs: 72 and 73, respectively).
NEDD8 is conjugated to its protein targets through a series of enzymatic reactions similar to other E1-E2-E3 ubiquitin-like (UBL) conjugation cascades. First, NEDD8 is activated in an ATP-dependent manner by an E1 enzyme, NEDD8 activating enzyme (NAE), which is a heterodimer of NAE1 and UBA3 subunits.
As used herein, the terms “NAE,” “NEDD8 activating enzyme,” “NEDD8 E 1 activating enzyme,” and “NEDD8 E1 enzyme” refer to a protein capable of catalyzing the transfer of NEDDS's C terminus to the catalytic cysteine of NEDD8 E2, forming a thiolester-linked E2-NEDD8 intermediate (Gong and Yeh (1999) J Biol Chem 274:12036-12042; and Liakopoulos et al. (1998) EMBO J 17:2208-2214; Osaka et al. (1998) Genes Dev 12:2263-2268, each of which are herein incorporated by reference in its entirety).
The only described NEDD8 E1 enzyme is a heterodimer of NAE1 (also referred to as APPBP1; amyloid beta precursor protein binding protein 1; and NEDD8-activating enzyme E1 regulatory subunit) and UBA3 (also referred to as NEDD8-activating enzyme E1 catalytic subunit or UBEC1) subunits. Nucleotide and amino acid sequences of NAE1 proteins are known in the art. Non-limiting examples of NAE1 sequences include Homo sapiens NAE1, the nucleotide and amino acid sequences of which are set forth in GenBank Ace. Nos. NM_001018159 and NP_001018169, respectively (and in SEQ ID NOs: 54 and 55, respectively); and Mus musculus NAE1, the nucleotide and amino acid sequences of which are set forth in GenBank Ace, Nos. NM_144931 and NP_659180, respectively (and in SEQ ID NOs: 56 and 57, respectively). In some embodiments of the presently disclosed invention, the NAE1 has the sequence set forth in SEQ ID NO: 55 or 57, or a biologically active fragment or variant thereof that is capable of interacting with UBA3.
Nucleotide and amino acid sequences of UBA3 proteins are also known in the art. Non-limiting examples of UBA3 sequences include Homo sapiens UBA3, the nucleotide and amino acid sequences of which are set forth in GenBank Ace. Nos. NM_003968 and NP_003959, respectively (and in SEQ ID NOs: 58 and 59, respectively); and Mus musculus UBA3, the nucleotide and amino acid sequences of which are set forth in GenBank Ace. Nos. NM_011666 and NP_035796, respectively (and in SEQ ID NOs: 60 and 61, respectively). The rat, Xenopus tropicalis, zebrafish (Danio rerio), Schizosaccharomyces pombe, and Saccharomyces cerevisiae UBA3 amino acid sequences are set forth in SEQ ID NOs: 74, 75, 76, 77, and 78, respectively. In some embodiments of the presently disclosed invention, the NEDD8 UBA3 protein has the sequence set forth in SEQ ID NO: 59, 61, 74, 75, 76, 77, or 78, or a biologically active variant thereof that is capable of interacting with NAE1 and catalyzing a reaction culminating in the generation of a thiolester-linked E2-NEDD8 intermediate.
Crystal structures of human Uba3 complexes with the NEDD8 E2 enzyme Ubc12 have been solved (Huang et al. (2004) Nat Struct Mol Biol 11(10):927-935; Huang et al. (2007) Nature 445(7126):394-398), which aided in identifying the Ubc12-interacting regions of NEDD8 E1 enzymes. FIG. 4 provides an alignment of UBA3 proteins from various organisms, highlighting the regions of human Uba3 that interact with human Ubc12's N-terminal extension and Uba3's ubiquitin-fold domain, which binds the catalytic domain of Ubc12.
NEDD8 E2 enzymes play central roles in the E1-E2-E3 NEDD8 conjugation cascade. As used herein, the terms “NEDD8 conjugating enzyme,” and “NEDD8 E2 enzyme” refer to a protein capable of transiently binding a NEDD8 E l enzyme for generation of a thiolester-linked E2 ̃UBL intermediate ( ̃ denotes covalent complex; -denotes noncovalent complex) and interacting with a NEDD8 E3 ligase as an E2 ̃UBL intermediate. The two known NEDD8 conjugating enzymes are UBC12, which is also known as UBE2M, and UBE2F. Nucleotide and amino acid sequences of UBC12 proteins are known in the art. Non-limiting examples of UBC12 sequences include Homo sapiens UBC12 (also known as UBE2M), the nucleotide and amino acid sequences of which are set forth in GenBank Acc. Nos. NM_003969 and NP_003960, respectively (and in SEQ ID NOs: 1 and 2, respectively); Mus musculus UBC12, the nucleotide and amino acid sequences of which are set forth in GenBank Ace. Nos. NM_145578 and NP_663553, respectively (and in SEQ ID NOs: 62 and 63, respectively); and Saccharomyces cerevisiae UBC12, the nucleotide and amino acid seqeunces of which are set forth in GenBank Acc. Nos. NM_001182194 and NP_013409, respectively (and in SEQ ID NOs: 5 and 6, respectively).
Nucleotide and amino acid sequences of UBE2F proteins are known in the art. Non-limiting examples of UBE2F sequences include Homo sapiens UBE2F, the nucleotide and amino acid sequences of which are set forth in GenBank Acc. Nos. NM_080678 and NP_542409, respectively (and in SEQ ID NOs: 3 and 4, respectively); and Mus musculus UBE2F, the nucleotide and amino acid sequences of which are set forth in GenBank Ace. Nos, NM_026454 and NP_080730, respectively (and in SEQ ID NOs: 64 and 65, respectively). In some embodiments of the presently disclosed invention, the NEDD8 E2 enzyme has the sequence set forth in SEQ ID NO: 2, 4, 6, 63, or 65, or a biologically active fragment or variant thereof capable of transiently binding a NEDD8 E1 enzyme for generation of a thiolester-linked E2 ̃UBL, intermediate and interacting with a NEDD8 E3 ligase as an E2 ̃UBL intermediate.
NEDD8 E2 enzymes have an about 20 to about 29-residue N-terminal extension upstream of its about 150-residue conserved E2 core domain, which contains the E2 catalytic cysteine. Ubc12's N-terminal sequence is conserved across species, but is not found in other E2 enzymes and is therefore, unique to the NEDD8 pathway. NEDD8 E2 enzymes interact with both NEDD8 E1 and co-E3 proteins via its unique N-terminal extension. As disclosed herein, acetylation of the amino terminus of the N-terminal extension of NEDD8 E2 enzymes enhances binding of yeast Ubc12 to NEDD8 co-E3 proteins and is necessary for binding of mammalian NEDD8 E2 enzymes to co-E3 proteins, but does not affect binding of NEDD8 E2 enzymes to NEDD8 E1 proteins.
Unlike other ubiquitin like proteins known to date, NEDD8 transfer from NEDD8 E2 enzymes to target proteins involves a “dual E3” mechanism (Scott et al. (2010) Mol Cell 39(5):784-796): a RING E3 ligase, Rbx1, is essential for target protein neddylation; an auxiliary co-E3, Dcn1, contains a “potentiating neddylation” domain (Dcn1P) thought to bind different regions of Ubc12 and target proteins from Rbx1 (Kim et al. (2008) J Biol Chem 283(48):33211-33220; Scott et al. (2010) Mol Cell 39(5):784-796; Kurz et al. (2005) Nature 435(7046):1257-1261; and Huang et al. (2011) J Biol Chem, each of which is herein incorporated by reference in its entirety).
As used herein, the terms “NEDD8 E3 ligase” and “NEDD8 E3 enzyme” refer to RING domain NEDD8 E3 ligases capable of interacting with an E2 ̃NEDD8 intermediate and catalyzing the transfer of NEDD8 from the E2 NEDD8 intermediate to an E3-associated target's lysine, producing an isopeptide-bonded target ̃NEDD8 complex; or HECT domain NEDD8 E3 ligases capable of forming an E3 ̃NEDD8 thiolester intermediate and catalyzing the subsequent transfer of NEDD8 onto a target's lysine. The only known NEDD8 E3 ligase is a RING domain E3 ligase. Nucleotide and amino acid sequences of NEDD8 E3 ligases are known in the art. Non-limiting examples of NEDD8 E3 ligase sequences include Homo sapiens Rbx1, the nucleotide and amino acid sequences of which are set forth in GenBank Ace. Nos. NM_014248 and NP_055063, respectively (and in SEQ ID NOs: 66 and 67, respectively); Mus musculus Rbx1, the nucleotide and amino acid sequences of which are set forth in GenBank Ace. Nos. NM_019712 and NP_062686, respectively (and in SEQ ID NOs: 68 and 69, respectively); and Saccharomyces cerevisiae Hrt1, the amino acid sequence of which is set forth in SEQ ID NO: 86. In some embodiments of the presently disclosed invention, the NEDD8 E3 ligase has the sequence set forth in SEQ ID NO: 67, 69, or 86, or a biologically active fragment or variant thereof that is capable of interacting with an E2 ̃NEDD8 intermediate and catalyzing the transfer of NEDD8 onto a target's lysine.
The amino terminus of Hrt1 recruits Cdc53 (yeast cullin ortholog) and the carboxy terminus of Hrt1 comprises the RING domain that activates Ubc12 ̃Rub1 (Rub1 is a NEDD8 ortholog).
As used herein, the terms “NEDD8 co-E3” and “NEDD8 co-E3 protein” refer to a protein capable of interacting with an E2 ̃NEDD8 intermediate and catalyzing the transfer of NEDD8 from the E2 ̃NEDD8 intermediate to an E3-associated target's lysine, wherein the protein does not comprise a RING or HECT domain. A structural model of the Dcn1-Cdc53-Hrt1-Ubc12 complex has been prepared and it suggests that Dcn1 restricts the otherwise flexible RING-Ubc12 ̃Rub1 into a catalytically competent orientation, bringing Ubc12's active site toward Cdc53 (Scott et al. (2010) Mol Cell 39:784-796, which is herein incorporated by reference in its entirety). Thus, the term “co-E3 activity” refers to the ability to interact with an E2 ̃NEDD8 intermediate and catalyze the transfer of NEDD8 from the E2 ̃NEDD8 intermediate to an E3-associated target's lysine in the absence of a RING or HECT domain; to enhance the activity of a RING or HECT domain NEDD8 E3 ligase; or to reduce nonspecific E2 NEDD8 discharge, which refers to the transfer of NEDD8 from an E2 catalytic cysteine to a nucleophile, such as a primary amine. Thus, in some embodiments, a NEDD8 co-E3 increases the catalytic rate (kcat) of the NEDD8 E3 ligase reaction. In particular embodiments, the kcat of a NEDD8 E3 ligase reaction is increased by about 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 50-fold, 100-fold, or greater by a NEDD8 co-E3 ligase.
Nucleotide and amino acid sequences of NEDD8 co-E3 proteins are known in the art. Non-limiting examples of NEDD8 co-E3 sequences include Saccharomyces cerevisiae Dcn1, the nucleotide and amino acid sequences of which are set forth in GenBank Ace. Nos. NM_001182015 and NP_013229, respectively (and in SEQ ID NOs: 47 and 48, respectively); Homo sapiens DCUN1D1 (which is also referred to herein as squamous cell carcinoma-related oncogene or SCCRO or Dcn1), the nucleotide and amino acid sequences of which are set forth in GenBank Ace. Nos. NM_020640 and NP_065691, respectively (and in SEQ ID NOs: 32 and 33, respectively); Mus musculus DCUN1D1 (also referred to herein as Dcn1), the nucleotide and amino acid sequences of which are set forth in GenBank Ace. Nos. NM_001205361 and NP_001192290, respectively (and in SEQ ID NOs: 70 and 71, respectively); Homo sapiens DCUN1D2 (also referred to herein as Dcn2), the nucleotide and amino acid sequences of which are set forth in GenBank Acc. Nos. NM_001014283 and NP_001014305, respectively (and in SEQ ID NOs: 35 and 36, respectively); Homo sapiens DCUN1D3 (also referred to herein as Den3), the nucleotide and amino acid sequences of which are set forth in GenBank Acc. Nos. NM_173475.2 and NP_775746.1, respectively (and in SEQ ID NOs: 38 and 39, respectively); Homo sapiens DCUN1D4 (also referred to herein as Dcn4), the nucleotide and amino acid sequences of which are set forth in GenBank Acc. Nos. NM_001040402 and NP_001035492, respectively (and in SEQ ID NOs: 41 and 42, respectively); and Homo sapiens DCUN1D5 (also referred to herein as Dcn5), the nucleotide and amino acid sequences of which are set forth in GenBank Acc. Nos. NM_032299.3 and NP_115675, respectively (and in SEQ ID NOs: 44 and 45, respectively). In some embodiments of the presently disclosed invention, the NEDD8 co-E3 protein has the sequence set forth in SEQ ID NO: 33, 36, 39, 42, 45, 48, or 71, or a biologically active fragment or variant thereof having co-E3 activity.
Dcn1 and other known NEDD8 co-E3 ligases comprise an amino-terminal ubiquitin associated (UBA) domain (residues 57-69 of yeast Dcn1, the sequence of which is set forth in SEQ ID NO: 48), followed by a disordered linker and a carboxy-terminal “potentiating neddylation” (PONY) domain. The PONY domain of co-E3 ligases, such as Dcn1, is sufficient for binding a NEDD8 E2 (Ubc12) and Cdc53/cullin proteins and for enhancing Cdc53 ̃Rub1 levels (Kurz et al. (2008) Mol Cell 29:23-35). The PONY domain of Homo sapiens DCUN1D1, DCUN1D2, DCUN1D3, DCUN1D4, DCUN1D5, and Saccharomyces cerevisiae Dcn1 is set forth in SEQ ID NOs: 34, 37, 40, 43, 46, and 49, respectively. An alignment of the potentiating neddylation domain from S. cerevisiae Dent, and human Dcn1, Dcn2, Dcn3, Dcn4, and Dcn5 is provided in FIG. 6, which highlights those residues that form contacts with acetylated S. cerevisiae Ubc121-24 or acetylated human Ubc121-15 or with cullin. A further description of the interactions between NEDD8 E2 and co-E3 ligases is provided elsewhere herein.
The most well-characterized neddylated proteins are cullin proteins. As used herein, the term “cullin” refers to a protein belonging to a family of hydrophobic proteins that serve as scaffolds for ubiquitin ligases, such as the Skp, cullin, F-box containing (SCF) ubiquitin ligase. There are 7 known cullin proteins, cullin 1 (also referred to herein as CUL1), cullin 2 (also referred to herein as CUL2), cullin 3 (also referred to herein as CUL3), cullin 4A (also referred to herein as CUL4A), cullin 4B (also referred to herein as CUL4B), cullin 5 (also referred to herein as CUL5), and cullin 7 (also referred to herein as CUL7). The amino acid sequences of human CUL1, CUL2, CUL3, CUL4A, CUL4B, CUL5, and CUL7 are set forth herein as SEQ ID NOs: 79, 80, 81, 82, 83, 84, and 85, respectively. The term “cullin” also refers to the S. cerevisiae Cdc53 ortholog. The yeast Cdc53 protein has a C-terminal domain comprised of several subdomains: a four-helix bundle (4HB) that connects to the N-terminal domain, an α/β-subdomain that binds Rbx1's N-terminal strand, and a C-terminal winged-helix subdomain (WHB) that contains the Rub1 acceptor Lys760. Cdc53's N-terminal region binds Skp1-F box protein complexes, which recruit substrates for ubiquitination and the Cdc53's C-terminal region binds the RING protein Hrt1 (Kamura et al. (1999) Science 284:657-661; Ohta et al. (1999) Mol Cell 3:535-541; Seol et al. (1999) Genes Dev 13:1614-1626; Skowyra et al. (1999) Science 284:662-665; Tan et al. (1999) Mol Cell 3:527-533; each of which are herein incorporated by reference in its entirety). The WHB subdomain of cullin proteins are important for Dcn1 binding (Kurz et al. (2008) Mol Cell 29:23-35).
The presently disclosed invention involves biologically active fragments and variants of the various NEDD8 E1, E2, E3, or co-E3 proteins, or of peptides that bind to the E2-binding pocket of co-E3 proteins and optionally inhibit its activity. As used herein “biologically active” fragments or variants of a polypeptide or a nucleotide sequence encoding the same that is useful in the methods of the invention retains substantially the same function as the respective native sequence. For example, a biologically active fragment or variant of a NEDD8 E2 enzyme is one that retains the ability to transiently bind a NEDD8 E1 enzyme for generation of a thiolester-linked E2-UBL intermediate and interact with a NEDD8 E3 ligase as an E2-UBL intermediate. Such nucleotide sequence fragments can comprise at least about 10, at least about 15, at least about 20, at least about 50, at least about 60, at least about 80, at least about 100, at least about 150, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1,000, at least about 1,500, at least about 2,000, at least about 2,500, at least about 3,000, at least about 3,500, at least about 4,000, at least about 4,500, at least about 5,000 contiguous nucleotides or up to the entire contiguous nucleotides of the nucleotide sequence of interest. Biologically active polypeptide fragments can comprise at least about 5, at least about 10, at least about 15, at least about 20, at least about 50, at least about 60, at least about 80, at least about 100, at least about 150, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1,000 continguous amino acid residues or up to the entire contiguous amino acid residues of the polypeptide. Methods for obtaining such fragments are known in the art and are described in further detail elsewhere herein.
By “variant” is intended substantially similar sequences. Thus, for nucleotide sequences or amino acid sequences, variants include sequences that are functionally equivalent to the nucleotide sequence of interest. Variant nucleotide sequences also include synthetically derived nucleotide sequences that have been generated, for example, by site directed mutagenesis but which still retain the function of the native sequence. Generally, nucleotide sequence variants or amino acid sequence variants of the invention will have at least 40%, at least about 50%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to its respective native nucleotide or amino acid sequence. As discussed elsewhere herein, variants of the nucleotide sequences can encode amino acid sequences that differ conservatively because of the degeneracy of the genetic code. Methods of determining sequence identity are also discussed elsewhere herein.
With respect to the amino acid sequences for the various full length polypeptides, variants include those polypeptides that are derived from the native polypeptides by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native polypeptide; deletion or addition of one or more amino acids at one or more sites in the native polypeptide; or substitution of one or more amino acids at one or more sites in the native polypeptide. Such variants may result from, for example, genetic polymorphism or from human manipulation. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York); Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods Enzymol. 154:367-382; Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.); U.S. Pat. No. 4,873,192; and the references cited therein; herein incorporated by reference. Guidance as to appropriate amino acid substitutions that may not affect biological activity of the various vector polypeptide may be found in the model of Dayhoff et al. (1978) Atlas of Polypeptide Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be preferred. The deletions, insertions, and substitutions of the protein sequences encompassed herein are not expected to produce radical changes in the characteristics of the protein. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays.
By “sequence identity” is intended the same nucleotides or amino acid residues are found within the variant sequence and a reference sequence when a specified, contiguous segment of the nucleotide sequence or amino acid sequence of the variant is aligned and compared to the nucleotide sequence or amino acid sequence of the reference sequence. Methods for sequence alignment and for determining identity between sequences are well known in the art. With respect to optimal alignment of two nucleotide sequences, the contiguous segment of the variant nucleotide sequence may have additional nucleotides or deleted nucleotides with respect to the reference nucleotide sequence. Likewise, for purposes of optimal alignment of two amino acid sequences, the contiguous segment of the variant amino acid sequence may have additional amino acid residues or deleted amino acid residues with respect to the reference amino acid sequence. The contiguous segment used for comparison to the reference nucleotide sequence or reference amino acid sequence will comprise at least 20 contiguous nucleotides, or amino acid residues, and may be 30, 40, 50, 100, or more nucleotides or amino acid residues. Corrections for increased sequence identity associated with inclusion of gaps in the variant's nucleotide sequence or amino acid sequence can be made by assigning gap penalties. Methods of sequence alignment are well known in the art.
The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, percent identity of an amino acid sequence can be determined using the Smith-Waterman homology search algorithm using an affine 6 gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix 62. Alternatively, percent identity of a nucleotide sequence is determined using the Smith-Waterman homology search algorithm using a gap open penalty of 25 and a gap extension penalty of 5. Such a determination of sequence identity can be performed using, for example, the DeCypher Hardware Accelerator from TimeLogic Version G. The Smith-Waterman homology search algorithm is taught in Smith and Waterman (1981) Adv. Appl. Math 2:482-489, herein incorporated by reference. Alternatively, the alignment program GCG Gap (Wisconsin Genetic Computing Group, Suite Version 10.1) using the default parameters may be used. The GCG Gap program applies the Needleman and Wunch algorithm and for the alignment of nucleotide sequences with an open gap penalty of 3 and an extend gap penalty of 1 may be used. Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990) J. Mol. Biol. 2/5:403. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength 12, to obtain nucleotide sequences having sufficient sequence identity. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences having sufficient sequence identity. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov. Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988) CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
Provided herein (see Examples I and Table 24) and in Scott et al. (2011) Science 334:674-678 and supporting online material, which can be found at www.sciencemag.org/content/suppl/2011/09/21/science.1209307.DC1.html (Scott et al. and its supporting online material is herein incorporated by reference in its entirety), is a description of the production and analysis of crystals of four NEDD8 E2/co-E3 complexes: (1) the PONY domain of S. cerevisiae Dcn1 and N-terminally acetylated S. cerevisiae Ubc121-24; (2) human Cul1WHB, the PONY domain of hDcn1 (Dcn1P), and N-terminally acetylated human Ubc121-15; (3) human Cul1WHB, human Dcn1P, and N-terminally acetylated human Ubc121-12 comprising a hydrophobic staple between residues 5 and 9; and (4) the PONY domain of human DCUN1D3 and N-terminally acetylated human Ube2f1-25. The solved structures of (1) the PONY domain of S. cerevisiae Dcn1 and N-terminally acetylated S. cerevisiae Ubc121-24; (2) human Cul1WHB, the PONY domain of hDcn1 (Dcn1P), and N-terminally acetylated human Ubc121-15; and (3) human Cul1WHB, human Dcn1P, and N-terminally acetylated human Ubc121-12 comprising a hydrophobic staple between residues 5 and 9 have been deposited in the Structural Bioinformatics Research Collaboratory for Structural Bioinformatics Protein Data Bank as accession numbers 3TDI, 3TDU, and 3TDZ, respectively, and each of which are herein incorporated by reference in its entirety.
As used herein, the terms “molecular structure” refer to the arrangement of atoms within a particular object (e.g., polypeptide). A three-dimensional molecular structure of a polypeptide is a representation of the tertiary or quaternary structure of the polypeptide.
As used herein, the term “atomic coordinates” refers to mathematical coordinates (represented as “X,” “Y” and “Z” values) that describe the positions of atoms in a crystal of a polypeptide with respect to a chosen crystallographic origin. As used herein, the term “crystallographic origin” refers to a reference point in the crystal unit cell with respect to the crystallographic symmetry operation. These atomic coordinates can be used to generate a three-dimensional representation of the molecular structure of the polypeptide. When referring to an E2-binding pocket having a particular set of atomic coordinates of a particular table provided herein, this refers to both the atom itself as well as its “X”, “Y”, and “Z” values.
Analysis of the four solved three-dimensional molecular structures led to the identification of a novel NEDD8 E2-binding pocket within NEDD8 co-E3 proteins. As used herein, an “E2-binding pocket” or “NEDD8 E2-binding pocket” of a NEDD8 co-E3 protein is comprised of the atoms of the NEDD8 co-E3 protein that interact with a NEDD8 E2 protein, and in some embodiments, additional atoms of the amino acids comprising the interacting atoms. Those atoms of the NEDD8 co-E3 protein that interact with a NEDD8 E2 protein are those atoms that form a bond (e.g., hydrogen bond, van der Waals interactions), with an atom of a NEDD8 E2 protein.
In some embodiments, the E2-binding pocket of a NEDD8 co-E3 protein comprises the atoms of Table 1, which are found in amino acid residues Ile90, Leu93, Val94, Ile97, Leu104, Glu105, Asp106, Leu107, Thr109, Leu110, Ala113, Leu121, Glu122, Leu173, Ile174, Leu175, Asp176, Gln189, Tyr190, and Leu193 of S. cerevisiae Dcn1 (SEQ ID NO: 48), or a structural variant thereof. In particular embodiments, the E2-binding pocket of a NEDD8 co-E3 protein comprises at least one of the following amino acid residues: an isoleucine corresponding to position 90 of SEQ ID NO: 48, a leucine corresponding to position 93 of SEQ ID NO: 48, a valine corresponding to position 94 of SEQ ID NO: 48, an isoleucine corresponding to position 97 of SEQ ID NO: 48, a leucine corresponding to position 104 of SEQ ID NO: 48, a glutamic acid corresponding to position 105 of SEQ ID NO: 48, an aspartic acid corresponding to position 106 of SEQ ID NO: 48, a leucine corresponding to position 107 of SEQ ID NO: 48, a threonine corresponding to position 109 of SEQ ID NO: 48, a leucine corresponding to position 110 of SEQ ID NO: 48, an alanine corresponding to position 113 of SEQ ID NO: 48, a leucine corresponding to position 121 of SEQ ID NO: 48, a glutamic acid corresponding to position 122 of SEQ ID NO: 48, a leucine corresponding to position 173 of SEQ ID NO: 48, an isoleucine corresponding to position 174 of SEQ ID NO: 48, a leucine corresponding to position 175 of SEQ ID NO: 48, an aspartic acid corresponding to position 176 of SEQ ID NO: 48, a glutamine corresponding to position 189 of SEQ ID NO: 48, a tyrosine corresponding to position 190 of SEQ ID NO: 48, and a leucine corresponding to position 193 of SEQ ID NO: 48.
As used herein, an amino acid residue of a NEDD8 co-E3 protein at the position corresponding to a particular amino acid residue of SEQ ID NO: 33, 36, 39, 42, 45, or 48 refers to the amino acid residue within the NEDD8 co-E3 protein that appears opposite the amino acid residue at a particular position in SEQ ID NO: 33, 36, 39, 42, 45, or 48 when the NEDD8 co-E3 protein sequence is aligned with SEQ ID NO: 33, 36, 39, 42, 45, or 48 for maximum homology using an alignment program, such as one known in the art (e.g., the GAP program in the GCG software package, using either a BLOSUM62 matrix or a PAM250 matrix).
In some of these embodiments, the E2-binding pocket comprises the atomic coordinates of Table 2 or 3, or a structural variant thereof. In particular embodiments, the co-E3 protein has the amino acid sequence set forth in SEQ ID NO: 48.
In certain embodiments, the E2-binding pocket of a NEDD8 co-E3 protein comprises the atoms of Table 1 and Table 4. The atoms in Table 4 are found in amino acid residues Asn84, Asp89, Asp91, Gly101, Tyr102, Asn103, and Glu186 of SEQ ID NO: 48. Thus, in some embodiments, the E2-binding pocket of a NEDD8 co-E3 protein comprises at least one of the following amino acid residues: an isoleucine corresponding to position 90 of SEQ ID NO: 48, a leucine corresponding to position 93 of SEQ ID NO: 48, a valine corresponding to position 94 of SEQ ID NO: 48, an isoleucine corresponding to position 97 of SEQ ID NO: 48, a leucine corresponding to position 104 of SEQ ID NO: 48, a glutamic acid corresponding to position 105 of SEQ ID NO: 48, an aspartic acid corresponding to position 106 of SEQ ID NO: 48, a leucine corresponding to position 107 of SEQ ID NO: 48, a threonine corresponding to position 109 of SEQ ID NO: 48, a leucine corresponding to position 110 of SEQ ID NO: 48, an alanine corresponding to position 113 of SEQ ID NO: 48, a leucine corresponding to position 121 of SEQ ID NO: 48, a glutamic acid corresponding to position 122 of SEQ ID NO: 48, a leucine corresponding to position 173 of SEQ ID NO: 48, an isoleucine corresponding to position 174 of SEQ ID NO: 48, a leucine corresponding to position 175 of SEQ ID NO: 48, an aspartic acid corresponding to position 176 of SEQ ID NO: 48, a glutamine corresponding to position 189 of SEQ ID NO: 48, a tyrosine corresponding to position 190 of SEQ ID NO: 48, a leucine corresponding to position 193 of SEQ ID NO: 48, an asparagine corresponding to position 84 of SEQ ID NO: 48; an aspartic acid corresponding to position 89 of SEQ ID NO: 48, an aspartic acid corresponding to position 91 of SEQ ID NO: 48, a glycine corresponding to position 101 of SEQ ID NO: 48, a tyrosine corresponding to position 102 of SEQ ID NO: 48, an asparagine corresponding to position 103 of SEQ ID NO: 48, and a glutamic acid corresponding to position 186 of SEQ ID NO: 48.
In some of these embodiments, the E2-binding pocket comprises the atomic coordinates of Table 2 and 5 or a structural variant thereof. In other embodiments, the E2-binding pocket comprises the atomic coordinates of Table 3 and 6, or a structural variant thereof.







TABLE 1







Minimal contacts between S. cerevisiae Dcn1


and S. cerevisiae Ubc12 with yUbc12 Chain C


interactions with yDcn1P Chain A and yUbc12


Chain D interactions with yDcn1P Chain B shown.











Distance


yUbc12 Atom
yDcn1 Atom
(Å)
















Acetyl-Met
1C
O
Leu
104A
CB
4.01





Leu
104A
O
4.1





Leu
104A
CD2
4.17


Acetyl-Met
1C
C
Leu
104A
O
3.82





Tyr
190A
OH
4.08


Acetyl-Met
1C
CA
Leu
104A
O
3.58





Tyr
190A
OH
4.44





Thr
109A
OG1
4.21


Acetyl-Met
1C
N
Leu
104A
C
3.94





Leu
104A
O
2.73





Thr
109A
OG1
3.31





Thr
109A
CB
4.46


Acetyl-Met
1C
CT1
Tyr
190A
CE1
3.9





Leu
104A
O
3.61





Tyr
190A
OH
4.34





Thr
109A
OG1
3.44





Leu
110A
CD2
4.36





Leu
110A
CG
4.16


Acetyl-Met
1C
OT
Tyr
190A
CD1
4.33





Tyr
190A
CE1
3.29





Tyr
190A
CZ
3.96





Tyr
190A
OH
3.71





Thr
109A
OG1
4.26





Leu
110A
CD2
3.57





Leu
110A
CG
3.72





Leu
110A
CD1
4.26


Acetyl-Met
1C
CT2
Tyr
190A
CE1
4.3





Leu
107A
CD2
3.9





Leu
104A
O
3.55





Glu
105A
C
4.32





Glu
105A
O
4.27





Asp
106A
C
4.13





Asp
106A
O
3.63





Leu
107A
N
4.37





Leu
107A
CA
4.1





Thr
109A
OG1
3.29





Leu
110A
CG
4.07





Leu
110A
CD1
4.36


Acetyl-Met
1C
CB
Leu
104A
CB
4.45





Leu
104A
O
3.65





Thr
109A
OG1
3.9





Thr
109A
CB
4.38


Acetyl-Met
1C
CG
Leu
104A
O
4.32





Thr
109A
OG1
3.11





Ile
97A
CD1
4.01





Thr
109A
CB
3.36





Thr
109A
CG2
4.25





Thr
109A
C
4.41





Thr
109A
O
4.36


Acetyl-Met
1C
SD
Leu
121A
CD1
3.76





Thr
109A
OG1
4.44





Leu
121A
CG
4.44





Leu
121A
CD2
3.83





Thr
109A
O
4.39


Acetyl-Met
1C
CE
Leu
121A
CD1
4.02





Thr
109A
OG1
4.42





Leu
110A
CD2
3.5





Leu
173A
CD1
3.68





Leu
110A
N
4.36





Leu
110A
CA
4.16





Leu
110A
CG
4.29





Leu
121A
CD2
4.07





Thr
109A
C
4.27





Ala
113A
CB
3.74





Thr
109A
O
3.9


Leu
2C
N
Tyr
190A
CE1
4.48





Tyr
190A
CZ
4.15





Leu
104A
O
4.35





Tyr
190A
OH
3.13


Leu
2C
CA
Tyr
190A
OH
3.59


Leu
2C
CB
Gln
189A
NE2
4.2





Tyr
190A
CE1
4.43





Tyr
190A
CZ
3.85





Tyr
190A
CE2
4.43





Tyr
190A
OH
3.31


Leu
2C
CG
Gln
189A
NE2
4.49





Tyr
190A
CE1
3.97





Tyr
190A
CZ
3.87





Leu
193A
CD1
4.27





Tyr
190A
OH
3.75


Leu
2C
CD1
Gln
189A
CB
4.19





Gln
189A
NE2
3.5





Tyr
190A
CE1
4.16





Tyr
190A
CZ
4.07





Tyr
190A
CE2
4.35





Leu
193A
CD1
3.87





Tyr
190A
OH
4.38


Leu
2C
CD2
Leu
193A
CD1
4.35





Leu
104A
O
4.32





Glu
105A
N
4.5





Glu
105A
CA
3.87





Glu
105A
CB
3.94





Glu
105A
CG
3.74


Leu
2C
C
Tyr
190A
OH
3.89


Lys
3C
N
Tyr
190A
OH
3.59


Lys
3C
CB
Leu
121A
CD1
4.45





Tyr
190A
OH
4.29


Lys
3C
CD
Ile
174A
CA
4.25





Ile
174A
C
4.32





Ile
174A
O
3.82





Leu
173A
O
3.41





Leu
173A
C
4.44


Lys
3C
CE
Ile
174A
C
4.41





Ile
174A
O
3.77





Glu
122A
OE2
3.24





Leu
175A
O
4.18





Asp
176A
OD1
3.87





Leu
173A
O
3.62





Glu
122A
CD
4.13


Lys
3C
NZ
Asp
176A
N
4.04





Asp
176A
CB
4.24





Asp
176A
CG
3.45





Asp
176A
OD2
4.25





Ile
174A
C
4.41





Ile
174A
O
3.45





Glu
122A
OE2
3.71





Leu
175A
C
4.28





Leu
175A
O
4.15





Asp
176A
CA
3.81





Asp
176A
OD1
2.45


Leu
4C
CB
Val
94A
CG2
4.49





Leu
104A
CD2
4.41


Leu
4C
CG
Val
94A
CG2
4.16


Leu
4C
CD1
Leu
121A
CD1
3.91





Ile
90A
CG2
3.86





Leu
121A
CB
4.03





Leu
121A
CG
4.3





Leu
93A
CD2
4.49





Leu
121A
CD2
4.38


Leu
4C
CD2
Val
94A
CG2
4.26





Leu
93A
CD2
4.31





Ile
97A
CD1
4.05





Leu
93A
CG
4.35


Acetyl-Met
1D
O
Leu
104B
CD2
4.46





Tyr
190B
OH
4.22





Leu
104B
O
4.38





Leu
104B
CB
3.84





Leu
104B
CG
4.35





Leu
104B
CD1
4.14


Acetyl-Met
1D
C
Tyr
190B
OH
3.32





Leu
104B
C
4.47





Leu
104B
O
3.71





Leu
104B
CB
4.09





Tyr
190B
CZ
4.45


Acetyl-Met
1D
CA
Tyr
190B
OH
3.45





Leu
104B
O
3.61





Thr
109B
OG1
4.13


Acetyl-Met
1D
N
Tyr
190B
OH
3.67





Leu
104B
C
4.01





Leu
104B
O
2.84





Thr
109B
OG1
3.38





Tyr
190B
CZ
4.37





Tyr
190B
CE1
4.31


Acetyl-Met
1D
CT1
Leu
110B
CG
3.8





Leu
110B
CD1
4.17





Leu
110B
CD2
4.41





Tyr
190B
OH
3.58





Leu
104B
O
3.81





Thr
109B
OG1
3.55





Tyr
190B
CZ
3.97





Tyr
190B
CE1
3.52


Acetyl-Met
1D
OT
Leu
110B
CB
4.48





Leu
110B
CG
3.18





Leu
110B
CD1
3.64





Leu
110B
CD2
3.44





Tyr
190B
OH
3.24





Thr
109B
OG1
4.24





Tyr
190B
CZ
3.66





Tyr
190B
CD1
4.35





Tyr
190B
CE1
3.17


Acetyl-Met
1D
CT2
Leu
110B
CG
3.84





Leu
110B
CD1
3.84





Leu
107B
CA
3.96





Leu
104B
O
3.81





Thr
109B
OG1
3.62





Asp
106B
C
4.18





Asp
106B
O
3.68





Leu
107B
N
4.31





Leu
107B
CD2
3.95





Tyr
190B
CE1
3.87





Leu
193B
CD1
4.19


Acetyl-Met
1D
CB
Thr
109B
CB
4.21





Leu
104B
CD2
4.23





Leu
104B
C
4.45





Leu
104B
O
3.72





Thr
109B
OG1
3.58





Leu
104B
CA
4.28





Leu
104B
CB
4.19


Acetyl-Met
1D
CG
Thr
109B
C
4.33





Thr
109B
O
4.2





Ile
97B
CD1
3.98





Thr
109B
CB
3.55





Thr
109B
OG1
3.19


Acetyl-Met
1D
SD
Leu
121B
CD2
4.09





Leu
121B
CD1
3.91


Acetyl-Met
1D
CE
Ala
113B
CB
3.73





Thr
109B
C
4.42





Thr
109B
O
3.96





Leu
121B
CD2
4.21





Leu
173B
CD1
3.7





Leu
110B
CA
4.26





Leu
110B
CG
4.24





Leu
110B
CD2
3.58





Leu
121B
CD1
4.35


Leu
2D
N
Tyr
190B
OH
2.85





Leu
104B
O
3.88





Tyr
190B
CZ
3.74





Tyr
190B
CE2
4.34


Leu
2D
CA
Tyr
190B
OH
3.65





Tyr
190B
CZ
4.37


Leu
2D
CB
Tyr
190B
OH
3.86





Tyr
190B
CZ
4.08





Tyr
190B
CE2
3.87


Leu
2D
CG
Tyr
190B
OH
4.39





Tyr
190B
CZ
4.31





Tyr
190B
CE2
4.21





Leu
193B
CD1
4.05


Leu
2D
CD1
Tyr
190B
CE2
4.27





Tyr
190B
CD2
4.41





Gln
189B
O
4.16





Leu
193B
CD1
3.98





Gln
189B
CB
4.05





Gln
189B
C
4.36


Leu
2D
CD2
Leu
104B
C
4.33





Leu
104B
O
4.17





Glu
105B
CA
4





Glu
105B
N
4.22





Glu
105B
CB
4.46





Glu
105B
CG
3.87





Leu
193B
CD1
4.37


Leu
2D
C
Tyr
190B
OH
3.82


Lys
3D
N
Tyr
190B
OH
3.01





Tyr
190B
CZ
3.98





Tyr
190B
CE2
4.04


Lys
3D
CA
Tyr
190B
OH
3.92


Lys
3D
CB
Leu
121B
CD1
4.22





Tyr
190B
OH
3.63


Lys
3D
CG
Leu
173B
O
4.25





Leu
121B
CD1
4


Lys
3D
CD
Leu
173B
O
4.3





Ile
174B
C
4.29





Ile
174B
CA
4.31





Ile
174B
O
3.58





Asp
176B
OD1
4


Lys
3D
CE
Leu
173B
O
4.42





Glu
122B
OE2
4.11





Ile
174B
C
4.47





Ile
174B
O
3.64





Asp
176B
CG
4.26





Asp
176B
OD1
3.08


Lys
3D
NZ
Leu
173B
O
3.4





Leu
175B
O
3.56





Glu
122B
CD
3.93





Glu
122B
OE2
2.93





Ile
174B
C
4.15





Leu
175B
C
4.08





Ile
174B
O
3.64





Asp
176B
N
4.29





Asp
176B
CA
4.18





Asp
176B
OD1
3.72


Leu
4D
N
Leu
121B
CD1
4.4


Leu
4D
CG
Ile
90B
CG2
4.02





Ile
90B
O
4.47





Val
94B
CG2
3.81


Leu
4D
CD1
Ile
90B
CG1
4.29





Leu
121B
CB
4.16





Leu
93B
CD2
4.43





Leu
121B
CD2
4.33





Ile
90B
CB
4.4





Ile
90B
CG2
3.7





Leu
121B
CG
4.39





Leu
121B
CD1
4.07


Leu
4D
CD2
Leu
93B
CB
4.15





Leu
93B
CG
3.79





Leu
93B
CD2
3.77





Ile
97B
CD1
4.02





Val
94B
N
4.34





Val
94B
CG2
3.86


Leu
4D
O
Ile
90B
CG2
3.87
















TABLE 2







Atomic coordinates of amino acid residues of


yDcn1 Chain B essential for binding yUbc12.


















Atom

Atom





Occu-
B-



No.

Name


X
Y
Z
pancy
factor





















ATOM
187
N
ILE
B
90
−17.000
−3.153
−25.375
1.00
26.78


ATOM
188
CA
ILE
B
90
−15.892
−2.790
−24.488
1.00
27.28


ATOM
189
CB
ILE
B
90
−14.690
−2.236
−25.299
1.00
27.75


ATOM
190
CG1
ILE
B
90
−14.046
−3.360
−26.125
1.00
28.57


ATOM
191
CD1
ILE
B
90
−13.276
−2.873
−27.351
1.00
31.07


ATOM
192
CG2
ILE
B
90
−13.656
−1.614
−24.375
1.00
28.76


ATOM
193
C
ILE
B
90
−16.331
−1.797
−23.407
1.00
27.14


ATOM
194
O
ILE
B
90
−15.941
−1.928
−22.242
1.00
27.17


ATOM
209
N
LEU
B
93
−18.305
−3.687
−20.756
1.00
23.07


ATOM
210
CA
LEU
B
93
−17.487
−4.475
−19.843
1.00
22.86


ATOM
211
CB
LEU
B
93
−16.484
−5.377
−20.591
1.00
22.77


ATOM
212
CG
LEU
B
93
−15.645
−6.359
−19.739
1.00
23.37


ATOM
213
CD1
LEU
B
93
−16.488
−7.155
−18.740
1.00
23.06


ATOM
214
CD2
LEU
B
93
−14.825
−7.312
−20.618
1.00
23.28


ATOM
215
C
LEU
B
93
−16.802
−3.612
−18.784
1.00
22.47


ATOM
216
O
LEU
B
93
−16.760
−3.987
−17.614
1.00
22.13


ATOM
217
N
VAL
B
94
−16.280
−2.461
−19.207
1.00
22.50


ATOM
218
CA
VAL
B
94
−15.661
−1.472
−18.311
1.00
22.44


ATOM
219
CB
VAL
B
94
−15.067
−0.282
−19.111
1.00
22.56


ATOM
220
CG1
VAL
B
94
−14.565
0.820
−18.189
1.00
21.37


ATOM
221
CG2
VAL
B
94
−13.932
−0.771
−20.001
1.00
23.01


ATOM
222
C
VAL
B
94
−16.636
−0.969
−17.241
1.00
22.81


ATOM
223
O
VAL
B
94
−16.310
−0.957
−16.049
1.00
22.30


ATOM
244
N
ILE
B
97
−17.365
−3.889
−14.797
1.00
23.81


ATOM
245
CA
ILE
B
97
−16.271
−4.009
−13.840
1.00
24.10


ATOM
246
CB
ILE
B
97
−14.901
−3.980
−14.553
1.00
23.81


ATOM
247
CG1
ILE
B
97
−14.670
−5.292
−15.301
1.00
21.97


ATOM
248
CD1
ILE
B
97
−13.777
−5.147
−16.491
1.00
19.97


ATOM
249
CG2
ILE
B
97
−13.760
−3.721
−13.556
1.00
23.53


ATOM
250
C
ILE
B
97
−16.347
−2.919
−12.769
1.00
24.99


ATOM
251
O
ILE
B
97
−16.207
−3.208
−11.577
1.00
24.71


ATOM
302
N
LEU
B
104
−9.556
−1.736
−13.282
1.00
30.61


ATOM
303
CA
LEU
B
104
−9.073
−2.533
−14.405
1.00
30.52


ATOM
304
CB
LEU
B
104
−8.981
−1.689
−15.675
1.00
30.40


ATOM
305
CG
LEU
B
104
−10.217
−0.909
−16.135
1.00
30.20


ATOM
306
CD1
LEU
B
104
−9.854
−0.018
−17.301
1.00
28.88


ATOM
307
CD2
LEU
B
104
−11.395
−1.826
−16.495
1.00
30.08


ATOM
308
C
LEU
B
104
−7.719
−3.189
−14.112
1.00
30.86


ATOM
309
O
LEU
B
104
−7.334
−4.139
−14.789
1.00
31.23


ATOM
310
N
GLU
B
105
−7.008
−2.677
−13.107
1.00
30.82


ATOM
311
CA
GLU
B
105
−5.735
−3.255
−12.660
1.00
30.75


ATOM
312
CB
GLU
B
105
−4.947
−2.250
−11.801
1.00
31.01


ATOM
313
CG
GLU
B
105
−4.454
−1.002
−12.547
1.00
34.35


ATOM
314
CD
GLU
B
105
−5.495
0.131
−12.635
1.00
38.44


ATOM
315
OE1
GLU
B
105
−6.630
−0.025
−12.121
1.00
39.33


ATOM
316
OE2
GLU
B
105
−5.166
1.192
−13.223
1.00
39.74


ATOM
317
C
GLU
B
105
−5.939
−4.552
−11.871
1.00
29.84


ATOM
318
O
GLU
B
105
−4.991
−5.313
−11.687
1.00
29.75


ATOM
319
N
ASP
B
106
−7.164
−4.787
−11.393
1.00
28.79


ATOM
320
CA
ASP
B
106
−7.466
−5.960
−10.575
1.00
27.99


ATOM
321
CB
ASP
B
106
−8.806
−5.800
−9.845
1.00
28.43


ATOM
322
CG
ASP
B
106
−8.778
−4.721
−8.757
1.00
30.42


ATOM
323
OD1
ASP
B
106
−7.717
−4.520
−8.116
1.00
32.67


ATOM
324
OD2
ASP
B
106
−9.836
−4.082
−8.527
1.00
31.35


ATOM
325
C
ASP
B
106
−7.492
−7.233
−11.420
1.00
27.00


ATOM
326
O
ASP
B
106
−8.260
−7.336
−12.384
1.00
26.52


ATOM
327
N
LEU
B
107
−6.651
−8.199
−11.051
1.00
25.92


ATOM
328
CA
LEU
B
107
−6.592
−9.494
−11.756
1.00
25.10


ATOM
329
CB
LEU
B
107
−5.474
−10.388
−11.204
1.00
25.01


ATOM
330
CG
LEU
B
107
−4.012
−10.030
−11.483
1.00
25.41


ATOM
331
CD1
LEU
B
107
−3.132
−11.160
−11.002
1.00
26.14


ATOM
332
CD2
LEU
B
107
−3.759
−9.781
−12.962
1.00
25.10


ATOM
333
C
LEU
B
107
−7.926
−10.248
−11.743
1.00
24.17


ATOM
334
O
LEU
B
107
−8.231
−10.968
−12.688
1.00
23.94


ATOM
340
N
THR
B
109
−10.630
−9.254
−12.532
1.00
20.74


ATOM
341
CA
THR
B
109
−11.358
−8.910
−13.749
1.00
20.00


ATOM
342
CB
THR
B
109
−11.009
−7.506
−14.241
1.00
20.13


ATOM
343
OG1
THR
B
109
−9.607
−7.447
−14.529
1.00
21.02


ATOM
344
CG2
THR
B
109
−11.372
−6.445
−13.185
1.00
19.14


ATOM
345
C
THR
B
109
−11.098
−9.920
−14.870
1.00
19.91


ATOM
346
O
THR
B
109
−11.932
−10.086
−15.768
1.00
19.52


ATOM
347
N
LEU
B
110
−9.940
−10.587
−14.809
1.00
19.41


ATOM
348
CA
LEU
B
110
−9.612
−11.680
−15.716
1.00
18.82


ATOM
349
CB
LEU
B
110
−8.118
−11.996
−15.665
1.00
18.81


ATOM
350
CG
LEU
B
110
−7.233
−11.177
−16.602
1.00
19.63


ATOM
351
CD1
LEU
B
110
−5.757
−11.366
−16.250
1.00
18.67


ATOM
352
CD2
LEU
B
110
−7.519
−11.548
−18.066
1.00
18.09


ATOM
353
C
LEU
B
110
−10.410
−12.941
−15.406
1.00
18.59


ATOM
354
O
LEU
B
110
−10.817
−13.646
−16.324
1.00
17.95


ATOM
369
N
ALA
B
113
−13.765
−12.239
−16.984
1.00
18.55


ATOM
370
CA
ALA
B
113
−13.760
−12.421
−18.436
1.00
18.75


ATOM
371
CB
ALA
B
113
−12.557
−11.717
−19.068
1.00
18.05


ATOM
372
C
ALA
B
113
−13.772
−13.909
−18.784
1.00
19.11


ATOM
373
O
ALA
B
113
−14.497
−14.348
−19.687
1.00
18.94


ATOM
434
N
LEU
B
121
−11.230
−9.617
−26.513
1.00
30.79


ATOM
435
CA
LEU
B
121
−11.866
−8.449
−25.908
1.00
31.55


ATOM
436
CB
LEU
B
121
−10.860
−7.659
−25.075
1.00
31.43


ATOM
437
CG
LEU
B
121
−10.332
−8.355
−23.817
1.00
31.12


ATOM
438
CD1
LEU
B
121
−9.213
−7.527
−23.191
1.00
30.04


ATOM
439
CD2
LEU
B
121
−11.457
−8.632
−22.807
1.00
30.44


ATOM
440
C
LEU
B
121
−12.509
−7.548
−26.950
1.00
32.31


ATOM
441
O
LEU
B
121
−13.418
−6.783
−26.638
1.00
32.69


ATOM
442
N
GLU
B
122
−12.036
−7.659
−28.188
1.00
33.07


ATOM
443
CA
GLU
B
122
−12.507
−6.842
−29.293
1.00
33.86


ATOM
444
CB
GLU
B
122
−11.527
−6.931
−30.474
1.00
34.09


ATOM
445
CG
GLU
B
122
−10.065
−6.525
−30.148
1.00
35.66


ATOM
446
CD
GLU
B
122
−9.214
−7.651
−29.515
1.00
37.96


ATOM
447
OE1
GLU
B
122
−9.750
−8.745
−29.236
1.00
38.95


ATOM
448
OE2
GLU
B
122
−7.993
−7.440
−29.300
1.00
38.39


ATOM
449
C
GLU
B
122
−13.922
−7.244
−29.727
1.00
34.10


ATOM
450
O
GLU
B
122
−14.689
−6.403
−30.199
1.00
34.37


ATOM
874
N
LEU
B
173
−5.876
−13.081
−24.295
1.00
27.16


ATOM
875
CA
LEU
B
173
−6.626
−11.828
−24.339
1.00
27.91


ATOM
876
CB
LEU
B
173
−7.349
−11.582
−23.011
1.00
27.86


ATOM
877
CG
LEU
B
173
−8.606
−12.381
−22.684
1.00
26.53


ATOM
878
CD1
LEU
B
173
−9.208
−11.801
−21.423
1.00
23.89


ATOM
879
CD2
LEU
B
173
−9.605
−12.329
−23.848
1.00
25.91


ATOM
880
C
LEU
B
173
−5.762
−10.612
−24.635
1.00
28.87


ATOM
881
O
LEU
B
173
−6.252
−9.630
−25.203
1.00
29.01


ATOM
882
N
ILE
B
174
−4.494
−10.655
−24.222
1.00
29.78


ATOM
883
CA
ILE
B
174
−3.571
−9.545
−24.473
1.00
30.66


ATOM
884
CB
ILE
B
174
−2.274
−9.654
−23.640
1.00
30.37


ATOM
885
CG1
ILE
B
174
−2.571
−9.533
−22.145
1.00
30.97


ATOM
886
CD1
ILE
B
174
−1.443
−10.039
−21.251
1.00
31.49


ATOM
887
CG2
ILE
B
174
−1.287
−8.569
−24.026
1.00
30.73


ATOM
888
C
ILE
B
174
−3.230
−9.489
−25.958
1.00
31.61


ATOM
889
O
ILE
B
174
−2.929
−8.415
−26.494
1.00
31.77


ATOM
890
N
LEU
B
175
−3.279
−10.649
−26.616
1.00
32.57


ATOM
891
CA
LEU
B
175
−3.011
−10.740
−28.046
1.00
33.68


ATOM
892
CB
LEU
B
175
−3.232
−12.164
−28.553
1.00
33.40


ATOM
893
CG
LEU
B
175
−2.038
−13.108
−28.646
1.00
33.23


ATOM
894
CD1
LEU
B
175
−2.475
−14.462
−29.171
1.00
32.15


ATOM
895
CD2
LEU
B
175
−0.973
−12.517
−29.552
1.00
33.47


ATOM
896
C
LEU
B
175
−3.915
−9.793
−28.806
1.00
34.64


ATOM
897
O
LEU
B
175
−5.136
−9.836
−28.634
1.00
35.03


ATOM
898
N
ASP
B
176
−3.314
−8.918
−29.614
1.00
35.77


ATOM
899
CA
ASP
B
176
−4.084
−8.053
−30.510
1.00
36.75


ATOM
900
CB
ASP
B
176
−3.204
−6.954
−31.120
1.00
37.22


ATOM
901
CG
ASP
B
176
−2.718
−5.939
−30.098
1.00
38.70


ATOM
902
OD1
ASP
B
176
−3.105
−6.022
−28.908
1.00
41.31


ATOM
903
OD2
ASP
B
176
−1.935
−5.044
−30.492
1.00
40.73


ATOM
904
C
ASP
B
176
−4.670
−8.929
−31.614
1.00
36.97


ATOM
905
O
ASP
B
176
−4.183
−10.046
−31.831
1.00
37.27


ATOM
1001
N
GLN
B
189
3.796
−4.772
−18.447
1.00
20.32


ATOM
1002
CA
GLN
B
189
2.672
−4.221
−17.680
1.00
21.04


ATOM
1003
CB
GLN
B
189
1.788
−3.313
−18.553
1.00
21.59


ATOM
1004
CG
GLN
B
189
2.471
−2.017
−19.012
1.00
24.86


ATOM
1005
CD
GLN
B
189
1.627
−1.176
−19.980
1.00
30.95


ATOM
1006
OE1
GLN
B
189
0.584
−1.617
−20.484
1.00
32.81


ATOM
1007
NE2
GLN
B
189
2.091
0.057
−20.240
1.00
34.12


ATOM
1008
C
GLN
B
189
1.828
−5.307
−17.005
1.00
20.86


ATOM
1009
O
GLN
B
189
1.480
−5.169
−15.829
1.00
20.70


ATOM
1010
N
TYR
B
190
1.512
−6.387
−17.725
1.00
20.44


ATOM
1011
CA
TYR
B
190
0.788
−7.510
−17.097
1.00
20.64


ATOM
1012
CB
TYR
B
190
0.072
−8.398
−18.121
1.00
20.85


ATOM
1013
CG
TYR
B
190
−1.295
−7.875
−18.511
1.00
22.90


ATOM
1014
CD1
TYR
B
190
−2.454
−8.460
−18.007
1.00
24.80


ATOM
1015
CE1
TYR
B
190
−3.720
−7.982
−18.364
1.00
25.31


ATOM
1016
CZ
TYR
B
190
−3.825
−6.906
−19.230
1.00
25.68


ATOM
1017
OH
TYR
B
190
−5.069
−6.425
−19.583
1.00
25.69


ATOM
1018
CE2
TYR
B
190
−2.681
−6.306
−19.742
1.00
25.12


ATOM
1019
CD2
TYR
B
190
−1.430
−6.789
−19.384
1.00
23.39


ATOM
1020
C
TYR
B
190
1.634
−8.354
−16.138
1.00
20.10


ATOM
1021
O
TYR
B
190
1.133
−8.764
−15.094
1.00
19.57


ATOM
1045
N
LEU
B
193
1.535
−6.975
−12.748
1.00
20.07


ATOM
1046
CA
LEU
B
193
0.316
−7.313
−12.019
1.00
20.06


ATOM
1047
CB
LEU
B
193
−0.922
−7.167
−12.909
1.00
20.10


ATOM
1048
CG
LEU
B
193
−1.217
−5.770
−13.476
1.00
20.30


ATOM
1049
CD1
LEU
B
193
−2.506
−5.798
−14.258
1.00
19.96


ATOM
1050
CD2
LEU
B
193
−1.263
−4.673
−12.390
1.00
20.81


ATOM
1051
C
LEU
B
193
0.395
−8.711
−11.423
1.00
20.19


ATOM
1052
O
LEU
B
193
0.190
−8.887
−10.221
1.00
20.50
















TABLE 3







Atomic coordinates of amino acid residues of


yDcn1 Chain A essential for binding yUbc12.


















Atom

Atom





Occu-
B-



No.

Name


X
Y
Z
pancy
factor





















ATOM
1906
N
ILE
A
90
−2.222
−45.427
−20.314
1.00
21.48


ATOM
1907
CA
ILE
A
90
−3.339
−45.857
−19.489
1.00
22.02


ATOM
1908
CB
ILE
A
90
−4.434
−46.463
−20.379
1.00
22.47


ATOM
1909
CG1
ILE
A
90
−5.253
−45.340
−21.002
1.00
23.62


ATOM
1910
CD1
ILE
A
90
−6.077
−44.572
−19.976
1.00
26.80


ATOM
1911
CG2
ILE
A
90
−5.389
−47.303
−19.578
1.00
23.20


ATOM
1912
C
ILE
A
90
−2.935
−46.825
−18.384
1.00
21.85


ATOM
1913
O
ILE
A
90
−3.369
−46.679
−17.243
1.00
21.90


ATOM
1928
N
LEU
A
93
−1.200
−44.808
−15.777
1.00
19.63


ATOM
1929
CA
LEU
A
93
−2.179
−44.094
−14.947
1.00
19.61


ATOM
1930
CB
LEU
A
93
−3.210
−43.349
−15.811
1.00
19.52


ATOM
1931
CG
LEU
A
93
−4.211
−42.444
−15.074
1.00
18.80


ATOM
1932
CD1
LEU
A
93
−3.517
−41.460
−14.123
1.00
16.51


ATOM
1933
CD2
LEU
A
93
−5.095
−41.703
−16.067
1.00
18.64


ATOM
1934
C
LEU
A
93
−2.874
−45.028
−13.952
1.00
19.66


ATOM
1935
O
LEU
A
93
−2.998
−44.694
−12.775
1.00
19.34


ATOM
1936
N
VAL
A
94
−3.316
−46.191
−14.436
1.00
20.02


ATOM
1937
CA
VAL
A
94
−3.895
−47.231
−13.576
1.00
20.55


ATOM
1938
CB
VAL
A
94
−4.275
−48.515
−14.381
1.00
20.23


ATOM
1939
CG1
VAL
A
94
−4.684
−49.648
−13.453
1.00
19.91


ATOM
1940
CG2
VAL
A
94
−5.409
−48.223
−15.353
1.00
20.95


ATOM
1941
C
VAL
A
94
−2.968
−47.587
−12.408
1.00
21.13


ATOM
1942
O
VAL
A
94
−3.399
−47.571
−11.247
1.00
21.68


ATOM
1963
N
ILE
A
97
−2.551
−44.631
−9.955
1.00
19.78


ATOM
1964
CA
ILE
A
97
−3.723
−44.543
−9.089
1.00
20.30


ATOM
1965
CB
ILE
A
97
−5.053
−44.675
−9.910
1.00
20.09


ATOM
1966
CG1
ILE
A
97
−5.296
−43.410
−10.745
1.00
19.51


ATOM
1967
CD1
ILE
A
97
−6.192
−43.624
−11.960
1.00
19.12


ATOM
1968
CG2
ILE
A
97
−6.261
−44.945
−8.997
1.00
20.08


ATOM
1969
C
ILE
A
97
−3.635
−45.556
−7.938
1.00
21.00


ATOM
1970
O
ILE
A
97
−3.840
−45.204
−6.761
1.00
20.37


ATOM
2021
N
LEU
A
104
−10.173
−47.353
−8.911
1.00
29.83


ATOM
2022
CA
LEU
A
104
−10.542
−46.612
−10.123
1.00
30.31


ATOM
2023
CB
LEU
A
104
−10.528
−47.522
−11.352
1.00
30.63


ATOM
2024
CG
LEU
A
104
−9.261
−48.272
−11.763
1.00
31.62


ATOM
2025
CD1
LEU
A
104
−8.933
−49.439
−10.812
1.00
33.32


ATOM
2026
CD2
LEU
A
104
−9.442
−48.785
−13.180
1.00
32.12


ATOM
2027
C
LEU
A
104
−11.905
−45.940
−10.024
1.00
30.38


ATOM
2028
O
LEU
A
104
−12.200
−45.008
−10.772
1.00
30.61


ATOM
2029
N
GLU
A
105
−12.724
−46.408
−9.089
1.00
30.37


ATOM
2030
CA
GLU
A
105
−14.089
−45.925
−8.944
1.00
30.32


ATOM
2031
CB
GLU
A
105
−15.027
−47.060
−8.505
1.00
30.79


ATOM
2032
CG
GLU
A
105
−14.852
−48.382
−9.278
1.00
34.03


ATOM
2033
CD
GLU
A
105
−13.910
−49.374
−8.588
1.00
37.92


ATOM
2034
OE1
GLU
A
105
−13.067
−48.946
−7.770
1.00
40.04


ATOM
2035
OE2
GLU
A
105
−14.018
−50.591
−8.863
1.00
39.49


ATOM
2036
C
GLU
A
105
−14.181
−44.755
−7.969
1.00
29.38


ATOM
2037
O
GLU
A
105
−15.248
−44.154
−7.827
1.00
29.38


ATOM
2038
N
ASP
A
106
−13.076
−44.453
−7.286
1.00
28.35


ATOM
2039
CA
ASP
A
106
−13.009
−43.299
−6.390
1.00
27.37


ATOM
2040
CB
ASP
A
106
−11.772
−43.351
−5.483
1.00
27.73


ATOM
2041
CG
ASP
A
106
−11.780
−44.528
−4.520
1.00
28.81


ATOM
2042
OD1
ASP
A
106
−12.877
−44.964
−4.089
1.00
31.71


ATOM
2043
OD2
ASP
A
106
−10.674
−45.011
−4.184
1.00
28.03


ATOM
2044
C
ASP
A
106
−12.944
−42.039
−7.238
1.00
26.37


ATOM
2045
O
ASP
A
106
−12.138
−41.952
−8.177
1.00
26.21


ATOM
2046
N
LEU
A
107
−13.788
−41.067
−6.903
1.00
25.02


ATOM
2047
CA
LEU
A
107
−13.857
−39.806
−7.646
1.00
24.02


ATOM
2048
CB
LEU
A
107
−15.111
−39.011
−7.251
1.00
24.13


ATOM
2049
CG
LEU
A
107
−16.464
−39.654
−7.601
1.00
24.37


ATOM
2050
CD1
LEU
A
107
−17.625
−38.816
−7.098
1.00
24.63


ATOM
2051
CD2
LEU
A
107
−16.630
−39.928
−9.101
1.00
24.56


ATOM
2052
C
LEU
A
107
−12.589
−38.955
−7.544
1.00
23.14


ATOM
2053
O
LEU
A
107
−12.312
−38.151
−8.430
1.00
23.15


ATOM
2059
N
THR
A
109
−9.811
−39.869
−8.275
1.00
20.00


ATOM
2060
CA
THR
A
109
−9.090
−40.122
−9.527
1.00
19.48


ATOM
2061
CB
THR
A
109
−9.400
−41.516
−10.126
1.00
19.37


ATOM
2062
OG1
THR
A
109
−10.783
−41.600
−10.504
1.00
19.30


ATOM
2063
CG2
THR
A
109
−9.065
−42.606
−9.129
1.00
20.66


ATOM
2064
C
THR
A
109
−9.367
−39.047
−10.576
1.00
18.77


ATOM
2065
O
THR
A
109
−8.517
−38.779
−11.418
1.00
18.55


ATOM
2066
N
LEU
A
110
−10.557
−38.449
−10.525
1.00
18.32


ATOM
2067
CA
LEU
A
110
−10.897
−37.323
−11.405
1.00
18.09


ATOM
2068
CB
LEU
A
110
−12.408
−37.051
−11.408
1.00
18.21


ATOM
2069
CG
LEU
A
110
−13.322
−38.008
−12.173
1.00
19.07


ATOM
2070
CD1
LEU
A
110
−14.784
−37.727
−11.833
1.00
19.91


ATOM
2071
CD2
LEU
A
110
−13.098
−37.862
−13.661
1.00
20.22


ATOM
2072
C
LEU
A
110
−10.154
−36.050
−11.012
1.00
17.74


ATOM
2073
O
LEU
A
110
−9.798
−35.262
−11.877
1.00
17.47


ATOM
2088
N
ALA
A
113
−6.659
−36.664
−12.409
1.00
16.15


ATOM
2089
CA
ALA
A
113
−6.724
−36.421
−13.847
1.00
16.33


ATOM
2090
CB
ALA
A
113
−7.934
−37.130
−14.448
1.00
15.86


ATOM
2091
C
ALA
A
113
−6.769
−34.929
−14.143
1.00
16.46


ATOM
2092
O
ALA
A
113
−6.073
−34.432
−15.042
1.00
16.39


ATOM
2153
N
LEU
A
121
−8.648
−39.338
−21.818
1.00
26.23


ATOM
2154
CA
LEU
A
121
−7.960
−40.441
−21.168
1.00
26.53


ATOM
2155
CB
LEU
A
121
−8.935
−41.261
−20.341
1.00
26.04


ATOM
2156
CG
LEU
A
121
−9.410
−40.537
−19.073
1.00
25.47


ATOM
2157
CD1
LEU
A
121
−10.506
−41.328
−18.368
1.00
23.26


ATOM
2158
CD2
LEU
A
121
−8.260
−40.242
−18.117
1.00
22.91


ATOM
2159
C
LEU
A
121
−7.210
−41.300
−22.176
1.00
27.40


ATOM
2160
O
LEU
A
121
−6.244
−41.976
−21.832
1.00
27.21


ATOM
2161
N
GLU
A
122
−7.652
−41.223
−23.429
1.00
28.34


ATOM
2162
CA
GLU
A
122
−7.059
−41.940
−24.547
1.00
29.67


ATOM
2163
CB
GLU
A
122
−7.893
−41.694
−25.815
1.00
30.04


ATOM
2164
CG
GLU
A
122
−9.327
−42.285
−25.788
1.00
32.62


ATOM
2165
CD
GLU
A
122
−10.340
−41.459
−24.962
1.00
36.03


ATOM
2166
OE1
GLU
A
122
−9.945
−40.458
−24.326
1.00
37.15


ATOM
2167
OE2
GLU
A
122
−11.546
−41.817
−24.951
1.00
37.17


ATOM
2168
C
GLU
A
122
−5.604
−41.538
−24.807
1.00
29.90


ATOM
2169
O
GLU
A
122
−4.828
−42.329
−25.341
1.00
29.76


ATOM
2593
N
LEU
A
173
−14.257
−36.100
−20.020
1.00
27.24


ATOM
2594
CA
LEU
A
173
−13.462
−37.319
−20.047
1.00
28.20


ATOM
2595
CB
LEU
A
173
−12.752
−37.534
−18.702
1.00
27.88


ATOM
2596
CG
LEU
A
173
−11.564
−36.653
−18.295
1.00
26.35


ATOM
2597
CD1
LEU
A
173
−10.879
−37.249
−17.070
1.00
24.58


ATOM
2598
CD2
LEU
A
173
−10.565
−36.496
−19.415
1.00
23.46


ATOM
2599
C
LEU
A
173
−14.284
−38.561
−20.404
1.00
29.58


ATOM
2600
O
LEU
A
173
−13.745
−39.509
−20.986
1.00
30.13


ATOM
2601
N
ILE
A
174
−15.566
−38.577
−20.028
1.00
30.42


ATOM
2602
CA
ILE
A
174
−16.469
−39.669
−20.419
1.00
31.46


ATOM
2603
CB
ILE
A
174
−17.863
−39.576
−19.712
1.00
31.45


ATOM
2604
CG1
ILE
A
174
−17.714
−39.524
−18.182
1.00
32.09


ATOM
2605
CD1
ILE
A
174
−18.987
−39.100
−17.425
1.00
31.01


ATOM
2606
CG2
ILE
A
174
−18.727
−40.771
−20.069
1.00
31.27


ATOM
2607
C
ILE
A
174
−16.639
−39.721
−21.958
1.00
32.27


ATOM
2608
O
ILE
A
174
−16.727
−40.805
−22.548
1.00
32.29


ATOM
2609
N
LEU
A
175
−16.662
−38.548
−22.595
1.00
33.10


ATOM
2610
CA
LEU
A
175
−16.824
−36.438
−24.046
1.00
33.94


ATOM
2611
CB
LEU
A
175
−16.591
−37.000
−24.504
1.00
33.73


ATOM
2612
CG
LEU
A
175
−17.754
−36.016
−24.433
1.00
32.38


ATOM
2613
CD1
LEU
A
175
−17.305
−34.695
−25.015
1.00
31.73


ATOM
2614
CD2
LEU
A
175
−18.961
−36.553
−25.170
1.00
30.45


ATOM
2615
C
LEU
A
175
−15.899
−39.346
−24.836
1.00
35.08


ATOM
2616
O
LEU
A
175
−14.676
−39.224
−24.732
1.00
35.38


ATOM
2617
N
ASP
A
176
−16.491
−40.256
−25.612
1.00
36.30


ATOM
2618
CA
ASP
A
176
−15.745
−41.072
−26.566
1.00
37.58


ATOM
2619
CB
ASP
A
176
−16.638
−42.160
−27.185
1.00
37.78


ATOM
2620
CG
ASP
A
176
−16.713
−43.424
−26.336
1.00
38.93


ATOM
2621
OD1
ASP
A
176
−15.702
−43.778
−25.687
1.00
40.88


ATOM
2622
OD2
ASP
A
176
−17.783
−44.073
−26.324
1.00
39.22


ATOM
2623
C
ASP
A
176
−15.208
−40.161
−27.664
1.00
38.32


ATOM
2624
O
ASP
A
176
−15.816
−39.123
−27.955
1.00
38.43


ATOM
2720
N
GLN
A
189
−24.108
−44.371
−14.531
1.00
23.07


ATOM
2721
CA
GLN
A
189
−22.967
−44.901
−13.784
1.00
23.84


ATOM
2722
CB
GLN
A
189
−21.967
−45.602
−14.716
1.00
24.30


ATOM
2723
CG
GLN
A
189
−22.352
−47.016
−15.106
1.00
26.75


ATOM
2724
CD
GLN
A
189
−21.292
−47.707
−15.960
1.00
31.60


ATOM
2725
OE1
GLN
A
189
−21.601
−48.637
−16.715
1.00
34.94


ATOM
2726
NE2
GLN
A
189
−20.037
−47.268
−15.842
1.00
32.62


ATOM
2727
C
GLN
A
189
−22.245
−43.822
−12.980
1.00
23.49


ATOM
2728
O
GLN
A
189
−21.936
−44.017
−11.802
1.00
23.77


ATOM
2729
N
TYR
A
190
−21.980
−42.684
−13.609
1.00
22.91


ATOM
2730
CA
TYR
A
190
−21.298
−41.606
−12.912
1.00
22.41


ATOM
2731
CB
TYR
A
190
−20.578
−40.677
−13.886
1.00
22.75


ATOM
2732
CG
TYR
A
190
−19.344
−41.317
−14.475
1.00
25.31


ATOM
2733
CD1
TYR
A
190
−18.109
−41.216
−13.839
1.00
27.94


ATOM
2734
CE1
TYR
A
190
−16.965
−41.818
−14.389
1.00
30.54


ATOM
2735
CZ
TYR
A
190
−17.072
−42.532
−15.580
1.00
30.87


ATOM
2736
OH
TYR
A
190
−15.967
−43.134
−16.144
1.00
33.77


ATOM
2737
CE2
TYR
A
190
−18.292
−42.651
−16.215
1.00
29.70


ATOM
2738
CD2
TYR
A
190
−19.416
−42.047
−15.664
1.00
28.05


ATOM
2739
C
TYR
A
190
−22.212
−40.836
−11.971
1.00
21.59


ATOM
2740
O
TYR
A
190
−21.766
−40.423
−10.891
1.00
21.41


ATOM
2764
N
LEU
A
193
−22.165
−42.386
−8.662
1.00
20.37


ATOM
2765
CA
LEU
A
193
−20.961
−42.081
−7.894
1.00
20.05


ATOM
2766
CB
LEU
A
193
−19.711
−42.257
−8.753
1.00
20.28


ATOM
2767
CG
LEU
A
193
−19.352
−43.673
−9.200
1.00
20.34


ATOM
2768
CD1
LEU
A
193
−18.137
−43.611
−10.088
1.00
20.81


ATOM
2769
CD2
LEU
A
193
−19.125
−44.614
−8.014
1.00
19.15


ATOM
2770
C
LEU
A
193
−21.011
−40.669
−7.325
1.00
19.95


ATOM
2771
O
LEU
A
193
−20.753
−40.470
−6.136
1.00
20.12
















TABLE 4







Additional contacts that contribute to, but are not essential,


for contact between S. cerevisiae Dcn1 and S. cerevisiae Ubc12


with yUbc12 Chain C interactions with yDcn1P Chain A and yUbc12


Chain D interactions with yDcn1P Chain B shown.











Distance


yUbc12 Atom
yDcn1 Atom
(Å)
















Arg
5C
NE
Leu
104A
CD2
4.07


Arg
5C
CZ
Leu
104A
CB
4.43





Glu
105A
CD
4.44





Glu
105A
OE2
3.95





Leu
104A
CD2
4.28


Arg
5C
NH1
Glu
105A
CG
4.02





Glu
105A
CD
4.28





Glu
105A
OE2
3.96


Arg
5C
NH2
Leu
104A
CB
4.21





Glu
105A
CG
4.21





Glu
105A
CD
3.64





Glu
105A
OE1
4.3





Glu
105A
OE2
3.05





Leu
104A
CD2
4.39





Leu
104A
CG
4.46





Leu
104A
CD1
4.13


Leu
7C
CB
Ile
90A
CD1
4.44





Ile
90A
CG2
4.39


Leu
7C
CD1
Leu
121A
CB
4.19


Leu
7C
CD2
Glu
122A
OE2
4.34





Glu
122A
CG
3.85





Glu
122A
CD
4.36


Leu
7C
C
Ile
90A
CD1
3.57


Leu
7C
O
Ile
90A
CD1
3.4


Gln
8C
N
Ile
90A
CD1
3.49


Gln
8C
CA
Ile
90A
CD1
3.4


Gln
8C
CB
Ile
90A
CD1
4.18





Asp
91A
OD1
4.43


Gln
8C
CG
Ile
90A
CD1
3.79





Val
94A
CG2
4.28





Asp
91A
OD1
3.49


Gln
8C
CD
Val
94A
CG1
4.46





Val
94A
CG2
3.58





Asp
91A
OD1
3.87





Val
94A
CB
4.37


Gln
8C
OE1
Val
94A
CG1
4.46





Val
94A
CG2
3.72


Gln
8C
NE2
Val
94A
CG1
3.83





Val
94A
CG2
3.53





Asp
91A
OD1
3.24





Asp
91A
CG
4.36





Asp
91A
CA
4.34





Val
94A
CB
3.84


Lys
11C
CB
Ile
90A
CD1
4.03


Lys
11C
CD
Ile
90A
CD1
4.14





Asp
89A
OD2
4.33





Asp
91A
CG
4.43





Asp
91A
OD2
3.44


Lys
11C
CE
Asp
89A
CG
4.47





Asp
89A
OD2
3.48





Asp
91A
OD2
3.64


Lys
11C
NZ
Asp
89A
CG
4.01





Asp
89A
OD2
2.97





Asp
91A
CG
3.92





Asp
91A
OD2
2.7





Asn
84A
CB
4.47


Arg
5D
N
Leu
104B
CD1
4.25


Arg
5D
CA
Leu
104B
CD1
4.37


Arg
5D
CB
Leu
104B
CD1
3.71


Arg
5D
CG
Leu
104B
CD1
4.36


Arg
5D
NE
Leu
104B
CD1
3.9


Arg
5D
CZ
Leu
104B
CB
4.48





Leu
104B
CD1
3.74





Glu
105B
OE2
4.2


Arg
5D
NH1
Leu
104B
CD1
4.31





Glu
105B
OE2
4.16


Arg
5D
NH2
Leu
104B
CB
3.97





Leu
104B
CG
4.04





Leu
104B
CD1
3.67





Glu
105B
OE2
3.4





Glu
105B
CD
4





Glu
105B
OE1
4.12


Gln
6D
CD
Glu
186B
CD
4.47





Glu
186B
OE2
3.46


Gln
6D
OE1
Glu
186B
CG
3.8





Glu
186B
CD
3.69





Glu
186B
OE2
2.9


Gln
6D
NE2
Asp
176B
OD1
4.18





Glu
186B
CD
4.41





Glu
186B
OE2
3.25


Leu
7D
CB
Ile
90B
CD1
3.77


Leu
7D
CG
Ile
90B
CD1
4.25


Leu
7D
CD1
Leu
121B
CB
4.01





Leu
121B
C
4.43





Ile
90B
CD1
3.76





Glu
122B
CG
4.4


Leu
7D
CD2
Glu
122B
CG
4.4


Gln
8D
CG
Ile
90B
CG2
4.17





Asp
91B
OD1
4.03


Gln
8D
CD
Asp
91B
OD1
4.34





Val
94B
CG2
4.02


Gln
8D
OE1
Val
94B
CG2
4.29


Gln
8D
NE2
Asp
91B
OD1
3.6





Val
94B
CB
4.02





Val
94B
CG2
3.75





Val
94B
CG1
3.87
















TABLE 5







Atomic coordinates of residues that contribute to, but are


not required for, binding of yDcn1 Chain B to yUbc12.


















Atom

Atom





Occu-
B-



No.

Name


X
Y
Z
pancy
factor





















ATOM
136
N
ASN
B
84
−24.546
−0.988
−25.260
1.00
25.93


ATOM
137
CA
ASN
B
84
−24.488
0.253
−26.016
1.00
27.03


ATOM
138
CB
ASN
B
84
−23.072
0.833
−25.998
1.00
27.10


ATOM
139
CG
ASN
B
84
−22.586
1.127
−24.609
1.00
26.68


ATOM
140
OD1
ASN
B
84
−23.153
1.954
−23.903
1.00
27.53


ATOM
141
ND2
ASN
B
84
−21.525
0.454
−24.205
1.00
26.15


ATOM
142
C
ASN
B
84
−24.887
−0.019
−27.445
1.00
27.74


ATOM
143
O
ASN
B
84
−24.248
−0.823
−28.120
1.00
27.97


ATOM
179
N
ASP
B
89
−20.480
−4.359
−25.414
1.00
26.21


ATOM
180
CA
ASP
B
89
−19.176
−4.073
−25.996
1.00
26.19


ATOM
181
CB
ASP
B
89
−19.314
−2.957
−27.045
1.00
26.08


ATOM
182
CG
ASP
B
89
−19.779
−1.625
−26.445
1.00
26.39


ATOM
183
OD1
ASP
B
89
−19.976
−1.535
−25.210
1.00
26.46


ATOM
184
OD2
ASP
B
89
−19.945
−0.655
−27.218
1.00
27.17


ATOM
185
C
ASP
B
89
−18.124
−3.716
−24.930
1.00
26.36


ATOM
186
O
ASP
B
89
−18.336
−3.935
−23.735
1.00
25.92


ATOM
195
N
ASP
B
91
−17.152
−0.819
−23.793
1.00
26.91


ATOM
196
CA
ASP
B
91
−17.667
0.172
−22.850
1.00
26.57


ATOM
197
CB
ASP
B
91
−18.385
1.308
−23.586
1.00
27.05


ATOM
198
CG
ASP
B
91
−17.432
2.176
−24.392
1.00
29.32


ATOM
199
OD1
ASP
B
91
−16.274
2.369
−23.951
1.00
31.34


ATOM
200
OD2
ASP
B
91
−17.840
2.667
−25.469
1.00
31.93


ATOM
201
C
ASP
B
91
−18.579
−0.476
−21.814
1.00
25.69


ATOM
202
O
ASP
B
91
−18.575
−0.092
−20.643
1.00
25.22


ATOM
278
N
GLY
B
101
−17.275
−3.210
−8.645
1.00
29.34


ATOM
279
CA
GLY
B
101
−16.450
−2.623
−7.591
1.00
29.68


ATOM
280
C
GLY
B
101
−14.959
−2.810
−7.784
1.00
29.86


ATOM
281
O
GLY
B
101
−14.203
−2.788
−6.815
1.00
30.12


ATOM
282
N
TYR
B
102
−14.530
−2.981
−9.033
1.00
29.95


ATOM
283
CA
TYR
B
102
−13.125
−3.256
−9.329
1.00
29.83


ATOM
284
CB
TYR
B
102
−12.976
−4.620
−10.014
1.00
29.64


ATOM
285
CG
TYR
B
102
−13.264
−5.790
−9.097
1.00
28.75


ATOM
286
CD1
TYR
B
102
−12.267
−6.325
−8.285
1.00
27.79


ATOM
287
CE1
TYR
B
102
−12.529
−7.385
−7.429
1.00
27.33


ATOM
288
CZ
TYR
B
102
−13.798
−7.929
−7.382
1.00
28.21


ATOM
289
OH
TYR
B
102
−14.063
−8.983
−6.538
1.00
28.62


ATOM
290
CE2
TYR
B
102
−14.812
−7.417
−8.175
1.00
28.92


ATOM
291
CD2
TYR
B
102
−14.539
−6.349
−9.029
1.00
29.04


ATOM
292
C
TYR
B
102
−12.471
−2.173
−10.169
1.00
30.22


ATOM
293
O
TYR
B
102
−13.155
−1.397
−10.842
1.00
30.20


ATOM
294
N
ASN
B
103
−11.141
−2.110
−10.079
1.00
30.62


ATOM
295
CA
ASN
B
103
−10.307
−1.343
−11.000
1.00
30.76


ATOM
296
CB
ASN
B
103
−9.066
−0.790
−10.286
1.00
30.89


ATOM
297
CG
ASN
B
103
−9.406
0.188
−9.160
1.00
32.03


ATOM
298
OD1
ASN
B
103
−10.086
1.193
−9.372
1.00
34.05


ATOM
299
ND2
ASN
B
103
−8.912
−0.096
−7.960
1.00
32.51


ATOM
300
C
ASN
B
103
−9.874
−2.289
−12.114
1.00
30.71


ATOM
301
O
ASN
B
103
−9.821
−3.509
−11.911
1.00
30.68


ATOM
980
N
GLU
B
186
3.220
−4.286
−23.322
1.00
25.28


ATOM
981
CA
GLU
B
186
1.962
−4.813
−22.773
1.00
25.53


ATOM
982
CB
GLU
B
186
1.005
−5.255
−23.886
1.00
26.28


ATOM
983
CG
GLU
B
186
0.428
−4.141
−24.769
1.00
30.25


ATOM
984
CD
GLU
B
186
−0.261
−4.683
−26.032
1.00
35.54


ATOM
985
OE1
GLU
B
186
−0.215
−5.914
−26.273
1.00
38.14


ATOM
986
OE2
GLU
B
186
−0.848
−3.877
−26.794
1.00
38.37


ATOM
987
C
GLU
B
186
2.231
−5.998
−21.836
1.00
24.41


ATOM
988
O
GLU
B
186
1.635
−6.093
−20.756
1.00
24.41
















TABLE 6







Atomic coordinates of residues that contribute to, but are


not required for, binding of yDcn1 Chain A to yUbc12.


















Atom

Atom





Occu-
B-



No.

Name


X
Y
Z
pancy
factor





















ATOM
1855
N
ASN
A
84
5.480
−47.125
−19.771
1.00
24.51


ATOM
1856
CA
ASN
A
84
5.351
−48.370
−20.509
1.00
24.71


ATOM
1857
CB
ASN
A
84
3.911
−48.902
−20.440
1.00
25.23


ATOM
1858
CG
ASN
A
84
3.565
−49.475
−19.082
1.00
26.13


ATOM
1859
CD1
ASN
A
84
4.293
−50.299
−18.541
1.00
30.17


ATOM
1860
ND2
ASN
A
84
2.447
−49.049
−18.529
1.00
27.12


ATOM
1861
C
ASN
A
84
5.729
−48.075
−21.938
1.00
24.19


ATOM
1862
O
ASN
A
84
5.204
−47.140
−22.528
1.00
24.25


ATOM
1898
N
ASP
A
89
1.132
−44.006
−20.314
1.00
21.24


ATOM
1899
CA
ASP
A
89
−0.152
−44.370
−20.921
1.00
21.52


ATOM
1900
CB
ASP
A
89
0.045
−45.548
−21.893
1.00
21.94


ATOM
1901
CG
ASP
A
89
0.661
−46.781
−21.218
1.00
23.83


ATOM
1902
CD1
ASP
A
89
0.856
−46.789
−19.967
1.00
27.18


ATOM
1903
OD2
ASP
A
89
0.951
−47.755
−21.946
1.00
27.23


ATOM
1904
C
ASP
A
89
−1.171
−44.759
−19.860
1.00
21.03


ATOM
1905
O
ASP
A
89
−0.986
−44.504
−18.677
1.00
20.38


ATOM
1914
N
ASP
A
91
−2.097
−47.801
−18.722
1.00
22.01


ATOM
1915
CA
ASP
A
91
−1.539
−48.709
−17.724
1.00
21.76


ATOM
1916
CB
ASP
A
91
−0.654
−49.764
−18.384
1.00
22.52


ATOM
1917
CG
ASP
A
91
−1.404
−50.617
−19.386
1.00
23.98


ATOM
1918
OD1
ASP
A
91
−2.482
−51.150
−19.045
1.00
25.33


ATOM
1919
OD2
ASP
A
91
−0.903
−50.762
−20.520
1.00
26.75


ATOM
1920
C
ASP
A
91
−0.745
−47.940
−16.676
1.00
21.29


ATOM
1921
O
ASP
A
91
−0.916
−48.190
−15.480
1.00
21.45


ATOM
1997
N
GLY
A
101
−2.947
−45.057
−3.801
1.00
22.73


ATOM
1998
CA
GLY
A
101
−3.770
−45.643
−2.749
1.00
23.50


ATOM
1999
C
GLY
A
101
−5.257
−45.694
−3.055
1.00
24.28


ATOM
2000
O
GLY
A
101
−6.068
−45.834
−2.140
1.00
24.21


ATOM
2001
N
TYR
A
102
−5.616
−45.604
−4.334
1.00
25.12


ATOM
2002
CA
TYR
A
102
−7.025
−45.504
−4.736
1.00
26.13


ATOM
2003
CB
TYR
A
102
−7.305
−44.158
−5.419
1.00
25.91


ATOM
2004
CG
TYR
A
102
−7.145
−43.006
−4.469
1.00
26.01


ATOM
2005
CD1
TYR
A
102
−8.209
−42.587
−3.663
1.00
26.06


ATOM
2006
CE1
TYR
A
102
−8.056
−41.536
−2.767
1.00
26.07


ATOM
2007
CZ
TYR
A
102
−6.827
−40.904
−2.664
1.00
25.39


ATOM
2008
OH
TYR
A
102
−6.657
−39.872
−1.780
1.00
25.43


ATOM
2009
CE2
TYR
A
102
−5.758
−41.306
−3.446
1.00
26.78


ATOM
2010
CD2
TYR
A
102
−5.919
−42.351
−4.346
1.00
25.73


ATOM
2011
C
TYR
A
102
−7.529
−46.640
−5.612
1.00
26.90


ATOM
2012
O
TYR
A
102
−6.748
−47.385
−6.199
1.00
27.17


ATOM
2013
N
ASN
A
103
−8.852
−46.760
−5.649
1.00
28.08


ATOM
2014
CA
ASN
A
103
−9.578
−47.619
−6.565
1.00
29.35


ATOM
2015
CB
ASN
A
103
−10.792
−48.231
−5.854
1.00
29.88


ATOM
2016
CG
ASN
A
103
−10.399
−49.134
−4.687
1.00
32.31


ATOM
2017
OD1
ASN
A
103
−9.440
−49.903
−4.782
1.00
35.88


ATOM
2018
ND2
ASN
A
103
−11.140
−49.045
−3.585
1.00
33.38


ATOM
2019
C
ASN
A
103
−10.045
−46.753
−7.729
1.00
29.59


ATOM
2020
O
ASN
A
103
−10.294
−45.555
−7.552
1.00
29.55


ATOM
2699
N
GLU
A
186
−23.763
−44.689
−19.329
1.00
26.90


ATOM
2700
CA
GLU
A
186
−22.562
−44.138
−18.718
1.00
26.47


ATOM
2701
CB
GLU
A
186
−21.512
−43.836
−19.791
1.00
26.96


ATOM
2702
CG
GLU
A
186
−20.845
−45.096
−20.328
1.00
29.85


ATOM
2703
CD
GLU
A
186
−20.272
−44.939
−21.736
1.00
34.58


ATOM
2704
OE1
GLU
A
186
−20.423
−43.852
−22.339
1.00
37.35


ATOM
2705
OE2
GLU
A
186
−19.666
−45.912
−22.245
1.00
36.39


ATOM
2706
C
GLU
A
186
−22.862
−42.917
−17.846
1.00
25.40


ATOM
2707
O
GLU
A
186
−22.228
−42.733
−16.803
1.00
25.32









In other embodiments, the E2-binding pocket of a NEDD8 co-E3 protein comprises the atoms of Table 7, which are found in amino acid residues Ile83, Ile86, Gln87, Pro97, Ala98, Ser99, Val102, Leu103, Ala106, Gln114, Cys115, Phe117, Phe164, Asn167, Met177, Ala180, Tyr181, and Leu184 of human Dcn1 (SEQ ID NO: 33), or a structural variant thereof. In particular embodiments, the E2-binding pocket of a NEDD8 co-E3 protein comprises at least one of the following amino acid residues: an isoleucine corresponding to position 83 of SEQ ID NO: 33, an isoleucine corresponding to position 86 of SEQ ID NO: 33, a glutamine corresponding to position 87 of SEQ ID NO: 33, a proline corresponding to position 97 of SEQ ID NO: 33, an alanine corresponding to position 98 of SEQ ID NO: 33, a serine corresponding to position 99 of SEQ ID NO: 33, a valine corresponding to position 102 of SEQ ID NO: 33, a leucine corresponding to position 103 of SEQ ID NO: 33, an alanine corresponding to position 106 of SEQ ID NO: 33, a glutamine corresponding to position 114 of SEQ ID NO: 33, a cysteine corresponding to position 115 of SEQ ID NO: 33, a phenylalanine corresponding to position 117 of SEQ ID NO: 33, a phenylalanine corresponding to position 164 of SEQ ID NO: 33, an asparagine corresponding to position 167 of SEQ ID NO: 33, a methionine corresponding to position 177 of SEQ ID NO: 33, an alanine corresponding to position 180 of SEQ ID NO: 33, a tyrosine corresponding to position 181 of SEQ ID NO: 33, and a leucine corresponding to position 184 of SEQ ID NO: 33.
In some of these embodiments, the E2-binding pocket comprises the atomic coordinates of Table 8, 9, 10, or 11, or a structural variant thereof. In particular embodiments, the co-E3 protein has the amino acid sequence set forth in SEQ ID NO: 33.
In certain embodiments, the E2-binding pocket of a NEDD8 co-E3 protein comprises the atoms of Tables 7 and 12. The atoms in Table 12 are found in amino acid residues Asp84, Ala94, Leu95, and Asp96 of SEQ ID NO: 33. Thus, in some embodiments, the E2-binding pocket of a NEDD8 co-E3 protein comprises at least one of the following amino acid residues: an isoleucine corresponding to position 83 of SEQ ID NO: 33, an isoleucine corresponding to position 86 of SEQ ID NO: 33, a glutamine corresponding to position 87 of SEQ ID NO: 33, a proline corresponding to position 97 of SEQ ID NO: 33, an alanine corresponding to position 98 of SEQ ID NO: 33, a serine corresponding to position 99 of SEQ ID NO: 33, a valine corresponding to position 102 of SEQ ID NO: 33, a leucine corresponding to position 103 of SEQ ID NO; 33, an alanine corresponding to position 106 of SEQ ID NO: 33, a glutamine corresponding to position 114 of SEQ ID NO: 33, a cysteine corresponding to position 115 of SEQ ID NO: 33, a phenylalanine corresponding to position 117 of SEQ ID NO: 33, a phenylalanine corresponding to position 164 of SEQ ID NO: 33, an asparagine corresponding to position 167 of SEQ ID NO: 33, a methionine corresponding to position 177 of SEQ ID NO: 33, an alanine corresponding to position 180 of SEQ ID NO; 33, a tyrosine corresponding to position 18I of SEQ ID NO: 33, a leucine corresponding to position 184 of SEQ ID NO: 33, an aspartic acid corresponding to position 84 of SEQ ID NO: 33, an alanine corresponding to position 94 of SEQ ID NO: 33, a leucine corresponding to position 95 of SEQ ID NO: 33, and an aspartic acid corresponding to position 96 of SEQ ID NO: 33.
In some of these embodiments, the E2-binding pocket comprises the atomic coordinates of Tables 8 and 13; Tables 9 and 14; Tables 10 and 15; or Tables 11 and 16; or a structural variant thereof.







TABLE 7







Minimal contacts between human Dcn1 and human Ubc12 with


hUbc12 Chain E interactions with hDcn1P Chain B and hUbc12 Chain


F interactions with hDcn1P ChainA shown.











Distance


hUbc12 Atom
hDcn1 Atom
(Å)
















Acetyl-Met
1E
O
Pro
97B
O
4.28





Cys
115B
SG
4.31


Acetyl-Met
1E
C
Gln
114B
OE1
3.79





Tyr
181B
OH
4.2





Pro
97B
O
4.19





Cys
115B
SG
4.24


Acetyl-Met
1E
CA
Gln
114B
OE1
4.13





Tyr
181B
OH
4.11





Pro
97B
C
4.46





Pro
97B
O
3.62





Cys
115B
SG
4.27


Acetyl-Met
1E
N
Ala
98B
CA
3.95





Tyr
181B
OH
4.3





Val
102B
CG1
4.46





Pro
97B
C
3.72





Pro
97B
O
2.7





Ala
98B
N
4.26


Acetyl-Met
1E
CT1
Phe
164B
CE2
4.29





Tyr
181B
CE1
4.26





Tyr
181B
CZ
4.38





Tyr
181B
OH
3.64





Pro
97B
O
3.59


Acetyl-Met
1E
OT
Leu
103B
CD1
4.34





Phe
164B
CE2
3.32





Phe
164B
CD2
4.31





Gln
114B
CB
4.29





Tyr
181B
CE1
3.45





Tyr
181B
CZ
3.44





Tyr
181B
OH
2.61





Phe
164B
CZ
4.04


Acetyl-Met
1E
CT2
Leu
103B
CD1
4.33





Ser
99B
O
3.61





Val
102B
C
4.33





Leu
103B
N
3.67





Leu
103B
CA
3.91





Leu
103B
CB
3.76





Ala
98B
CA
4.24





Ala
98B
C
4.2





Ala
98B
O
4.47





Leu
184B
CD2
4.07





Val
102B
CB
4.36





Val
102B
CG1
4.25





Pro
97B
O
3.58


Acetyl-Met
1E
CB
Pro
97B
CB
4.35





Pro
97B
C
4.29





Pro
97B
O
3.55





Cys
115B
SG
3.98





Ile
86B
CG2
4.42


Acetyl-Met
1E
CG
Val
102B
CG1
3.87





Pro
97B
C
4.45





Pro
97B
O
3.53





Ile
86B
CG2
3.63


Acetyl-Met
1E
SD
Phe
164B
CZ
3.9





Val
102B
CG1
4.47





Gln
114B
CA
4.31





Gln
114B
C
4.36





Cys
115B
N
3.4





Cys
115B
CA
4.02





Ile
86B
CG2
4.1


Acetyl-Met
1E
CE
Phe
117B
CZ
3.7





Ala
106B
CB
4.48





Phe
164B
CZ
4.24





Val
102B
CG1
3.71





Phe
117B
CE1
4.04





Phe
117B
CE2
4.49





Ile
86B
CD1
4.34





Ile
86B
CG2
4.02


Ile
2E
N
Gln
114B
CD
4.32





Gln
114B
OE1
3.24





Tyr
181B
CZ
4.13





Tyr
181B
OH
3.28


Ile
2E
CA
Gln
114B
OE1
3.88





Tyr
181B
OH
4.11


Ile
2E
CB
Gln
114B
OE1
4.23





Tyr
181B
CZ
3.8





Tyr
181B
OH
3.75





Tyr
181B
CE2
3.7





Tyr
181B
CD2
4.42





Met
177B
SD
4.01


Ile
2E
CG1
Tyr
181B
CG
4.48





Tyr
181B
CD1
4.32





Tyr
181B
CE1
3.85





Tyr
181B
CZ
3.51





Tyr
181B
OH
3.76





Tyr
181B
CE2
3.69





Tyr
181B
CD2
4.16


Ile
2E
CD1
Ala
98B
CA
4.32





Ala
98B
CB
3.64





Ala
98B
O
4.16


Ile
2E
CG2
Ala
180B
CB
3.66





Met
177B
SD
3.73


Ile
2E
C
Gln
114B
OE1
3.83


Lys
3E
N
Gln
114B
CD
3.92





Gln
114B
OE1
2.87





Gln
114B
NE2
4.33





Cys
115B
SG
4.07


Lys
3E
CA
Gln
114B
CD
4.5





Gln
114B
OE1
3.64





Met
177B
CE
4.45





Cys
115B
SG
4.16


Lys
3E
CB
Gln
114B
CD
3.78





Gln
114B
OE1
3.3





Gln
114B
NE2
3.92





Met
177B
CE
3.86





Cys
115B
SG
4.11


Lys
3E
CG
Met
177B
CE
3.63


Lys
3E
CD
Met
177B
CE
3.52


Lys
3E
NZ
Asn
167B
OD1
4.22


Lys
3E
C
Cys
115B
SG
3.72


Lys
3E
O
Cys
115B
SG
4.34


Leu
4E
N
Cys
115B
SG
3.31


Leu
4E
CA
Cys
115B
SG
3.54


Leu
4E
CB
Cys
115B
SG
3.87


Leu
4E
CG
Cys
115B
SG
4.49





Ile
83B
O
4.35


Leu
4E
CD1
Cys
115B
CA
4.29





Cys
115B
CB
3.97





Cys
115B
SG
3.82





Ile
86B
CB
4.13





Ile
86B
CG2
4.13





Ile
83B
O
3.71





Ile
83B
CG2
3.74





Ile
83B
CA
3.86





Ile
83B
CB
3.73





Ile
83B
C
4.24


Leu
4E
CD2
Pro
97B
CB
3.78





Pro
97B
CG
4.09





Ile
86B
CG2
4.22





Ile
83B
O
4.37





Gln
87B
N
4.12





Gln
87B
CA
4.12





Gln
87B
CB
4.21





Gln
87B
OE1
3.57





Gln
87B
CD
4.31


Acetyl-Met
1F
O
Pro
97A
O
4.39





Cys
115A
SG
4.36


Acetyl-Met
1F
C
Pro
97A
O
4.33





Cys
115A
SG
4.23





Gln
114A
OE1
3.8





Tyr
181A
OH
4.28


Acetyl-Met
1F
CA
Pro
97A
C
4.48





Pro
97A
O
3.73





Cys
115A
SG
4.23





Gln
114A
OE1
4.14





Tyr
181A
OH
4.21


Acetyl-Met
1F
N
Pro
97A
C
3.72





Pro
97A
O
2.79





Ala
98A
N
4.21





Ala
98A
CA
3.88





Tyr
181A
OH
4.37


Acetyl-Met
1F
CT1
Pro
97A
O
3.65





Ala
98A
CA
4.5





Tyr
181A
CE1
4.26





Tyr
181A
CZ
4.41





Phe
164A
CE2
4.2





Tyr
181A
OH
3.69


Acetyl-Met
1F
OT
Leu
103A
CD1
4.46





Tyr
181A
CE1
3.54





Tyr
181A
CZ
3.57





Phe
164A
CZ
3.92





Phe
164A
CE2
3.18





Phe
164A
CD2
4.18





Gln
114A
CB
4.17





Tyr
181A
OH
2.74


Acetyl-Met
1F
CT2
Ala
98A
C
4.16





Ala
98A
O
4.35





Pro
97A
O
3.6





Ala
98A
CA
4.19





Ser
99A
O
3.64





Val
102A
CB
4.44





Val
102A
CG1
4.33





Leu
103A
N
3.83





Leu
184A
CD2
4.05





Leu
103A
CA
4.04





Leu
103A
CB
3.81





Leu
103A
CD1
4.45





Tyr
181A
CE1
4.49


Acetyl-Met
1F
CB
Pro
97A
C
4.26





Pro
97A
O
3.6





Pro
97A
CB
4.31





Cys
115A
SG
3.95





Ile
86A
CG2
4.45


Acetyl-Met
1F
CG
Pro
97A
C
4.4





Pro
97A
O
3.53





Val
102A
CG1
3.85





Ile
86A
CG2
3.75


Acetyl-Met
1F
SD
Val
102A
CG1
4.44





Phe
164A
CZ
3.86





Ile
86A
CG2
4.17





Cys
115A
CA
4





Gln
114A
CA
4.31





Gln
114A
C
4.35





Cys
115A
N
3.38


Acetyl-Met
1F
CE
Val
102A
CG1
3.7





Phe
117A
CE1
4.13





Phe
117A
CZ
3.69





Phe
117A
CE2
4.4





Phe
164A
CZ
4.26





Ile
86A
CD1
4.29





Ile
86A
CG2
4.09


Ile
2F
N
Tyr
181A
CZ
4.22





Gln
114A
CD
4.28





Gln
114A
OE1
3.26





Tyr
181A
OH
3.35


Ile
2F
CA
Gln
114A
OE1
3.92





Tyr
181A
OH
4.16


Ile
2F
CB
Tyr
181A
CZ
3.88





Met
177A
SD
4.02





Gln
114A
OE1
4.25





Tyr
181A
OH
3.76





Tyr
181A
CE2
3.77


Ile
2F
CG1
Tyr
181A
CG
4.5





Tyr
181A
CD1
4.36





Tyr
181A
CE1
3.85





Tyr
181A
CZ
3.43





Tyr
181A
CD2
4.11





Ala
180A
CB
4.43





Tyr
181A
OH
3.65





Tyr
181A
CE2
3.58


Ile
2F
CD1
Ala
98A
CB
3.66





Ala
98A
O
4.19





Ala
98A
CA
4.3


Ile
2F
CG2
Met
177A
SD
3.78





Ala
180A
CB
3.75


Ile
2F
C
Gln
114A
OE1
3.86


Lys
3F
N
Cys
115A
SG
4.11





Gln
114A
CD
3.96





Gln
114A
OE1
2.89





Gln
114A
NE2
4.36


Lys
3F
CA
Cys
115A
SG
4.14





Gln
114A
OE1
3.66


Lys
3F
CB
Cys
115A
SG
3.94





Gln
114A
CD
3.88





Gln
114A
OE1
3.27





Gln
114A
NE2
4.16


Lys
3F
CG
Met
177A
SD
3.99





Met
177A
CE
3.2





Gln
114A
CD
3.91





Gln
114A
OE1
3.67





Gln
114A
NE2
3.69


Lys
3F
CD
Met
177A
CE
3.67





Gln
114A
CD
4.1





Gln
114A
OE1
4.25





Gln
114A
NE2
3.78


Lys
3F
NZ
Asn
167A
OD1
3.99


Lys
3F
C
Cys
115A
SG
3.8


Lys
3F
O
Cys
115A
SG
4.5


Leu
4F
N
Cys
115A
SG
3.34


Leu
4F
CA
Cys
115A
SG
3.58


Leu
4F
CB
Cys
115A
SG
3.82


Leu
4F
CG
Gln
87A
OE1
4.29


Leu
4F
CD1
Cys
115A
SG
4.09





Ile
86A
CB
4.08





Ile
86A
CG2
3.97





Cys
115A
CB
4.28





Ile
83A
C
4.24





Ile
83A
O
3.66





Ile
83A
CA
3.96





Ile
83A
CB
3.75





Ile
83A
CG2
4.05


Leu
4F
CD2
Gln
87A
CB
4.26





Gln
87A
CD
4.2





Gln
87A
OE1
3.46





Pro
97A
CB
3.63





Pro
97A
CG
3.88





Gln
87A
N
4.16





Gln
87A
CA
4.12





Ile
86A
CG2
4.09
















TABLE 8







Atomic coordinates of amino acid residues of hDcn1 Chain A essential


for binding hUbc12 from structure with stapled hUbc12 peptide.


















Atom

Atom





Occu-
B-



No.

Name


X
Y
Z
pancy
factor





















ATOM
198
N
ILE
A
83
13.031
40.450
4.212
1.00
11.06


ATOM
199
CA
ILE
A
83
12.692
41.331
5.343
1.00
11.10


ATOM
200
CB
ILE
A
83
12.045
40.580
6.548
1.00
11.11


ATOM
201
CG1
ILE
A
83
13.025
39.622
7.224
1.00
12.43


ATOM
202
CD1
ILE
A
83
12.509
39.060
8.572
1.00
12.78


ATOM
203
CG2
ILE
A
83
10.770
39.869
6.106
1.00
11.68


ATOM
204
C
ILE
A
83
13.839
42.249
5.794
1.00
10.27


ATOM
205
O
ILE
A
83
13.610
43.411
6.117
1.00
9.79


ATOM
218
N
ILE
A
86
14.182
44.958
3.139
1.00
9.70


ATOM
219
CA
ILE
A
86
13.051
45.879
3.287
1.00
10.08


ATOM
220
CB
ILE
A
86
11.691
45.130
3.541
1.00
10.44


ATOM
221
CG1
ILE
A
86
11.201
44.403
2.280
1.00
10.75


ATOM
222
CD1
ILE
A
86
10.930
45.295
1.085
1.00
12.22


ATOM
223
CG2
ILE
A
86
10.611
46.083
4.067
1.00
10.11


ATOM
224
C
ILE
A
86
13.311
46.862
4.423
1.00
9.98


ATOM
225
O
ILE
A
86
13.098
48.064
4.264
1.00
9.90


ATOM
226
N
GLN
A
87
13.764
46.349
5.567
1.00
10.21


ATOM
227
CA
GLN
A
87
14.082
47.199
6.708
1.00
10.45


ATOM
228
CB
GLN
A
87
14.505
46.369
7.922
1.00
10.47


ATOM
229
CG
GLN
A
87
14.872
47.217
9.154
1.00
10.24


ATOM
230
CD
GLN
A
87
13.657
47.835
9.835
0.67
9.76


ATOM
231
OE1
GLN
A
87
12.747
47.136
10.263
1.00
11.23


ATOM
232
NE2
GLN
A
87
13.650
49.151
9.947
1.00
9.80


ATOM
233
C
GLN
A
87
15.190
48.197
6.361
1.00
10.65


ATOM
234
O
GLN
A
87
15.079
49.379
6.676
1.00
10.43


ATOM
306
N
PRO
A
97
8.934
51.727
8.623
1.00
12.49


ATOM
307
CA
PRO
A
97
8.458
50.775
7.613
1.00
12.43


ATOM
308
CB
PRO
A
97
9.098
49.454
8.041
1.00
12.32


ATOM
309
CG
PRO
A
97
10.354
49.872
8.779
1.00
11.89


ATOM
310
CD
PRO
A
97
9.949
51.127
9.513
1.00
12.41


ATOM
311
C
PRO
A
97
6.935
50.663
7.572
1.00
12.50


ATOM
312
O
PRO
A
97
6.386
50.382
6.524
1.00
12.36


ATOM
313
N
ALA
A
98
6.279
50.895
8.710
1.00
12.89


ATOM
314
CA
ALA
A
98
4.831
50.836
8.815
1.00
13.39


ATOM
315
CB
ALA
A
98
4.426
50.451
10.252
1.00
13.37


ATOM
316
C
ALA
A
98
4.138
52.141
8.396
1.00
13.61


ATOM
317
O
ALA
A
98
2.931
52.264
8.554
1.00
14.04


ATOM
318
N
SER
A
99
4.889
53.105
7.864
1.00
13.57


ATOM
319
CA
SER
A
99
4.318
54.420
7.534
1.00
13.68


ATOM
320
CB
SER
A
99
5.396
55.509
7.572
1.00
13.92


ATOM
321
OG
SER
A
99
6.281
55.392
6.464
1.00
14.94


ATOM
322
C
SER
A
99
3.577
54.467
6.189
1.00
13.30


ATOM
323
O
SER
A
99
3.791
53.623
5.310
1.00
13.12


ATOM
338
N
VAL
A
102
5.854
54.647
2.979
1.00
9.89


ATOM
339
CA
VAL
A
102
6.354
53.352
2.502
1.00
9.86


ATOM
340
CB
VAL
A
102
6.884
52.478
3.682
1.00
10.15


ATOM
341
CG1
VAL
A
102
7.180
51.047
3.233
1.00
9.36


ATOM
342
CG2
VAL
A
102
8.139
53.106
4.290
1.00
8.80


ATOM
343
C
VAL
A
102
5.284
52.604
1.692
1.00
9.91


ATOM
344
O
VAL
A
102
5.589
51.953
0.692
1.00
10.20


ATOM
345
N
LEU
A
103
4.031
52.702
2.123
1.00
9.82


ATOM
346
CA
LEU
A
103
2.926
52.077
1.388
1.00
9.65


ATOM
347
CB
LEU
A
103
1.623
52.189
2.187
1.00
9.74


ATOM
348
CG
LEU
A
103
0.366
51.488
1.645
1.00
9.65


ATOM
349
CD1
LEU
A
103
0.615
50.022
1.289
1.00
9.76


ATOM
350
CD2
LEU
A
103
−0.762
51.606
2.661
1.00
8.19


ATOM
351
C
LEU
A
103
2.767
52.696
−0.006
1.00
9.65


ATOM
352
O
LEU
A
103
2.483
51.993
−0.990
1.00
9.39


ATOM
369
N
ALA
A
106
5.491
51.324
−2.295
1.00
11.19


ATOM
370
CA
ALA
A
106
5.107
49.981
−2.730
1.00
11.36


ATOM
371
CB
ALA
A
106
4.610
49.160
−1.550
1.00
11.21


ATOM
372
C
ALA
A
106
4.047
50.032
−3.832
1.00
11.22


ATOM
373
O
ALA
A
106
4.085
49.236
−4.769
1.00
11.65


ATOM
436
N
GLN
A
114
3.331
42.667
0.750
1.00
11.54


ATOM
437
CA
GLN
A
114
4.091
43.403
1.767
1.00
11.10


ATOM
438
CB
GLN
A
114
3.200
43.832
2.955
1.00
10.82


ATOM
439
CG
GLN
A
114
2.418
42.702
3.641
1.00
9.56


ATOM
440
CD
GLN
A
114
1.970
43.064
5.062
1.00
9.99


ATOM
441
OE1
GLN
A
114
0.868
42.701
5.494
1.00
10.07


ATOM
442
NE2
GLN
A
114
2.818
43.780
5.787
1.00
5.79


ATOM
443
C
GLN
A
114
5.289
42.596
2.254
1.00
10.86


ATOM
444
O
GLN
A
114
5.268
41.370
2.216
1.00
10.95


ATOM
445
N
CYS
A
115
6.324
43.312
2.689
1.00
11.28


ATOM
446
CA
CYS
A
115
7.530
42.756
3.322
1.00
11.28


ATOM
447
CB
CYS
A
115
7.177
41.891
4.536
1.00
11.40


ATOM
448
SG
CYS
A
115
6.269
42.812
5.794
1.00
11.83


ATOM
449
C
CYS
A
115
8.493
42.025
2.382
1.00
11.75


ATOM
450
O
CYS
A
115
9.382
41.294
2.836
1.00
11.65


ATOM
460
N
PHE
A
117
10.183
42.611
−2.011
1.00
11.84


ATOM
461
CA
PHE
A
117
10.100
43.012
−3.413
1.00
11.80


ATOM
462
CB
PHE
A
117
10.823
44.337
−3.678
1.00
11.71


ATOM
463
CG
PHE
A
117
10.223
45.539
−3.003
1.00
11.95


ATOM
464
CD1
PHE
A
117
8.855
45.793
−3.052
1.00
12.60


ATOM
465
CE1
PHE
A
117
8.321
46.933
−2.440
1.00
12.58


ATOM
466
CZ
PHE
A
117
9.163
47.836
−1.789
1.00
11.89


ATOM
467
CE2
PHE
A
117
10.532
47.605
−1.757
1.00
12.68


ATOM
468
CD2
PHE
A
117
11.057
46.465
−2.368
1.00
12.57


ATOM
469
C
PHE
A
117
10.803
41.964
−4.252
1.00
11.62


ATOM
470
O
PHE
A
117
11.867
41.482
−3.871
1.00
11.77


ATOM
852
N
PHE
A
164
−2.039
43.997
−1.681
1.00
11.20


ATOM
853
CA
PHE
A
164
−0.671
44.121
−1.150
1.00
11.38


ATOM
854
CB
PHE
A
164
−0.271
45.597
−1.193
1.00
11.09


ATOM
855
CG
PHE
A
164
1.083
45.896
−0.624
1.00
11.82


ATOM
856
CD1
PHE
A
164
2.238
45.586
−1.333
1.00
10.42


ATOM
857
CE1
PHE
A
164
3.504
45.881
−0.801
1.00
11.27


ATOM
858
CZ
PHE
A
164
3.611
46.506
0.436
1.00
10.73


ATOM
859
CE2
PHE
A
164
2.466
46.842
1.144
1.00
11.29


ATOM
860
CD2
PHE
A
164
1.203
46.535
0.612
1.00
10.92


ATOM
861
C
PHE
A
164
−0.571
43.567
0.276
1.00
11.74


ATOM
862
O
PHE
A
164
0.378
42.852
0.617
1.00
11.82


ATOM
877
N
ASN
A
167
−2.074
37.844
2.624
1.00
18.39


ATOM
878
CA
ASN
A
167
−1.150
36.746
2.375
1.00
20.42


ATOM
879
CB
ASN
A
167
−1.347
35.618
3.392
1.00
20.66


ATOM
880
CG
ASN
A
167
−1.107
36.073
4.821
1.00
22.22


ATOM
881
OD1
ASN
A
167
−1.802
35.643
5.744
1.00
24.64


ATOM
882
ND2
ASN
A
167
−0.119
36.949
5.013
1.00
24.02


ATOM
883
C
ASN
A
167
−1.308
36.220
0.961
1.00
21.22


ATOM
884
O
ASN
A
167
−2.432
36.089
0.479
1.00
21.40


ATOM
951
N
MET
A
177
−4.843
42.866
10.516
1.00
10.02


ATOM
952
CA
MET
A
177
−4.125
43.512
9.425
1.00
10.50


ATOM
953
CB
MET
A
177
−3.652
42.449
8.431
1.00
10.73


ATOM
954
CG
MET
A
177
−2.496
42.886
7.552
1.00
13.40


ATOM
955
SD
MET
A
177
−0.990
43.167
8.501
1.00
15.69


ATOM
956
CE
MET
A
177
−0.624
41.511
9.064
1.00
15.67


ATOM
957
C
MET
A
177
−5.002
44.547
8.705
1.00
10.32


ATOM
958
O
MET
A
177
−4.536
45.635
8.370
1.00
10.56


ATOM
972
N
ALA
A
180
−5.365
47.632
10.862
1.00
9.44


ATOM
973
CA
ALA
A
180
−4.089
48.350
10.775
1.00
9.83


ATOM
974
CB
ALA
A
180
−2.915
47.382
10.914
1.00
9.97


ATOM
975
C
ALA
A
180
−3.960
49.171
9.486
1.00
9.72


ATOM
976
O
ALA
A
180
−3.606
50.344
9.539
1.00
9.95


ATOM
977
N
TYR
A
181
−4.275
48.574
8.341
1.00
9.95


ATOM
978
CA
TYR
A
181
−4.206
49.302
7.069
1.00
10.52


ATOM
979
CB
TYR
A
181
−4.174
48.352
5.862
1.00
10.51


ATOM
980
CG
TYR
A
181
−2.795
47.759
5.628
1.00
10.25


ATOM
981
CD1
TYR
A
181
−1.812
48.485
4.963
1.00
11.37


ATOM
982
CE1
TYR
A
181
−0.533
47.965
4.764
1.00
12.31


ATOM
983
CZ
TYR
A
181
−0.221
46.695
5.237
1.00
12.09


ATOM
984
OH
TYR
A
181
1.050
46.188
5.040
1.00
10.57


ATOM
985
CE2
TYR
A
181
−1.178
45.951
5.911
1.00
11.41


ATOM
986
CD2
TYR
A
181
−2.456
46.490
6.103
1.00
10.74


ATOM
987
C
TYR
A
181
−5.287
50.388
6.927
1.00
10.90


ATOM
988
O
TYR
A
181
−5.014
51.471
6.394
1.00
11.09


ATOM
1011
N
LEU
A
184
−4.270
53.352
8.754
1.00
13.42


ATOM
1012
CA
LEU
A
184
−3.243
54.021
7.973
1.00
14.11


ATOM
1013
CB
LEU
A
184
−2.224
52.997
7.460
1.00
14.21


ATOM
1014
CG
LEU
A
184
−0.841
53.458
7.012
1.00
15.69


ATOM
1015
CD1
LEU
A
184
−0.110
54.189
8.135
1.00
15.46


ATOM
1016
CD2
LEU
A
184
−0.033
52.252
6.522
1.00
15.38


ATOM
1017
C
LEU
A
184
−3.825
54.840
6.810
1.00
14.10


ATOM
1018
O
LEU
A
184
−3.521
56.020
6.690
1.00
14.39
















TABLE 9







Atomic coordinates of amino acid residues of hDcn1 Chain B essential


for binding hUbc12 from structure with stapled hUbc12 peptide.


















Atom

Atom





Occu-
B-



No.

Name


X
Y
Z
pancy
factor





















ATOM
2412
N
ILE
B
83
−30.405
108.348
−21.310
1.00
22.06


ATOM
2413
CA
ILE
B
83
−29.529
107.901
−22.393
1.00
21.85


ATOM
2414
CB
ILE
B
83
−30.307
107.192
−23.536
1.00
21.90


ATOM
2415
CG1
ILE
B
83
−31.335
108.117
−24.185
1.00
21.89


ATOM
2416
CD1
ILE
B
83
−31.892
107.564
−25.477
1.00
21.91


ATOM
2417
CG2
ILE
B
83
−30.979
105.921
−23.005
1.00
21.93


ATOM
2418
C
ILE
B
83
−28.560
108.983
−22.902
1.00
21.52


ATOM
2419
O
ILE
B
83
−27.395
108.687
−23.175
1.00
21.21


ATOM
2432
N
ILE
B
86
−25.810
109.354
−20.278
1.00
20.01


ATOM
2433
CA
ILE
B
86
−24.882
108.227
−20.411
1.00
19.96


ATOM
2434
CB
ILE
B
86
−25.632
106.873
−20.671
1.00
20.05


ATOM
2435
CG1
ILE
B
86
−26.467
106.442
−19.450
1.00
20.17


ATOM
2436
CD1
ILE
B
86
−25.690
106.246
−18.153
1.00
19.80


ATOM
2437
CG2
ILE
B
86
−24.662
105.766
−21.113
1.00
19.91


ATOM
2438
C
ILE
B
86
−23.874
108.506
−21.530
1.00
19.93


ATOM
2439
O
ILE
B
86
−22.666
108.343
−21.341
1.00
19.67


ATOM
2440
N
GLN
B
87
−24.383
108.938
−22.684
1.00
20.03


ATOM
2441
CA
GLN
B
87
−23.539
109.293
−23.826
1.00
20.34


ATOM
2442
CB
GLN
B
87
−24.396
109.721
−25.025
1.00
20.22


ATOM
2443
CG
GLN
B
87
−23.607
110.009
−26.307
1.00
20.12


ATOM
2444
CD
GLN
B
87
−23.008
108.758
−26.930
0.99
20.10


ATOM
2445
OE1
GLN
B
87
−23.728
107.857
−27.361
1.00
19.31


ATOM
2446
NE2
GLN
B
87
−21.681
108.704
−26.989
1.00
20.30


ATOM
2447
C
GLN
B
87
−22.549
110.392
−23.452
1.00
20.46


ATOM
2448
O
GLN
B
87
−21.352
110.280
−23.726
1.00
20.60


ATOM
2520
N
PRO
B
97
−19.269
103.969
−25.674
1.00
22.67


ATOM
2521
CA
PRO
B
97
−20.254
103.509
−24.684
1.00
22.62


ATOM
2522
CB
PRO
B
97
−21.551
104.178
−25.141
1.00
22.57


ATOM
2523
CG
PRO
B
97
−21.104
105.391
−25.892
1.00
22.66


ATOM
2524
CD
PRO
B
97
−19.839
104.980
−26.585
1.00
22.59


ATOM
2525
C
PRO
B
97
−20.426
101.988
−24.639
1.00
22.66


ATOM
2526
O
PRO
B
97
−20.804
101.448
−23.598
1.00
22.58


ATOM
2527
N
ALA
B
98
−20.139
101.312
−25.752
1.00
22.54


ATOM
2528
CA
ALA
B
98
−20.255
99.858
−25.834
1.00
22.70


ATOM
2529
CB
ALA
B
98
−20.715
99.437
−27.234
1.00
22.63


ATOM
2530
C
ALA
B
98
−18.962
99.130
−25.453
1.00
22.71


ATOM
2531
O
ALA
B
98
−18.874
97.912
−25.585
1.00
23.00


ATOM
2532
N
SER
B
99
−17.966
99.872
−24.974
1.00
22.72


ATOM
2533
CA
SER
B
99
−16.659
99.291
−24.670
1.00
22.62


ATOM
2534
CB
SER
B
99
−15.563
100.356
−24.749
1.00
22.71


ATOM
2535
OG
SER
B
99
−15.605
101.204
−23.615
1.00
23.45


ATOM
2536
C
SER
B
99
−16.627
98.606
−23.306
1.00
22.39


ATOM
2537
O
SER
B
99
−17.471
98.871
−22.443
1.00
22.27


ATOM
2552
N
VAL
B
102
−16.397
100.871
−20.002
1.00
20.71


ATOM
2553
CA
VAL
B
102
−17.675
101.406
−19.530
1.00
20.51


ATOM
2554
CB
VAL
B
102
−18.519
101.986
−20.699
1.00
20.67


ATOM
2555
CG1
VAL
B
102
−19.911
102.406
−20.225
1.00
20.65


ATOM
2556
CG2
VAL
B
102
−17.805
103.177
−21.332
1.00
20.43


ATOM
2557
C
VAL
B
102
−18.460
100.357
−18.727
1.00
20.40


ATOM
2558
O
VAL
B
102
−19.110
100.693
−17.735
1.00
20.38


ATOM
2559
N
LEU
B
103
−18.334
99.094
−19.147
1.00
20.15


ATOM
2560
CA
LEU
B
103
−18.999
97.991
−18.399
1.00
20.02


ATOM
2561
CB
LEU
B
103
−18.941
96.682
−19.200
1.00
19.79


ATOM
2562
CG
LEU
B
103
−19.610
95.427
−18.617
1.00
20.45


ATOM
2563
CD1
LEU
B
103
−21.060
95.686
−18.213
1.00
20.61


ATOM
2564
CD2
LEU
B
103
−19.524
94.245
−19.594
1.00
20.05


ATOM
2565
C
LEU
B
103
−18.351
97.814
−17.017
1.00
19.81


ATOM
2566
O
LEU
B
103
−19.041
97.559
−16.027
1.00
19.65


ATOM
2583
N
ALA
B
106
−19.655
100.587
−14.795
1.00
20.13


ATOM
2584
CA
ALA
B
106
−21.017
100.219
−14.405
1.00
20.18


ATOM
2585
CB
ALA
B
106
−21.765
99.659
−15.593
1.00
20.03


ATOM
2586
C
ALA
B
106
−21.007
99.215
−13.249
1.00
20.37


ATOM
2587
O
ALA
B
106
−21.790
99.331
−12.297
1.00
20.20


ATOM
2650
N
GLN
B
114
−28.388
98.507
−17.786
1.00
19.92


ATOM
2651
CA
GLN
B
114
−27.641
99.225
−18.818
1.00
20.09


ATOM
2652
CB
GLN
B
114
−27.253
98.283
−19.977
1.00
19.77


ATOM
2653
CG
GLN
B
114
−28.425
97.614
−20.687
1.00
18.83


ATOM
2654
CD
GLN
B
114
−28.066
97.115
−22.078
1.00
18.37


ATOM
2655
OE1
GLN
B
114
−27.419
97.821
−22.857
1.00
18.08


ATOM
2656
NE2
GLN
B
114
−28.501
95.900
−22.402
1.00
16.31


ATOM
2657
C
GLN
B
114
−28.403
100.440
−19.347
1.00
20.26


ATOM
2658
O
GLN
B
114
−29.638
100.462
−19.325
1.00
20.37


ATOM
2659
N
CYS
B
115
−27.645
101.440
−19.803
1.00
20.71


ATOM
2660
CA
CYS
B
115
−28.163
102.672
−20.438
1.00
20.94


ATOM
2661
CB
CYS
B
115
−29.037
102.341
−21.650
1.00
21.06


ATOM
2662
SG
CYS
B
115
−28.203
101.301
−22.847
1.00
21.61


ATOM
2663
C
CYS
B
115
−28.878
103.664
−19.512
1.00
21.23


ATOM
2664
O
CYS
B
115
−29.614
104.542
−19.982
1.00
21.16


ATOM
2674
N
PHE
B
117
−28.223
105.377
−15.127
1.00
21.67


ATOM
2675
CA
PHE
B
117
−27.839
105.322
−13.715
1.00
21.68


ATOM
2676
CB
PHE
B
117
−26.477
105.984
−13.463
1.00
21.63


ATOM
2677
CG
PHE
B
117
−25.319
105.324
−14.166
1.00
22.09


ATOM
2678
CD1
PHE
B
117
−25.134
103.944
−14.111
1.00
21.98


ATOM
2679
CE1
PHE
B
117
−24.050
103.347
−14.754
1.00
22.16


ATOM
2680
CZ
PHE
B
117
−23.127
104.133
−15.437
1.00
21.74


ATOM
2681
CE2
PHE
B
117
−23.291
105.507
−15.483
1.00
21.59


ATOM
2682
CD2
PHE
B
117
−24.380
106.097
−14.842
1.00
21.55


ATOM
2683
C
PHE
B
117
−28.880
106.059
−12.886
1.00
21.55


ATOM
2684
O
PHE
B
117
−29.336
107.137
−13.271
1.00
21.64


ATOM
3066
N
PHE
B
164
−27.140
93.194
−15.243
1.00
19.87


ATOM
3067
CA
PHE
B
164
−26.985
94.525
−15.822
1.00
20.00


ATOM
3068
CB
PHE
B
164
−25.500
94.894
−15.809
1.00
20.10


ATOM
3069
CG
PHE
B
164
−25.185
96.227
−16.431
1.00
20.07


ATOM
3070
CD1
PHE
B
164
−25.504
97.413
−15.774
1.00
20.33


ATOM
3071
CE1
PHE
B
164
−25.201
98.652
−16.344
1.00
19.91


ATOM
3072
CZ
PHE
B
164
−24.556
98.709
−17.579
1.00
19.28


ATOM
3073
CE2
PHE
B
164
−24.227
97.532
−18.241
1.00
20.37


ATOM
3074
CD2
PHE
B
164
−24.542
96.295
−17.662
1.00
20.04


ATOM
3075
C
PHE
B
164
−27.543
94.604
−17.252
1.00
20.03


ATOM
3076
O
PHE
B
164
−28.208
95.577
−17.616
1.00
19.88


ATOM
3091
N
ASN
B
167
−33.402
93.223
−19.278
1.00
23.56


ATOM
3092
CA
ASN
B
167
−34.452
94.170
−18.910
1.00
25.06


ATOM
3093
CB
ASN
B
167
−35.630
94.094
−19.885
1.00
25.40


ATOM
3094
CG
ASN
B
167
−35.207
94.269
−21.327
1.00
26.53


ATOM
3095
OD1
ASN
B
167
−35.598
93.486
−22.193
1.00
28.48


ATOM
3096
ND2
ASN
B
167
−34.401
95.293
−21.595
1.00
27.88


ATOM
3097
C
ASN
B
167
−34.945
93.921
−17.488
1.00
25.57


ATOM
3098
O
ASN
B
167
−35.087
92.765
−17.079
1.00
25.68


ATOM
3165
N
MET
B
177
−28.572
90.183
−27.289
1.00
17.20


ATOM
3166
CA
MET
B
177
−27.921
90.921
−26.216
1.00
17.43


ATOM
3167
CB
MET
B
177
−28.969
91.422
−25.212
1.00
17.67


ATOM
3168
CG
MET
B
177
−28.531
92.623
−24.387
1.00
19.15


ATOM
3169
SD
MET
B
111
−28.144
94.055
−25.410
1.00
21.43


ATOM
3170
CE
MET
B
177
−29.768
94.544
−25.987
1.00
22.65


ATOM
3171
C
MET
B
177
−26.868
90.049
−25.528
1.00
17.09


ATOM
3172
O
MET
B
177
−25.758
90.510
−25.258
1.00
16.99


ATOM
3186
N
ALA
B
180
−23.753
89.661
−27.733
1.00
17.12


ATOM
3187
CA
ALA
B
180
−22.999
90.911
−27.738
1.00
17.35


ATOM
3188
CB
ALA
B
180
−23.929
92.104
−27.945
1.00
17.41


ATOM
3189
C
ALA
B
180
−22.183
91.081
−26.458
1.00
17.51


ATOM
3190
O
ALA
B
180
−21.041
91.537
−26.509
1.00
17.37


ATOM
3191
N
TYR
B
181
−22.763
90.710
−25.317
1.00
17.76


ATOM
3192
CA
TYR
B
181
−22.041
90.799
−24.045
1.00
18.12


ATOM
3193
CB
TYR
B
181
−22.995
90.874
−22.845
1.00
18.11


ATOM
3194
CG
TYR
B
181
−23.543
92.269
−22.622
1.00
18.19


ATOM
3195
CD1
TYR
B
181
−22.789
93.238
−21.952
1.00
18.71


ATOM
3196
CE1
TYR
B
181
−23.283
94.531
−21.749
1.00
19.16


ATOM
3197
CZ
TYR
B
181
−24.544
94.866
−22.224
1.00
19.33


ATOM
3198
OH
TYR
B
181
−25.035
96.146
−22.033
1.00
19.73


ATOM
3199
CE2
TYR
B
181
−25.309
93.921
−22.900
1.00
18.60


ATOM
3200
CD2
TYR
B
181
−24.805
92.629
−23.094
1.00
18.30


ATOM
3201
C
TYR
B
181
−20.989
89.707
−23.872
1.00
18.34


ATOM
3202
O
TYR
B
181
−19.907
89.973
−23.352
1.00
18.28


ATOM
3225
N
LEU
B
184
−18.023
90.608
−25.720
1.00
21.89


ATOM
3226
CA
LEU
B
184
−17.324
91.670
−25.010
1.00
22.22


ATOM
3227
CB
LEU
B
184
−18.328
92.723
−24.521
1.00
22.27


ATOM
3228
CG
LEU
B
184
−17.831
94.108
−24.104
1.00
22.26


ATOM
3229
CD1
LEU
B
184
−17.213
94.861
−25.272
1.00
21.91


ATOM
3230
CD2
LEU
B
184
−18.972
94.913
−23.499
1.00
22.24


ATOM
3231
C
LEU
B
184
−16.515
91.123
−23.836
1.00
22.40


ATOM
3232
O
LEU
B
184
−15.325
91.410
−23.714
1.00
22.83
















TABLE 10







Atomic coordinates of amino acid residues of


hDcn1 Chain A essential for binding hUbc12.


















Atom

Atom





Occu-
B-



No.

Name


X
Y
Z
pancy
factor




















ATOM
196
N
ILE
A
83
36.977
−14.686
−20.772
1.00
12.90


ATOM
197
CA
ILE
A
83
36.506
−15.088
−22.103
1.00
13.86


ATOM
198
CB
ILE
A
83
37.408
−14.550
−23.249
1.00
14.02


ATOM
199
CG1
ILE
A
83
38.829
−15.121
−23.160
1.00
14.11


ATOM
200
CD1
ILE
A
83
39.710
−14.759
−24.349
1.00
15.19


ATOM
201
CG2
ILE
A
83
37.426
−13.022
−23.244
1.00
14.59


ATOM
202
C
ILE
A
83
36.282
−16.597
−22.244
1.00
14.48


ATOM
203
O
ILE
A
83
35.350
−17.023
−22.926
1.00
14.64


ATOM
216
N
ILE
A
86
33.068
−17.241
−20.441
1.00
9.57


ATOM
217
CA
ILE
A
86
31.948
−16.711
−21.229
1.00
9.17


ATOM
218
CB
ILE
A
86
32.238
−15.296
−21.827
1.00
8.85


ATOM
219
CG1
ILE
A
86
32.344
−14.228
−20.724
1.00
9.45


ATOM
220
CD1
ILE
A
86
31.063
−13.939
−19.963
1.00
10.17


ATOM
221
CG2
ILE
A
86
31.154
−14.890
−22.848
1.00
9.18


ATOM
222
C
ILE
A
86
31.582
−17.703
−22.335
1.00
9.55


ATOM
223
O
ILE
A
86
30.403
−17.993
−22.547
1.00
9.03


ATOM
224
N
GLN
A
87
32.591
−18.224
−23.028
1.00
10.48


ATOM
225
CA
GLN
A
87
32.377
−19.248
−24.049
1.00
11.22


ATOM
226
CB
GLN
A
87
33.700
−19.650
−24.710
1.00
11.32


ATOM
227
CG
GLN
A
87
33.546
−20.663
−25.841
1.00
12.84


ATOM
228
CD
GLN
A
87
32.742
−20.115
−27.008
0.67
13.76


ATOM
229
OE1
GLN
A
87
33.143
−19.144
−27.646
1.00
16.05


ATOM
230
NE2
GLN
A
87
31.601
−20.736
−27.291
1.00
15.09


ATOM
231
C
GLN
A
87
31.679
−20.479
−23.467
1.00
11.20


ATOM
232
O
GLN
A
87
30.738
−21.002
−24.066
1.00
11.47


ATOM
304
H
PRO
A
97
27.365
−17.205
−28.903
1.00
9.39


ATOM
305
CA
PRO
A
97
27.770
−16.104
−28.025
1.00
9.60


ATOM
306
CB
PRO
A
97
29.295
−16.200
−28.024
1.00
9.57


ATOM
307
CG
PRO
A
97
29.575
−17.639
−28.292
1.00
9.43


ATOM
308
CD
PRO
A
97
28.486
−18.109
−29.214
1.00
9.60


ATOM
309
C
PRO
A
97
27.322
−14.723
−28.507
1.00
9.56


ATOM
310
O
PRO
A
97
27.104
−13.836
−27.683
1.00
9.53


ATOM
311
N
ALA
A
98
27.156
−14.566
−29.820
1.00
10.14


ATOM
312
CA
ALA
A
98
26.780
−13.293
−30.431
1.00
10.80


ATOM
313
CB
ALA
A
98
27.526
−13.116
−31.758
1.00
10.99


ATOM
314
C
ALA
A
98
25.270
−13.144
−30.640
1.00
11.25


ATOM
315
O
ALA
A
98
24.818
−12.229
−31.340
1.00
12.29


ATOM
316
N
SER
A
99
24.490
−14.032
−30.029
1.00
11.27


ATOM
317
CA
SER
A
99
23.039
−14.011
−30.210
1.00
11.21


ATOM
318
CB
SER
A
99
22.439
−15.384
−29.919
1.00
11.58


ATOM
319
OG
SER
A
99
22.566
−15.712
−28.553
1.00
12.43


ATOM
320
C
SER
A
99
22.370
−12.956
−29.335
1.00
10.50


ATOM
321
O
SER
A
99
22.918
−12.540
−28.313
1.00
9.98


ATOM
336
N
VAL
A
102
21.794
−13.908
−25.598
1.00
7.12


ATOM
337
CA
VAL
A
102
22.960
−13.673
−24.751
1.00
6.85


ATOM
338
CB
VAL
A
102
24.260
−14.153
−25.433
1.00
6.88


ATOM
339
CG1
VAL
A
102
25.492
−13.783
−24.603
1.00
7.40


ATOM
340
CG2
VAL
A
102
24.198
−15.661
−25.669
1.00
7.91


ATOM
341
C
VAL
A
102
23.037
−12.205
−24.314
1.00
6.55


ATOM
342
O
VAL
A
102
23.311
−11.926
−23.150
1.00
6.96


ATOM
343
N
LEU
A
103
22.789
−11.276
−25.237
1.00
6.21


ATOM
344
CA
LEU
A
103
22.751
−9.852
−24.891
1.00
6.05


ATOM
345
CB
LEU
A
103
22.576
−8.990
−26.146
1.00
6.39


ATOM
346
CG
LEU
A
103
22.643
−7.472
−25.936
1.00
5.17


ATOM
347
CD1
LEU
A
103
23.967
−7.059
−25.301
1.00
6.27


ATOM
348
CD2
LEU
A
103
22.475
−6.764
−27.258
1.00
6.33


ATOM
349
C
LEU
A
103
21.661
−9.529
−23.870
1.00
6.49


ATOM
350
O
LEU
A
103
21.872
−8.721
−22.956
1.00
6.43


ATOM
367
N
ALA
A
106
22.865
−10.651
−20.508
1.00
7.03


ATOM
368
CA
ALA
A
106
23.841
−9.666
−20.041
1.00
6.99


ATOM
369
CB
ALA
A
106
24.801
−9.293
−21.165
1.00
7.01


ATOM
370
C
ALA
A
106
23.169
−8.416
−19.464
1.00
6.61


ATOM
371
O
ALA
A
106
23.605
−7.879
−18.442
1.00
7.01


ATOM
446
N
GLN
A
114
30.971
−6.245
−22.517
1.00
7.54


ATOM
447
CA
GLN
A
114
30.955
−7.504
−23.257
1.00
8.01


ATOM
448
CB
GLN
A
114
30.775
−7.256
−24.758
1.00
7.85


ATOM
449
CG
GLN
A
114
31.857
−6.400
−25.406
1.00
7.92


ATOM
450
CD
GLN
A
114
31.964
−6.653
−26.896
1.00
8.14


ATOM
451
OE1
GLN
A
114
31.982
−7.801
−27.341
1.00
9.25


ATOM
452
NE2
GLN
A
114
32.060
−5.580
−27.674
1.00
7.38


ATOM
453
C
GLN
A
114
32.186
−8.380
−22.996
1.00
8.45


ATOM
454
O
GLN
A
114
33.298
−7.883
−22.747
1.00
8.88


ATOM
455
N
CYS
A
115
31.949
−9.691
−23.029
1.00
9.24


ATOM
456
CA
CYS
A
115
32.990
−10.723
−22.948
1.00
9.94


ATOM
457
CB
CYS
A
115
34.087
−10.498
−23.996
1.00
10.22


ATOM
458
SG
CYS
A
115
33.482
−10.519
−25.689
1.00
11.14


ATOM
459
C
CYS
A
115
33.596
−10.896
−21.559
1.00
10.57


ATOM
460
O
CYS
A
115
34.726
−11.387
−21.417
1.00
10.06


ATOM
470
N
PHE
A
117
31.900
−11.315
−17.158
1.00
10.88


ATOM
471
CA
PHE
A
117
31.016
−10.979
−16.047
1.00
10.54


ATOM
472
CB
PHE
A
117
29.925
−12.035
−15.829
1.00
10.31


ATOM
473
CG
PHE
A
117
28.955
−12.191
−16.974
1.00
10.63


ATOM
474
CD1
PHE
A
117
28.370
−11.086
−17.585
1.00
10.88


ATOM
475
CE1
PHE
A
117
27.466
−11.249
−18.632
1.00
11.40


ATOM
476
CZ
PHE
A
117
27.122
−12.526
−19.065
1.00
10.91


ATOM
477
CE2
PHE
A
117
27.689
−13.637
−18.454
1.00
11.21


ATOM
478
CD2
PHE
A
117
28.595
−13.465
−17.410
1.00
11.19


ATOM
479
C
PHE
A
117
31.842
−10.888
−14.774
1.00
10.24


ATOM
480
O
PHE
A
117
32.678
−11.748
−14.507
1.00
10.91


ATOM
868
N
PHE
A
164
27.612
−1.423
−22.879
1.00
8.55


ATOM
869
CA
PHE
A
164
28.065
−2.810
−22.824
1.00
8.10


ATOM
870
CB
PHE
A
164
26.836
−3.708
−22.983
1.00
8.03


ATOM
871
CG
PHE
A
164
27.133
−5.176
−22.982
1.00
7.71


ATOM
872
CD1
PHE
A
164
27.444
−5.836
−21.798
1.00
7.58


ATOM
873
CE1
PHE
A
164
27.695
−7.206
−21.787
1.00
8.30


ATOM
874
CZ
PHE
A
164
27.620
−7.932
−22.970
1.00
8.55


ATOM
875
CE2
PHE
A
164
27.296
−7.287
−24.158
1.00
7.42


ATOM
876
CD2
PHE
A
164
27.062
−5.912
−24.161
1.00
7.45


ATOM
877
C
PHE
A
164
29.089
−3.107
−23.920
1.00
8.08


ATOM
878
O
PHE
A
164
30.094
−3.790
−23.689
1.00
7.67


ATOM
893
N
ASN
A
167
34.518
0.021
−24.949
1.00
18.51


ATOM
894
CA
ASN
A
167
35.584
−0.198
−23.981
1.00
23.93


ATOM
895
CB
ASN
A
167
36.945
0.137
−24.598
1.00
24.56


ATOM
896
CG
ASN
A
167
37.254
−0.702
−25.827
1.00
27.69


ATOM
897
OD1
ASN
A
167
37.067
−1.920
−25.830
1.00
30.17


ATOM
898
ND2
ASN
A
167
37.740
−0.051
−26.877
1.00
29.71


ATOM
899
C
ASN
A
167
35.354
0.648
−22.732
1.00
27.76


ATOM
900
O
ASN
A
167
34.860
1.775
−22.833
1.00
27.47


ATOM
972
N
MET
A
177
32.057
−2.089
−34.346
1.00
9.67


ATOM
973
CA
MET
A
177
31.336
−2.605
−33.182
1.00
8.98


ATOM
974
CB
MET
A
177
32.086
−2.279
−31.885
1.00
9.19


ATOM
975
CG
MET
A
177
31.772
−3.243
−30.739
1.00
10.77


ATOM
976
SD
MET
A
177
32.121
−4.976
−31.150
1.00
13.17


ATOM
977
CE
MET
A
177
33.883
−4.902
−31.440
1.00
14.81


ATOM
978
C
MET
A
177
29.899
−2.081
−33.121
1.00
8.33


ATOM
979
O
MET
A
177
28.973
−2.845
−32.850
1.00
7.85


ATOM
993
N
ALA
A
180
27.819
−3.852
−35.736
1.00
7.21


ATOM
994
CA
ALA
A
180
27.550
−5.215
−35.276
1.00
7.23


ATOM
995
CB
ALA
A
180
28.814
−5.842
−34.679
1.00
7.51


ATOM
996
C
ALA
A
180
26.383
−5.261
−34.281
1.00
7.58


ATOM
997
O
ALA
A
180
25.485
−6.095
−34.406
1.00
7.82


ATOM
998
N
TYR
A
181
26.379
−4.344
−33.316
1.00
7.11


ATOM
999
CA
TYR
A
181
25.315
−4.309
−32.316
1.00
6.57


ATOM
1000
CB
TYR
A
181
25.759
−3.593
−31.036
1.00
7.02


ATOM
1001
CG
TYR
A
181
26.605
−4.491
−30.160
1.00
6.87


ATOM
1002
CD1
TYR
A
181
26.007
−5.394
−29.275
1.00
7.10


ATOM
1003
CE1
TYR
A
181
26.771
−6.228
−28.481
1.00
7.04


ATOM
1004
CZ
TYR
A
181
28.149
−6.176
−28.558
1.00
7.84


ATOM
1005
OH
TYR
A
181
28.901
−7.006
−27.768
1.00
9.69


ATOM
1006
CE2
TYR
A
181
28.775
−5.298
−29.426
1.00
7.94


ATOM
1007
CD2
TYR
A
181
27.998
−4.463
−30.227
1.00
7.63


ATOM
1008
C
TYR
A
181
24.009
−3.755
−32.876
1.00
6.27


ATOM
1009
O
TYR
A
181
22.947
−4.239
−32.515
1.00
6.90


ATOM
1032
N
LEU
A
184
22.293
−6.498
−34.554
1.00
8.61


ATOM
1033
CA
LEU
A
184
21.717
−7.379
−33.544
1.00
9.06


ATOM
1034
CB
LEU
A
184
22.740
−7.634
−32.429
1.00
9.47


ATOM
1035
CG
LEU
A
184
22.475
−8.763
−31.427
1.00
10.45


ATOM
1036
CD1
LEU
A
184
22.222
−10.090
−32.134
1.00
11.79


ATOM
1037
CD2
LEU
A
184
23.652
−8.883
−30.468
1.00
11.28


ATOM
1038
C
LEU
A
184
20.400
−6.841
−32.966
1.00
9.44


ATOM
1039
O
LEU
A
184
19.392
−7.552
−32.958
1.00
10.68
















TABLE 11







Atomic coordinates of amino acid residues of


hDcn1 Chain B essential for binding hUbc12.


















Atom

Atom





Occu-
B-



No.

Name


X
Y
Z
pancy
factor




















ATOM
1819
N
ILE
B
83
29.948
2.614
6.205
1.00
14.95


ATOM
1820
CA
ILE
B
83
28.894
3.066
7.119
1.00
15.72


ATOM
1821
CB
ILE
B
83
29.070
2.516
8.566
1.00
15.81


ATOM
1822
CG1
ILE
B
83
30.448
2.860
9.141
1.00
16.94


ATOM
1823
CD1
ILE
B
83
30.633
2.394
10.579
1.00
18.68


ATOM
1824
CG2
ILE
B
83
28.840
1.009
8.592
1.00
16.52


ATOM
1825
C
ILE
B
83
28.672
4.583
7.111
1.00
15.83


ATOM
1826
O
ILE
B
83
27.533
5.044
7.233
1.00
15.95


ATOM
1839
N
ILE
B
86
26.874
5.426
3.809
1.00
13.37


ATOM
1840
CA
ILE
B
86
25.480
4.984
3.892
1.00
12.69


ATOM
1841
CB
ILE
B
86
25.337
3.542
4.485
1.00
12.54


ATOM
1842
CG1
ILE
B
86
26.021
2.496
3.586
1.00
12.43


ATOM
1843
CD1
ILE
B
86
25.317
2.196
2.257
1.00
12.53


ATOM
1844
CG2
ILE
B
86
23.859
3.187
4.725
1.00
12.96


ATOM
1845
C
ILE
B
86
24.654
5.989
4.703
1.00
12.56


ATOM
1846
O
ILE
B
86
23.542
6.341
4.310
1.00
12.49


ATOM
1847
N
GLN
B
87
25.207
6.449
5.822
1.00
12.84


ATOM
1848
CA
GLN
B
87
24.552
7.470
6.634
1.00
13.49


ATOM
1849
CB
GLN
B
87
25.390
7.787
7.878
1.00
13.64


ATOM
1850
CG
GLN
B
87
24.756
8.801
8.836
1.00
15.44


ATOM
1851
CD
GLN
B
87
23.413
8.346
9.383
1.00
16.30


ATOM
1852
OE1
GLN
B
87
23.328
7.357
10.113
1.00
18.29


ATOM
1853
NE2
GLN
B
87
22.358
9.073
9.039
0.84
17.18


ATOM
1854
C
GLN
B
87
24.278
8.739
5.815
1.00
13.78


ATOM
1855
O
GLN
B
87
23.177
9.286
5.867
0.88
13.69


ATOM
1927
N
PRO
B
97
17.781
5.711
8.137
1.00
11.79


ATOM
1928
CA
PRO
B
97
18.531
4.583
7.573
1.00
11.68


ATOM
1929
CB
PRO
B
97
19.873
4.646
8.310
1.00
11.76


ATOM
1930
CG
PRO
B
97
20.002
6.061
8.735
1.00
11.97


ATOM
1931
CD
PRO
B
97
18.605
6.494
9.074
1.00
11.71


ATOM
1932
C
PRO
B
97
17.869
3.225
7.797
1.00
12.06


ATOM
1933
O
PRO
B
97
18.101
2.309
7.017
1.00
12.24


ATOM
1934
N
ALA
B
98
17.049
3.113
8.841
1.00
12.54


ATOM
1935
CA
ALA
B
98
16.374
1.860
9.181
1.00
12.70


ATOM
1936
CB
ALA
B
98
16.361
1.668
10.698
1.00
12.91


ATOM
1937
C
ALA
B
98
14.952
1.766
8.609
1.00
13.00


ATOM
1938
O
ALA
B
98
14.168
0.906
9.021
1.00
13.88


ATOM
1939
N
SER
B
99
14.622
2.641
7.663
1.00
13.24


ATOM
1940
CA
SER
B
99
13.279
2.672
7.082
1.00
13.23


ATOM
1941
CB
SER
B
99
12.953
4.062
6.531
1.00
13.54


ATOM
1942
OG
SER
B
99
13.704
4.337
5.363
1.00
14.52


ATOM
1943
C
SER
B
99
13.092
1.629
5.986
1.00
12.89


ATOM
1944
O
SER
B
99
14.056
1.196
5.349
1.00
12.57


ATOM
1959
H
VAL
B
102
14.549
2.518
2.526
1.00
9.33


ATOM
1960
CA
VAL
B
102
15.975
2.245
2.364
1.00
9.28


ATOM
1961
CB
VAL
B
102
16.792
2.715
3.583
1.00
9.50


ATOM
1962
CG1
VAL
B
102
18.242
2.256
3.483
1.00
9.82


ATOM
1963
CG2
VAL
B
102
16.730
4.243
3.694
1.00
9.83


ATOM
1964
C
VAL
B
102
16.217
0.765
2.040
1.00
8.85


ATOM
1965
O
VAL
B
102
17.042
0.444
1.186
1.00
9.10


ATOM
1966
N
LEU
B
103
15.477
−0.126
2.694
1.00
8.30


ATOM
1967
CA
LEU
B
103
15.550
−1.554
2.369
1.00
7.93


ATOM
1968
CB
LEU
B
103
14.711
−2.380
3.350
1.00
8.08


ATOM
1969
CG
LEU
B
103
14.821
−3.899
3.221
1.00
7.79


ATOM
1970
CD1
LEU
B
103
16.257
−4.353
3.403
1.00
8.56


ATOM
1971
CD2
LEU
B
103
13.939
−4.569
4.243
1.00
9.12


ATOM
1972
C
LEU
B
103
15.123
−1.847
0.925
1.00
7.70


ATOM
1973
O
LEU
B
103
15.727
−2.687
0.246
1.00
7.60


ATOM
1990
N
ALA
B
106
17.929
−0.799
−1.331
1.00
7.92


ATOM
1991
CA
ALA
B
106
18.975
−1.823
−1.244
1.00
7.82


ATOM
1992
CB
ALA
B
106
19.211
−2.221
0.201
1.00
7.95


ATOM
1993
C
ALA
B
106
18.642
−3.054
−2.099
1.00
7.83


ATOM
1994
O
ALA
B
106
19.513
−3.619
−2.767
1.00
7.89


ATOM
2063
N
GLN
B
114
23.763
−5.496
4.444
1.00
10.08


ATOM
2064
CA
GLN
B
114
23.384
−4.238
5.078
1.00
9.77


ATOM
2065
CB
GLN
B
114
22.454
−4.492
6.267
1.00
9.56


ATOM
2066
CG
GLN
B
114
23.081
−5.287
7.408
1.00
9.34


ATOM
2067
CD
GLN
B
114
22.379
−5.053
8.719
1.00
8.85


ATOM
2068
OE1
GLN
B
114
22.105
−3.910
9.098
1.00
9.42


ATOM
2069
NE2
GLN
B
114
22.098
−6.135
9.437
1.00
8.52


ATOM
2070
C
GLN
B
114
24.592
−3.416
5.528
1.00
10.01


ATOM
2071
O
GLN
B
114
25.608
−3.968
5.960
1.00
10.08


ATOM
2072
N
CYS
B
115
24.450
−2.095
5.401
1.00
10.57


ATOM
2073
CA
CYS
B
115
25.405
−1.099
5.915
1.00
11.33


ATOM
2074
CB
CYS
B
115
25.797
−1.391
7.369
1.00
11.41


ATOM
2075
SG
CYS
B
115
24.404
−1.367
8.516
1.00
13.01


ATOM
2076
C
CYS
B
115
26.639
−0.897
5.037
1.00
11.90


ATOM
2077
O
CYS
B
115
27.651
−0.352
5.488
1.00
12.13


ATOM
2087
N
PHE
B
117
27.457
−0.565
0.404
1.00
12.62


ATOM
2088
CA
PHE
B
117
27.233
−0.894
−0.999
1.00
12.39


ATOM
2089
CB
PHE
B
117
26.485
0.223
−1.740
1.00
12.42


ATOM
2090
CG
PHE
B
117
25.068
0.452
−1.285
1.00
12.77


ATOM
2091
CD1
PHE
B
117
24.193
−0.609
−1.057
1.00
12.77


ATOM
2092
CE1
PHE
B
117
22.874
−0.370
−0.656
1.00
12.83


ATOM
2093
CZ
PHE
B
117
22.423
0.936
−0.498
1.00
12.86


ATOM
2094
CE2
PHE
B
117
23.280
1.999
−0.743
1.00
13.41


ATOM
2095
CD2
PHE
B
117
24.594
1.752
−1.139
1.00
13.43


ATOM
2096
C
PHE
B
117
28.583
−1.046
−1.667
1.00
11.92


ATOM
2097
O
PHE
B
117
29.497
−0.262
−1.414
1.00
12.30


ATOM
2485
N
PHE
B
164
20.554
−10.249
3.036
1.00
10.13


ATOM
2486
CA
PHE
B
164
21.005
−8.872
3.207
1.00
9.30


ATOM
2487
CB
PHE
B
164
19.903
−7.944
2.685
1.00
8.96


ATOM
2488
CG
PHE
B
164
20.185
−6.479
2.852
1.00
8.28


ATOM
2489
CD1
PHE
BB
164
21.081
−5.824
2.011
1.00
7.37


ATOM
2490
CE1
PHE
B
164
21.325
−4.458
2.152
1.00
7.93


ATOM
2491
CZ
PHE
B
164
20.663
−3.732
3.132
1.00
7.52


ATOM
2492
CE2
PHE
B
164
19.767
−4.372
3.975
1.00
8.31


ATOM
2493
CD2
PHE
B
164
19.524
−5.739
3.831
1.00
7.61


ATOM
2494
C
PHE
B
164
21.330
−8.579
4.673
1.00
8.93


ATOM
2495
O
PHE
B
164
22.328
−7.921
4.972
1.00
8.93


ATOM
2510
N
ASN
B
167
25.423
−11.883
8.254
1.00
17.88


ATOM
2511
CA
AASN
B
167
26.822
−11.839
7.864
0.50
20.28


ATOM
2512
CA
BASN
B
167
26.827
−11.858
7.892
0.50
20.24


ATOM
2513
CB
AASN
B
167
27.727
−12.325
8.995
0.50
21.60


ATOM
2514
CB
BASN
B
167
27.634
−12.433
9.057
0.50
21.55


ATOM
2515
CG
AASN
B
167
27.856
−11.307
10.110
0.50
25.80


ATOM
2516
CG
BASN
B
167
29.119
−12.423
8.809
0.50
25.38


ATOM
2517
OD1
AASN
B
167
28.050
−10.117
9.859
0.50
28.22


ATOM
2518
OD1
BASN
B
167
29.601
−12.981
7.826
0.50
27.79


ATOM
2519
ND2
AASN
B
167
27.750
−11.770
11.350
0.50
28.23


ATOM
2520
ND2
BASN
B
167
29.863
−11.810
9.722
0.50
27.67


ATOM
2521
C
ASN
B
167
27.010
−12.683
6.608
1.00
20.72


ATOM
2522
O
ASN
B
167
26.555
−13.829
6.549
1.00
20.44


ATOM
2589
N
MET
B
177
18.700
−9.451
15.173
1.00
12.40


ATOM
2590
CA
MET
B
177
18.680
−8.891
13.824
1.00
11.50


ATOM
2591
CB
MET
B
177
19.985
−9.210
13.081
1.00
11.92


ATOM
2592
CG
MET
B
177
20.314
−8.240
11.947
1.00
13.23


ATOM
2593
SD
MET
B
177
20.326
−6.502
12.455
1.00
13.85


ATOM
2594
CE
MET
B
177
21.758
−6.458
13.517
1.00
14.84


ATOM
2595
C
MET
B
177
17.465
−9.394
13.045
1.00
10.79


ATOM
2596
O
MET
B
177
16.811
−8.624
12.346
1.00
10.53


ATOM
2610
N
ALA
B
180
14.364
−7.574
14.322
1.00
8.93


ATOM
2611
CA
ALA
B
180
14.340
−6.219
13.771
1.00
8.75


ATOM
2612
CB
ALA
B
180
15.696
−5.561
13.910
1.00
9.14


ATOM
2613
C
ALA
B
180
13.870
−6.189
12.314
1.00
8.69


ATOM
2614
O
ALA
B
180
13.040
−5.350
11.935
1.00
9.10


ATOM
2615
N
TYR
B
181
14.379
−7.107
11.497
1.00
8.07


ATOM
2616
CA
TYR
B
181
13.968
−7.133
10.097
1.00
7.64


ATOM
2617
CB
TYR
B
181
14.990
−7.869
9.224
1.00
7.89


ATOM
2618
CG
TYR
B
181
16.138
−6.973
8.845
1.00
6.99


ATOM
2619
CD1
TYR
B
181
16.023
−6.068
7.794
1.00
7.98


ATOM
2620
CE1
TYR
B
181
17.075
−5.234
7.450
1.00
7.54


ATOM
2621
CZ
TYR
B
181
18.255
−5.297
8.173
1.00
8.50


ATOM
2622
OH
TYR
B
181
19.290
−4.473
7.839
1.00
9.59


ATOM
2623
CE2
TYR
B
181
18.390
−6.176
9.224
1.00
8.12


ATOM
2624
CD2
TYR
B
181
17.332
−7.003
9.562
1.00
7.84


ATOM
2625
C
TYR
B
181
12.540
−7.633
9.894
1.00
7.55


ATOM
2626
O
TYR
B
181
11.829
−7.156
9.006
1.00
8.17


ATOM
2649
N
LEU
B
184
10.286
−4.821
10.528
1.00
10.96


ATOM
2650
CA
LEU
B
184
10.311
−3.937
9.370
1.00
11.71


ATOM
2651
CB
LEU
B
184
11.758
−3.707
8.917
1.00
12.41


ATOM
2652
CG
LEU
B
184
12.048
−2.593
7.910
1.00
13.96


ATOM
2653
CD1
LEU
B
184
11.550
−1.250
8.429
1.00
14.99


ATOM
2654
CD2
LEU
B
184
13.543
−2.532
7.604
1.00
14.64


ATOM
2655
C
LEU
B
184
9.466
−4.465
8.207
1.00
11.44


ATOM
2656
O
LEU
B
184
8.546
−3.785
7.754
1.00
12.30
















TABLE 12







Additional contacts that contribute to, but are not essential,


for contact between human Dcn1 and human Ubc12 with hUbc12 Chain


E interactions with hDcn1P Chain B and hUbc12 Chain F interactions


with hDcn1P Chain A shown.











Distance


hUbc12 Atom
hDcn1 Atom
(Å)
















Leu
7E
CB
Ile
83B
CG2
4.14





Ile
83B
CD1
4.33


Leu
7E
CD1
Cys
115B
CB
3.61





Cys
115B
SG
3.78





Ile
83B
CG2
3.94


Leu
7E
C
Ile
83B
CD1
4.08


Leu
7E
O
Ile
83B
CD1
4.15


Lys
8E
N
Ile
83B
CD1
3.91


Lys
8E
CA
Ile
83B
CD1
3.85


Lys
8E
CB
Ile
83B
CD1
4.35


Lys
8E
CG
Ile
83B
CB
4.37





Ile
83B
CG1
4.22





Ile
83B
CD1
3.66


Lys
8E
CD
Asp
84B
OD1
4.18


Lys
8E
CE
Gln
87B
OE1
4.21


Lys
8E
NZ
Gln
87B
CB
3.97





Gln
87B
OE1
3.02





Gln
87B
CG
3.92





Gln
87B
CD
3.85


Lys
11E
CE
Ile
83B
CG1
3.97





Ile
83B
CD1
3.27


Lys
11E
NZ
Ile
83B
CG1
3.51





Ile
83B
CD1
3.27





Asp
84B
CG
4.36





Asp
84B
OD2
3.58


Leu
7F
CB
Ile
83A
CD1
4.13





Ile
83A
CG2
4.15


Leu
7F
CD1
Cys
115A
SG
3.96





Cys
115A
CB
3.83





Ile
83A
CG2
4.06


Leu
7F
C
Ile
83A
CD1
3.82


Leu
7F
O
Ile
83A
CD1
3.63


Lys
8F
N
Ile
83A
CD1
4.02


Lys
8F
CA
Ile
83A
CD1
4.08


Lys
8F
CD
Gln
87A
CG
4.46





Gln
87A
CD
4.5





Gln
87A
OE1
3.73


Lys
8F
CE
Gln
87A
CG
4.42





Gln
87A
CD
3.95





Gln
87A
OE1
2.91


Lys
8F
NZ
Gln
87A
CB
4.08





Gln
87A
CG
3.4





Gln
87A
CD
2.7





Gln
87A
OE1
1.78





Gln
87A
NE2
3.69


Lys
11F
CB
Ile
83A
CD1
3.99


Lys
11F
CD
Asp
84A
OD2
4.28





Ile
83A
CG1
4.26





Ile
83A
CD1
3.82


Lys
11F
CE
Asp
84A
OD2
3.93


Lys
11F
NZ
Asp
84A
CG
3.65





Asp
84A
OD1
4.2





Asp
84A
OD2
2.91
















TABLE 13







Atomic coordinates of residues that contribute to, but are not required for, binding


of hDcn1 Chain A to hUbc12 from structure with stapled hUbc12 peptide.


















Atom

Atom





Occu-
B-



No.

Name


X
Y
Z
pancy
factor




















ATOM
206
N
ASP
A
84
15.064
41.735
5.788
1.00
10.17


ATOM
207
CA
ASP
A
84
16.219
42.546
6.174
1.00
10.55


ATOM
208
CB
ASP
A
84
17.461
41.675
6.366
1.00
11.23


ATOM
209
CG
ASP
A
84
17.375
40.805
7.603
1.00
13.04


ATOM
210
OD1
ASP
A
84
16.531
41.113
8.522
1.00
16.41


ATOM
211
OD2
ASP
A
84
18.111
39.803
7.658
1.00
15.83


ATOM
212
C
ASP
A
84
16.489
43.657
5.159
1.00
9.88


ATOM
213
O
ASP
A
84
16.754
44.791
5.545
1.00
9.65


ATOM
285
N
ALA
A
94
15.031
56.885
5.766
1.00
11.05


ATOM
286
CA
ALA
A
94
14.657
57.464
7.067
1.00
11.12


ATOM
287
CB
ALA
A
94
15.239
58.886
7.231
1.00
11.03


ATOM
288
C
ALA
A
94
13.133
57.443
7.302
1.00
11.47


ATOM
289
O
ALA
A
94
12.545
58.432
7.751
1.00
10.94


ATOM
290
N
LEU
A
95
12.504
56.311
6.972
1.00
11.82


ATOM
291
CA
LEU
A
95
11.064
56.133
7.181
1.00
12.39


ATOM
292
CB
LEU
A
95
10.330
55.975
5.843
1.00
12.25


ATOM
293
CG
LEU
A
95
10.424
57.112
4.823
1.00
12.44


ATOM
294
CD1
LEU
A
95
9.967
56.631
3.452
1.00
12.15


ATOM
295
CD2
LEU
A
95
9.632
58.326
5.278
1.00
12.56


ATOM
296
C
LEU
A
95
10.731
54.935
8.064
1.00
12.66


ATOM
297
O
LEU
A
95
11.381
53.895
7.995
1.00
13.03


ATOM
298
N
ASP
A
96
9.702
55.101
8.883
1.00
13.06


ATOM
299
CA
ASP
A
96
9.059
53.996
9.589
1.00
13.50


ATOM
300
CB
ASP
A
96
7.901
54.552
10.424
1.00
13.89


ATOM
301
CG
ASP
A
96
7.251
53.509
11.344
1.00
16.05


ATOM
302
OD1
ASP
A
96
7.257
52.290
11.044
1.00
17.16


ATOM
303
OD2
ASP
A
96
6.702
53.934
12.381
1.00
17.91


ATOM
304
C
ASP
A
96
8.539
53.014
8.531
1.00
12.97


ATOM
305
O
ASP
A
96
7.807
53.421
7.620
1.00
12.93
















TABLE 14







Atomic coordinates of residues that contribute to, but are not required for,


binding of hDcn1 Chain B to hUbc12 in structure of stapled hUbc12 peptide.


















Atom

Atom





Occu-
B-



No.

Name


X
Y
Z
pancy
factor




















ATOM
2420
N
ASP
B
84
−29.031
110.230
−22.980
1.00
21.25


ATOM
2421
CA
ASP
B
84
−28.182
111.358
−23.383
1.00
21.22


ATOM
2422
CB
ASP
B
84
−29.019
112.619
−23.623
1.00
21.50


ATOM
2423
CG
ASP
B
84
−29.913
112.501
−24.842
1.00
22.06


ATOM
2424
OD1
ASP
B
84
−29.581
111.722
−25.760
1.00
23.61


ATOM
2425
OD2
ASP
B
84
−30.952
113.192
−24.884
1.00
23.44


ATOM
2426
C
ASP
B
84
−27.088
111.651
−22.365
1.00
20.88


ATOM
2427
O
ASP
B
84
−25.970
112.013
−22.735
1.00
20.81


ATOM
2499
N
ALA
B
94
−14.009
109.936
−22.759
1.00
22.67


ATOM
2500
CA
ALA
B
94
−13.443
109.599
−24.075
1.00
22.83


ATOM
2501
CB
ALA
B
94
−12.005
110.127
−24.218
1.00
22.75


ATOM
2502
C
ALA
B
94
−13.517
108.097
−24.360
1.00
22.93


ATOM
2503
O
ALA
B
94
−12.552
107.489
−24.826
1.00
22.99


ATOM
2504
N
LEU
B
95
−14.680
107.514
−24.076
1.00
23.01


ATOM
2505
CA
LEU
B
95
−14.892
106.079
−24.232
1.00
23.06


ATOM
2506
CB
LEU
B
95
−15.078
105.407
−22.863
1.00
23.00


ATOM
2507
CG
LEU
B
95
−13.879
105.257
−21.920
1.00
23.07


ATOM
2508
CD1
LEU
B
95
−14.348
104.751
−20.567
1.00
23.09


ATOM
2509
CD2
LEU
B
95
−12.807
104.331
−22.492
1.00
23.19


ATOM
2510
C
LEU
B
95
−16.087
105.753
−25.121
1.00
23.04


ATOM
2511
O
LEU
B
95
−17.128
106.409
−25.048
1.00
23.06


ATOM
2512
N
ASP
B
96
−15.912
104.739
−25.966
1.00
23.04


ATOM
2513
CA
ASP
B
96
−17.017
104.098
−26.670
1.00
23.21


ATOM
2514
CB
ASP
B
96
−16.474
102.949
−27.538
1.00
23.36


ATOM
2515
CG
ASP
B
96
−17.524
102.342
−28.477
1.00
24.22


ATOM
2516
OD1
ASP
B
96
−18.723
102.277
−28.126
1.00
25.07


ATOM
2517
OD2
ASP
B
96
−17.132
101.898
−29.578
1.00
24.66


ATOM
2518
C
ASP
B
96
−17.984
103.568
−25.603
1.00
22.87


ATOM
2519
O
ASP
B
96
−17.567
102.833
−24.704
1.00
22.78
















TABLE 15







Atomic coordinates of residues that contribute to, but are


not required for, binding of hDcn1 Chain A to hUbc12.


















Atom

Atom





Occu-
B-



No.

Name


X
Y
Z
pancy
factor




















ATOM
204
N
ASP
A
84
37.117
−17.398
−21.586
1.00
15.30


ATOM
205
CA
ASP
A
84
36.947
−18.847
−21.612
1.00
15.98


ATOM
206
CB
ASP
A
84
38.173
−19.558
−21.028
1.00
17.35


ATOM
207
CG
ASP
A
84
39.362
−19.558
−21.982
1.00
21.48


ATOM
208
OD1
ASP
A
84
39.184
−19.251
−23.181
1.00
24.51


ATOM
209
OD2
ASP
A
84
40.482
−19.872
−21.530
1.00
24.38


ATOM
210
C
ASP
A
84
35.666
−19.254
−20.888
1.00
14.58


ATOM
211
O
ASP
A
84
34.915
−20.101
−21.374
1.00
14.36


ATOM
283
N
ALA
A
94
23.572
−23.460
−24.936
1.00
12.74


ATOM
284
CA
ALA
A
94
23.354
−23.863
−26.331
1.00
13.46


ATOM
285
CB
ALA
A
94
22.336
−25.015
−26.429
1.00
13.90


ATOM
286
C
ALA
A
94
22.937
−22.668
−27.195
1.00
13.29


ATOM
287
O
ALA
A
94
22.037
−22.762
−28.036
1.00
13.55


ATOM
288
N
LEU
A
95
23.604
−21.540
−26.966
1.00
12.35


ATOM
289
CA
LEU
A
95
23.344
−20.312
−27.704
1.00
11.64


ATOM
290
CB
LEU
A
95
22.743
−19.238
−26.788
1.00
11.73


ATOM
291
CG
LEU
A
95
21.370
−19.454
−26.147
1.00
12.12


ATOM
292
CD1
LEU
A
95
21.138
−18.429
−25.053
1.00
12.54


ATOM
293
CD2
LEU
A
95
20.245
−19.399
−27.176
1.00
13.08


ATOM
294
C
LEU
A
95
24.640
−19.790
−28.295
1.00
11.08


ATOM
295
O
LEU
A
95
25.702
−19.886
−27.675
1.00
11.12


ATOM
296
N
ASP
A
96
24.544
−19.231
−29.496
1.00
10.92


ATOM
297
CA
ASP
A
96
25.671
−18.539
−30.101
1.00
10.77


ATOM
298
CB
ASP
A
96
25.292
−18.026
−31.489
1.00
11.39


ATOM
299
CG
ASP
A
96
26.490
−17.540
−32.278
1.00
13.24


ATOM
300
OD1
ASP
A
96
27.084
−16.505
−31.903
1.00
14.15


ATOM
301
OD2
ASP
A
96
26.836
−18.197
−33.280
1.00
16.29


ATOM
302
C
ASP
A
96
26.060
−17.373
−29.187
1.00
9.82


ATOM
303
O
ASP
A
96
25.191
−16.644
−28.725
1.00
9.62
















TABLE 16







Atomic coordinates of residues that contribute to, but are


not required for, binding of hDcn1 Chain B to hUbc12.


















Atom

Atom





Occu-
B-



No.

Name


X
Y
Z
pancy
factor




















ATOM
1827
N
ASP
B
84
29.753
5.342
6.933
1.00
16.27


ATOM
1828
CA
ASP
B
84
29.678
6.800
6.878
1.00
16.75


ATOM
1829
CB
ASP
B
84
31.075
7.421
7.001
1.00
17.47


ATOM
1830
CG
ASP
B
84
31.691
7.209
8.374
1.00
19.75


ATOM
1831
OD1
ASP
B
84
31.009
7.478
9.388
1.00
21.39


ATOM
1832
OD2
ASP
B
84
32.863
6.777
8.441
1.00
21.09


ATOM
1833
C
ASP
B
84
28.984
7.274
5.602
1.00
16.12


ATOM
1834
O
ASP
B
84
28.155
8.184
5.640
1.00
16.27


ATOM
1906
N
ALA
B
94
16.681
12.075
2.790
1.00
15.25


ATOM
1907
CA
ALA
B
94
15.771
12.480
3.867
1.00
16.29


ATOM
1908
CB
ALA
B
94
14.856
13.631
3.416
1.00
16.50


ATOM
1909
C
ALA
B
94
14.962
11.288
4.394
1.00
16.33


ATOM
1910
O
ALA
B
94
13.767
11.399
4.687
1.00
16.79


ATOM
1911
N
LEU
B
95
15.642
10.148
4.503
1.00
15.53


ATOM
1912
CA
LEU
B
95
15.058
8.907
5.003
1.00
14.51


ATOM
1913
CB
LEU
B
95
15.049
7.834
3.907
1.00
14.56


ATOM
1914
CG
LEU
B
95
14.170
8.032
2.671
1.00
15.10


ATOM
1915
CD1
LEU
B
95
14.550
7.008
1.608
1.00
15.62


ATOM
1916
CD2
LEU
B
95
12.693
7.925
3.006
1.00
16.31


ATOM
1917
C
LEU
B
95
15.852
8.393
6.190
1.00
13.79


ATOM
1918
O
LEU
B
95
17.080
8.489
6.215
1.00
13.74


ATOM
1919
N
ASP
B
96
15.143
7.843
7.173
1.00
13.45


ATOM
1920
CA
ASP
B
96
15.789
7.160
8.286
1.00
13.09


ATOM
1921
CB
ASP
B
96
14.745
6.737
9.323
1.00
13.72


ATOM
1922
CG
ASP
B
96
15.365
6.262
10.624
1.00
15.39


ATOM
1923
OD1
ASP
B
96
16.013
5.196
10.631
1.00
16.12


ATOM
1924
OD2
ASP
B
96
15.186
6.952
11.648
1.00
17.96


ATOM
1925
C
ASP
B
96
16.518
5.939
7.721
1.00
12.21


ATOM
1926
O
ASP
B
96
15.954
5.217
6.904
1.00
12.01









In still other embodiments, the E2-binding pocket of a NEDD8 co-E3 protein comprises the atoms of Table 17, which are found in amino acid residues Glu108, Met111, Glu112, Pro122, Thr123, Glu124, Val127, Leu128, Ala131, Met139, Cys140, Phe142, Phe189, Ile204, Ala207, Leu208, and Leu211 of human Dcn3 (SEQ ID NO: 39), or a structural variant thereof. In particular embodiments, the E2-binding pocket of a NEDD8 co-E3 protein comprises at least one of the following amino acid residues: a glutamic acid corresponding to position 108 of SEQ ID NO: 39, a methionine corresponding to position 111 of SEQ ID NO: 39, a glutamic acid corresponding to position 112 of SEQ ID NO: 39, a proline corresponding to position 122 of SEQ ID NO: 39, a threonine corresponding to position 123 of SEQ ID NO: 39, a glutamic acid corresponding to position 124 of SEQ ID NO: 39, a valine corresponding to position 127 of SEQ ID NO: 39, a leucine corresponding to position 128 of SEQ ID NO: 39, an alanine corresponding to position 131 of SEQ ID NO: 39, a methionine corresponding to position 139 of SEQ ID NO: 39, a cysteine corresponding to position 140 of SEQ ID NO: 39, a phenylalanine corresponding to position 142 of SEQ ID NO: 39, a phenylalanine corresponding to position 189 of SEQ ID NO: 39, an isoleucine corresponding to position 204 of SEQ ID NO: 39, an alanine corresponding to position 207 of SEQ ID NO: 39, a leucine corresponding to position 208 of SEQ ID NO: 39, and a leucine corresponding to position 211 of SEQ ID NO: 39.
In some of these embodiments, the E2-binding pocket comprises the atomic coordinates of Table 18 or 19, or a structural variant thereof. In particular embodiments, the co-E3 protein has the amino acid sequence set forth in SEQ ID NO: 39.
In certain embodiments, the E2-binding pocket of a NEDD8 co-E3 protein comprises the atoms of Table 17 and 20. The atoms in Table 20 are found in amino acid residues Glu109, Val120, and Asp121. Thus, in some embodiments, the E2-binding pocket of a NEDD8 co-E3 protein comprises at least one of the following amino acid residues: a glutamic acid corresponding to position 108 of SEQ ID NO: 39, a methionine corresponding to position 111 of SEQ ID NO: 39, a glutamic acid corresponding to position 112 of SEQ ID NO: 39, a praline corresponding to position 122 of SEQ ID NO: 39, a threonine corresponding to position 123 of SEQ ID NO: 39, a glutamic acid corresponding to position 124 of SEQ ID NO: 39, a valine corresponding to position 127 of SEQ ID NO: 39, a leucine corresponding to position 128 of SEQ ID NO: 39, an alanine corresponding to position 131 of SEQ ID NO: 39, a methionine corresponding to position 139 of SEQ ID NO: 39, a cysteine corresponding to position 140 of SEQ ID NO: 39, a phenylalanine corresponding to position 142 of SEQ ID NO: 39, a phenylalanine corresponding to position 189 of SEQ ID NO: 39, an isoleucine corresponding to position 204 of SEQ ID NO: 39, an alanine corresponding to position 207 of SEQ ID NO: 39, a leucine corresponding to position 208 of SEQ ID NO: 39, a leucine corresponding to position 211 of SEQ ID NO: 39; a glutamic acid corresponding to position 109 of SEQ ID NO: 39; a valine corresponding to position 120 of SEQ ID NO: 39; and an aspartic acid at position 121 of SEQ ID NO: 39.
In some of these embodiments, the E2-binding pocket comprises the atomic coordinates of Tables 18 and 21; 19 and 22; or a structural variant thereof.







TABLE 17







Minimal contacts between human Dcn3 and human Ube2f with


hUbe2f Chain F interactions with hDcn3P Chain A and hUbe2f Chain


G interactions with hDcn3P Chain B shown.











Distance


hUbe2f Atom
hDcn3 Atom
(Å)
















Acetyl-Met
1F
OT
Phe
189A
CZ
3.75





Phe
189A
CE2
3.2





Phe
189A
CD2
4.22





Met
139A
CG
3.96





Met
139A
SD
3.8





Met
139A
CE
3.47


Acetyl-Met
1F
CT1
Phe
189A
CZ
4.37





Phe
189A
CE2
3.94





Pro
122A
O
3.76





Thr
123A
CA
4.33


Acetyl-Met
1F
CT2
Phe
189A
CE2
4.36





Glu
124A
O
4.33





Leu
128A
N
4.19





Leu
128A
CA
4.42





Leu
128A
CB
4.33





Leu
211A
CD2
3.48





Thr
123A
O
3.78





Pro
122A
O
3.88





Thr
123A
CA
4.03





Thr
123A
C
3.97


Acetyl-Met
1F
N
Val
127A
CG1
4.18





Pro
122A
C
3.76





Pro
122A
O
2.74





Thr
123A
N
4.26





Thr
123A
CA
3.88


Acetyl-Met
1F
CA
Pro
122A
O
3.67


Acetyl-Met
1F
C
Met
139A
SD
4.47





Cys
140A
SG
4.43





Pro
122A
O
4.36


Acetyl-Met
1F
O
Pro
122A
O
4.34





Thr
123A
CG2
3.93


Acetyl-Met
1F
CB
Cys
140A
SG
4.24





Pro
122A
CB
4.47





Pro
122A
C
4.36





Pro
122A
O
3.53


Acetyl-Met
1F
CG
Met
111A
CG
3.84





Met
111A
SD
4.19





Val
127A
CG1
3.64





Pro
122A
O
3.66


Acetyl-Met
1F
SD
Met
139A
C
4.43





Phe
189A
CE1
4.45





Phe
189A
CZ
3.73





Met
111A
CG
4.27





Met
111A
SD
4.17





Met
111A
CE
4.22





Cys
140A
N
3.38





Cys
140A
CA
3.92





Val
127A
CG1
4.4


Acetyl-Met
1F
CE
Ala
131A
CB
3.97





Phe
142A
CE1
3.9





Phe
142A
CZ
3.67





Phe
189A
CE1
4.4





Phe
189A
CZ
3.87





Met
111A
SD
3.76





Met
111A
CE
3.86





Cys
140A
N
4.49





Val
127A
O
4.46





Val
127A
CG1
3.89


Leu
2F
N
Met
139A
SD
4.06





Thr
123A
CG2
4.33


Leu
2F
CA
Met
139A
SD
4.46





Thr
123A
CG2
4.3


Leu
2F
CB
Met
139A
SD
3.92





Met
139A
CE
3.91





Ile
204A
CD1
4.16


Leu
2F
CG
Met
139A
CE
4.47





Ala
207A
CB
4.12


Leu
2F
CD1
Leu
211A
CD2
4.4





Thr
123A
O
4.2





Thr
123A
CA
4.46





Thr
123A
CB
4.18


Leu
2F
CD2
Ile
204A
CG2
4.38





Met
139A
CE
3.97





Ile
204A
CD1
4.21





Ile
204A
O
4.08





Ala
207A
CB
3.61





Ala
207A
C
4.4





Leu
208A
N
4.1





Leu
208A
CG
3.56





Leu
208A
CD2
3.57


Thr
3F
N
Met
139A
SD
4.08


Thr
3F
OG1
Met
139A
CB
3.82





Met
139A
CG
4.45





Met
139A
SD
3.76





Cys
140A
SG
3.59


Thr
3F
CG2
Met
139A
SD
4.28





Ile
204A
CD1
3.82


Thr
3F
C
Cys
140A
SG
3.89


Thr
3F
O
Cys
140A
SG
4.22


Leu
4F
N
Cys
140A
SG
3.56


Leu
4F
CA
Cys
140A
SG
3.65


Leu
4F
CB
Cys
140A
SG
3.93


Leu
4F
CG
Glu
108A
O
4.44


Leu
4F
CD1
Glu
108A
CB
3.63





Cys
140A
CB
4.21





Glu
108A
CA
3.73





Glu
108A
C
3.93





Met
111A
CB
4.3





Met
111A
CG
4.47





Cys
140A
SG
4.04





Glu
108A
O
3.31


Leu
4F
CD2
Glu
112A
N
4.28





Glu
112A
CA
4.01





Glu
112A
CB
3.92





Glu
112A
CG
4.34





Pro
122A
CB
3.54





Pro
122A
CG
3.87


Acetyl-Met
1G
OT
Met
139B
CE
3.53





Phe
189B
CZ
3.91





Phe
189B
CE2
3.36





Phe
189B
CD2
4.4





Met
139B
CG
4.22





Met
139B
SD
3.92


Acetyl-Met
1G
CT1
Pro
122B
O
3.69





Thr
123B
CA
4.42





Phe
189B
CZ
4.46





Phe
189B
CE2
4.03


Acetyl-Met
1G
CT2
Pro
122B
O
3.91





Thr
123B
CA
4.2





Thr
123B
C
4.12





Val
127B
CG1
4.37





Thr
123B
O
3.97





Glu
124B
O
4.2





Leu
211B
CD2
3.6





Phe
189B
CE2
4.4





Leu
128B
N
4.04





Leu
128B
CB
4.26





Leu
128B
CA
4.32


Acetyl-Met
1G
N
Pro
122B
C
3.69





Pro
122B
O
2.62





Thr
123B
N
4.23





Thr
123B
CA
3.95





Val
127B
CG1
4.16


Acetyl-Met
1G
CA
Pro
122B
O
3.5





Cys
140B
SG
4.5


Acetyl-Met
1G
C
Thr
123B
OG1
4.45





Pro
122B
O
4.19





Cys
140B
SG
4.38





Met
139B
SD
4.49


Acetyl-Met
1G
O
Thr
123B
OG1
3.96





Pro
122B
O
4.18





Cys
140B
SG
4.47


Acetyl-Met
1G
CB
Pro
122B
C
4.31





Pro
122B
O
3.34





Cys
140B
SG
4.02


Acetyl-Met
1G
CG
Met
111B
CG
3.92





Met
111B
SD
4.32





Pro
122B
O
3.43





Val
127B
CG1
3.6


Acetyl-Met
1G
SD
Met
111B
CG
4.29





Met
111B
SD
4.26





Met
111B
CE
4.35





Met
139B
C
4.46





Cys
140B
N
3.38





Cys
140B
CA
3.84





Val
127B
CG1
4.31





Phe
189B
CZ
3.79


Acetyl-Met
1G
CE
Met
111B
SD
3.99





Met
111B
CE
4.1





Val
127B
CG1
3.75





Ala
131B
CB
3.9





Phe
142B
CE1
3.96





Phe
142B
CZ
3.83





Phe
189B
CZ
3.76





Phe
189B
CE1
4.3


Leu
2G
N
Met
139B
CE
4.18





Met
139B
SD
3.68


Leu
2G
CA
Thr
123B
OG1
4.14





Met
139B
SD
4.37


Leu
2G
CB
Met
139B
CE
4.09





Met
139B
SD
4.17


Leu
2G
CG
Met
139B
CE
3.98





Leu
208B
CD2
4.16


Leu
2G
CD1
Ala
207B
CB
3.61





Met
139B
CE
4.12





Leu
208B
CD2
3.55





Ala
207B
C
4.32





Ile
204B
O
4





Leu
208B
N
4.14





Leu
208B
CG
3.7


Leu
2G
CD2
Thr
123B
CB
4.48





Thr
123B
OG1
3.87





Thr
123B
CG2
4.41





Ala
207B
CB
4.49





Thr
123B
O
4.27


Thr
3G
N
Met
139B
SD
4.04


Thr
3G
OG1
Met
139B
CB
3.84





Cys
140B
SG
3.5





Met
139B
SD
3.9


Thr
3G
CG2
Met
139B
SD
4.24


Thr
3G
C
Cys
140B
SG
3.97


Thr
3G
O
Cys
140B
SG
4.42


Leu
4G
N
Cys
140B
SG
3.52


Leu
4G
CA
Cys
140B
SG
3.62


Leu
4G
CB
Cys
140B
SG
3.85


Leu
4G
CG
Cys
140B
SG
4.46


Leu
4G
CD1
Glu
108B
C
4.11





Glu
108B
O
3.6





Met
111B
CB
4.3





Met
111B
CG
4.41





Glu
108B
CA
3.81





Glu
108B
CB
3.67





Cys
140B
CB
4.04





Cys
140B
SG
3.8


Leu
4G
CD2
Glu
112B
CG
4.47





Pro
122B
CG
3.78





Glu
112B
N
4.42





Glu
112B
CA
4.19





Glu
112B
CB
4.17





Pro
122B
CB
3.39
















TABLE 18







Atomic coordinates of amino acid residues essential


for human Dcn3 Chain A to bind hUbe2F.


















Atom

Atom





Occu-
B-



No.

Name


X
Y
Z
pancy
factor




















ATOM
187
N
GLU
A
108
16.684
2.422
47.251
1.00
32.01


ATOM
188
CA
GLU
A
108
17.898
3.239
47.429
1.00
32.03


ATOM
189
CB
GLU
A
108
17.705
4.680
46.928
1.00
32.27


ATOM
190
CG
GLU
A
108
16.456
5.392
47.441
1.00
33.27


ATOM
191
CD
GLU
A
108
15.314
5.376
46.434
1.00
35.06


ATOM
192
OE1
GLU
A
108
15.176
4.387
45.679
1.00
35.38


ATOM
193
OE2
GLU
A
108
14.551
6.363
46.399
1.00
36.14


ATOM
194
C
GLU
A
108
18.476
3.251
48.851
1.00
31.77


ATOM
195
O
GLU
A
108
19.693
3.258
49.018
1.00
31.35


ATOM
209
N
MET
A
111
19.991
−0.224
49.462
1.00
28.11


ATOM
210
CA
MET
A
111
21.137
−0.378
48.570
1.00
27.16


ATOM
211
CB
MET
A
111
20.793
0.211
47.202
1.00
27.24


ATOM
212
CG
MET
A
111
21.843
−0.028
46.128
1.00
27.61


ATOM
213
SD
MET
A
111
21.915
−1.768
45.692
1.00
29.58


ATOM
214
CE
MET
A
111
20.390
−1.963
44.779
1.00
25.97


ATOM
215
C
MET
A
111
22.400
0.294
49.111
1.00
26.60


ATOM
216
O
MET
A
111
23.516
−0.231
48.985
1.00
26.39


ATOM
217
N
GLU
A
112
22.213
1.471
49.694
1.00
25.97


ATOM
218
CA
GLU
A
112
23.315
2.248
50.250
1.00
25.38


ATOM
219
CB
GLU
A
112
22.847
3.680
50.561
1.00
24.79


ATOM
220
CG
GLU
A
112
23.924
4.609
51.094
1.00
24.86


ATOM
221
CD
GLU
A
112
24.277
4.342
52.556
1.00
25.41


ATOM
222
OE1
GLU
A
112
23.366
4.048
53.367
1.00
24.01


ATOM
223
OE2
GLU
A
112
25.477
4.428
52.895
1.00
26.77


ATOM
224
C
GLU
A
112
23.899
1.524
51.480
1.00
25.08


ATOM
225
O
GLU
A
112
25.118
1.373
51.588
1.00
25.12


ATOM
299
N
PRO
A
122
29.322
2.686
46.412
1.00
26.00


ATOM
300
CA
PRO
A
122
28.153
2.245
45.628
1.00
25.78


ATOM
301
CB
PRO
A
122
27.022
3.162
46.120
1.00
25.58


ATOM
302
CG
PRO
A
122
27.497
3.753
47.390
1.00
25.21


ATOM
303
CD
PRO
A
122
28.986
3.786
47.338
1.00
25.69


ATOM
304
C
PRO
A
122
28.301
2.388
44.099
1.00
25.85


ATOM
305
O
PRO
A
122
27.590
1.718
43.354
1.00
25.98


ATOM
306
N
THR
A
123
29.195
3.262
43.644
0.85
26.10


ATOM
307
CA
THR
A
123
29.356
3.551
42.211
1.00
26.12


ATOM
308
CB
THR
A
123
29.594
5.050
41.977
1.00
25.83


ATOM
309
OG1
THR
A
123
30.856
5.415
42.536
1.00
25.73


ATOM
310
CG2
THR
A
123
28.509
5.866
42.638
1.00
26.02


ATOM
311
C
THR
A
123
30.510
2.765
41.573
1.00
26.40


ATOM
312
O
THR
A
123
30.782
2.871
40.367
1.00
26.51


ATOM
313
N
GLU
A
124
31.174
1.964
42.388
1.00
26.52


ATOM
314
CA
GLU
A
124
32.391
1.277
41.977
1.00
27.12


ATOM
315
CB
GLU
A
124
33.253
1.064
43.218
1.00
27.17


ATOM
316
CG
GLU
A
124
34.192
−0.090
43.152
1.00
31.54


ATOM
317
CD
GLU
A
124
34.229
−0.832
44.442
1.00
34.97


ATOM
318
OE1
GLU
A
124
33.153
−0.981
45.040
1.00
38.54


ATOM
319
OE2
GLU
A
124
35.317
−1.249
44.864
1.00
35.84


ATOM
320
C
GLU
A
124
32.137
−0.034
41.195
1.00
26.48


ATOM
321
O
GLU
A
124
31.048
−0.614
41.258
1.00
26.29


ATOM
344
N
VAL
A
127
30.268
−3.281
42.243
1.00
24.00


ATOM
345
CA
VAL
A
127
28.822
−3.096
42.392
1.00
23.84


ATOM
346
CB
VAL
A
127
28.461
−1.735
43.066
1.00
23.94


ATOM
347
CG1
VAL
A
127
26.957
−1.516
43.092
1.00
23.56


ATOM
348
CG2
VAL
A
127
29.006
−1.696
44.485
1.00
23.26


ATOM
349
C
VAL
A
127
28.117
−3.264
41.046
1.00
23.76


ATOM
350
O
VAL
A
127
27.015
−3.820
40.989
1.00
23.37


ATOM
351
N
LEU
A
128
28.759
−2.798
39.970
1.00
23.82


ATOM
352
CA
LEU
A
128
28.271
−3.081
38.618
1.00
24.05


ATOM
353
CB
LEU
A
128
29.088
−2.342
37.537
1.00
24.16


ATOM
354
CG
LEU
A
128
28.768
−2.629
36.055
1.00
23.92


ATOM
355
CD1
LEU
A
128
27.382
−2.121
35.666
1.00
23.42


ATOM
356
CD2
LEU
A
128
29.834
−2.033
35.131
1.00
23.07


ATOM
357
C
LEU
A
128
28.230
−4.597
38.360
1.00
23.85


ATOM
358
O
LEU
A
128
27.226
−5.111
37.861
1.00
24.40


ATOM
375
N
ALA
A
131
25.273
−6.175
40.307
1.00
24.30


ATOM
376
CA
ALA
A
131
24.040
−5.888
39.589
1.00
24.39


ATOM
377
CB
ALA
A
131
23.979
−4.414
39.234
1.00
23.85


ATOM
378
C
ALA
A
131
23.907
−6.751
38.330
1.00
25.10


ATOM
379
O
ALA
A
131
22.824
−7.264
38.030
1.00
25.10


ATOM
440
N
MET
A
139
18.715
0.516
38.147
1.00
33.32


ATOM
441
CA
MET
A
139
19.661
1.079
39.119
1.00
32.93


ATOM
442
CB
MET
A
139
20.280
2.368
38.591
1.00
32.99


ATOM
443
CG
MET
A
139
21.560
2.151
37.852
1.00
33.71


ATOM
444
SD
MET
A
139
22.351
3.707
37.450
1.00
35.60


ATOM
445
CE
MET
A
139
23.571
3.131
36.258
1.00
32.49


ATOM
446
C
MET
A
139
19.038
1.357
40.483
1.00
32.55


ATOM
447
O
MET
A
139
17.924
1.889
40.563
1.00
32.23


ATOM
448
N
CYS
A
140
19.776
1.003
41.540
1.00
31.91


ATOM
449
CA
CYS
A
140
19.395
1.286
42.930
1.00
31.63


ATOM
450
CB
CYS
A
140
19.065
2.773
43.148
1.00
31.56


ATOM
451
SG
CYS
A
140
20.261
3.913
42.469
1.00
31.88


ATOM
452
C
CYS
A
140
18.235
0.433
43.412
1.00
31.60


ATOM
453
O
CYS
A
140
17.563
0.780
44.391
1.00
31.76


ATOM
463
N
PHE
A
142
17.106
−3.895
43.853
1.00
29.28


ATOM
464
CA
PHE
A
142
17.085
−5.342
43.679
1.00
28.66


ATOM
465
CB
PHE
A
142
18.143
−6.045
44.543
1.00
28.19


ATOM
466
CG
PHE
A
142
19.576
−5.670
44.235
1.00
27.80


ATOM
467
CD1
PHE
A
142
20.035
−5.556
42.922
1.00
26.71


ATOM
468
CE1
PHE
A
142
21.362
−5.235
42.649
1.00
25.52


ATOM
469
CZ
PHE
A
142
22.250
−5.032
43.693
1.00
26.49


ATOM
470
CE2
PHE
A
142
21.815
−5.161
45.013
1.00
26.54


ATOM
471
CD2
PHE
A
142
20.487
−5.481
45.277
1.00
27.03


ATOM
472
C
PHE
A
142
15.712
−5.805
44.158
1.00
28.73


ATOM
473
O
PHE
A
142
15.370
−5.604
45.327
1.00
28.41


ATOM
849
N
PHE
A
189
21.039
−1.501
32.229
1.00
30.93


ATOM
850
CA
PHE
A
189
21.160
−1.056
33.617
1.00
31.54


ATOM
851
CB
PHE
A
189
22.500
−1.553
34.166
1.00
31.55


ATOM
852
CG
PHE
A
189
22.696
−1.333
35.643
1.00
31.93


ATOM
853
CD1
PHE
A
189
21.763
−1.789
36.571
1.00
33.33


ATOM
854
CE1
PHE
A
189
21.968
−1.598
37.943
1.00
33.69


ATOM
855
CZ
PHE
A
189
23.126
−0.973
38.387
1.00
32.63


ATOM
856
CE2
PHE
A
189
24.069
−0.532
37.470
1.00
32.60


ATOM
857
CD2
PHE
A
189
23.853
−0.718
36.110
1.00
32.46


ATOM
858
C
PHE
A
189
21.058
0.466
33.745
1.00
32.32


ATOM
859
O
PHE
A
189
20.321
0.974
34.589
1.00
32.66


ATOM
954
N
ILE
A
204
25.618
9.706
32.247
1.00
34.61


ATOM
955
CA
ILE
A
204
25.508
8.298
32.657
1.00
34.09


ATOM
956
CB
ILE
A
204
24.018
7.839
32.683
1.00
34.26


ATOM
957
CG1
ILE
A
204
23.232
8.581
33.776
1.00
34.69


ATOM
958
CD1
ILE
A
204
23.623
8.214
35.217
1.00
35.98


ATOM
959
CG2
ILE
A
204
23.894
6.320
32.839
1.00
34.13


ATOM
960
C
ILE
A
204
26.319
7.391
31.725
1.00
33.50


ATOM
961
O
ILE
A
204
27.104
6.566
32.178
1.00
33.59


ATOM
975
N
ALA
A
207
29.988
7.778
32.451
1.00
29.61


ATOM
976
CA
ALA
A
207
30.345
7.131
33.705
1.00
29.37


ATOM
977
CB
ALA
A
207
29.407
7.586
34.815
1.00
29.46


ATOM
978
C
ALA
A
207
30.344
5.607
33.582
1.00
28.94


ATOM
979
O
ALA
A
207
31.266
4.929
34.063
1.00
28.63


ATOM
980
N
LEU
A
208
29.312
5.076
32.934
1.00
28.74


ATOM
981
CA
LEU
A
208
29.198
3.634
32.714
1.00
28.63


ATOM
982
CB
LEU
A
208
27.801
3.262
32.255
1.00
28.66


ATOM
983
CG
LEU
A
208
26.696
3.311
33.306
1.00
29.25


ATOM
984
CD1
LEU
A
208
25.366
2.970
32.646
1.00
29.90


ATOM
985
CD2
LEU
A
208
26.995
2.351
34.460
1.00
30.23


ATOM
986
C
LEU
A
208
30.233
3.097
31.725
1.00
28.50


ATOM
987
O
LEU
A
208
30.692
1.966
31.874
1.00
28.53


ATOM
1011
N
LEU
A
211
33.243
2.948
33.888
1.00
28.64


ATOM
1012
CA
LEU
A
211
33.102
1.803
34.773
1.00
28.57


ATOM
1013
CB
LEU
A
211
31.764
1.868
35.513
1.00
28.22


ATOM
1014
CG
LEU
A
211
31.691
1.163
36.865
1.00
27.92


ATOM
1015
CD1
LEU
A
211
32.732
1.716
37.830
1.00
26.69


ATOM
1016
CD2
LEU
A
211
30.304
1.334
37.429
1.00
26.94


ATOM
1017
C
LEU
A
211
33.255
0.447
34.064
1.00
28.71


ATOM
1018
O
LEU
A
211
34.032
−0.405
34.510
1.00
28.67
















TABLE 19







Atomic coordinates of amino acid residues essential


for human Dcn3 Chain B to bind hUbe2F.


















Atom

Atom





Occu-
B-



No.

Name


X
Y
Z
pancy
factor




















ATOM
1789
N
GLU
B
108
−2.928
−7.977
13.187
1.00
29.97


ATOM
1790
CA
GLU
B
108
−2.667
−8.790
11.989
1.00
30.11


ATOM
1791
CB
GLU
B
108
−2.220
−10.221
12.340
1.00
30.21


ATOM
1792
CG
GLU
B
108
−3.139
−10.971
13.304
1.00
32.70


ATOM
1793
CD
GLU
B
108
−2.758
−10.746
14.765
1.00
35.39


ATOM
1794
OE1
GLU
B
108
−1.786
−9.993
15.029
1.00
36.06


ATOM
1795
OE2
GLU
B
108
−3.431
−11.324
15.646
1.00
35.26


ATOM
1796
C
GLU
B
108
−3.813
−8.795
10.968
1.00
29.58


ATOM
1797
O
GLU
B
108
−3.574
−8.641
9.771
1.00
29.17


ATOM
1811
N
MET
B
111
−3.873
−5.424
9.369
1.00
26.60


ATOM
1812
CA
MET
B
111
−2.622
−5.223
8.640
1.00
26.08


ATOM
1813
CB
MET
B
111
−1.434
−5.761
9.444
1.00
25.97


ATOM
1814
CG
MET
B
111
−0.084
−5.508
8.793
1.00
26.18


ATOM
1815
SD
MET
B
111
0.323
−3.749
8.840
1.00
28.45


ATOM
1816
CE
MET
B
111
0.542
−3.511
10.610
1.00
25.99


ATOM
1817
C
MET
B
111
−2.676
−5.912
7.274
1.00
25.72


ATOM
1818
O
MET
B
111
−2.175
−5.375
6.272
1.00
25.80


ATOM
1819
N
GLU
B
112
−3.269
−7.106
7.250
1.00
24.77


ATOM
1820
CA
GLU
B
112
−3.437
−7.860
6.011
1.00
24.17


ATOM
1821
CB
GLU
B
112
−3.954
−9.276
6.294
1.00
23.71


ATOM
1822
CG
GLU
B
112
−3.966
−10.185
5.077
1.00
23.79


ATOM
1823
CD
GLU
B
112
−5.214
−10.001
4.225
1.00
24.84


ATOM
1824
OE1
GLU
B
112
−6.315
−9.845
4.793
1.00
23.82


ATOM
1825
OE2
GLU
B
112
−5.100
−10.013
2.982
1.00
25.84


ATOM
1826
C
GLU
B
112
−4.352
−7.104
5.033
1.00
23.85


ATOM
1827
O
GLU
B
112
−4.036
−6.998
3.851
1.00
24.18


ATOM
1901
N
PRO
B
122
2.307
−8.239
1.798
1.00
23.06


ATOM
1902
CA
PRO
B
122
2.578
−7.735
3.172
1.00
23.39


ATOM
1903
CB
PRO
B
122
1.678
−8.608
4.056
1.00
23.19


ATOM
1904
CG
PRO
B
122
0.682
−9.236
3.122
1.00
23.52


ATOM
1905
CD
PRO
B
122
1.369
−9.376
1.804
1.00
23.09


ATOM
1906
C
PRO
B
122
4.037
−7.894
3.620
1.00
23.35


ATOM
1907
O
PRO
B
122
4.438
−7.374
4.661
1.00
23.55


ATOM
1908
N
THR
B
123
4.802
−8.590
2.790
1.00
23.31


ATOM
1909
CA
THR
B
123
6.142
−9.068
3.052
1.00
23.09


ATOM
1910
CB
THR
B
123
6.134
−10.589
2.697
1.00
23.23


ATOM
1911
OG1
THR
B
123
6.033
−11.371
3.890
1.00
23.77


ATOM
1912
CG2
THR
B
123
7.327
−11.017
1.891
1.00
22.66


ATOM
1913
C
THR
B
123
7.189
−8.294
2.211
1.00
23.32


ATOM
1914
O
THR
B
123
8.400
−8.483
2.374
1.00
23.40


ATOM
1915
N
GLU
B
124
6.737
−7.419
1.314
1.00
23.26


ATOM
1916
CA
GLU
B
124
7.663
−6.765
0.384
1.00
23.57


ATOM
1917
CB
GLU
B
124
7.014
−6.506
−0.974
1.00
23.45


ATOM
1918
CG
GLU
B
124
6.055
−5.334
−1.019
1.00
25.47


ATOM
1919
CD
GLU
B
124
5.787
−4.870
−2.441
1.00
28.88


ATOM
1920
OE1
GLU
B
124
4.946
−5.505
−3.136
1.00
29.24


ATOM
1921
OE2
GLU
B
124
6.427
−3.870
−2.854
1.00
30.26


ATOM
1922
C
GLU
B
124
3.329
−5.505
0.945
1.00
23.68


ATOM
1923
O
GLU
B
124
7.937
−4.985
1.999
1.00
23.52


ATOM
1946
N
VAL
B
127
6.523
−2.296
2.424
1.00
24.05


ATOM
1947
CA
VAL
B
127
5.875
−2.439
3.725
1.00
23.81


ATOM
1948
CB
VAL
B
127
5.124
−3.796
3.862
1.00
23.86


ATOM
1949
CG1
VAL
B
127
4.638
−4.015
5.282
1.00
22.40


ATOM
1950
CG2
VAL
B
127
3.946
−3.842
2.892
1.00
23.69


ATOM
1951
C
VAL
B
127
6.911
−2.240
4.829
1.00
23.92


ATOM
1952
O
VAL
B
127
6.634
−1.571
5.839
1.00
24.19


ATOM
1953
N
LEU
B
128
8.107
−2.786
4.619
1.00
23.58


ATOM
1954
CA
LEU
B
128
9.239
−2.482
5.489
1.00
23.70


ATOM
1955
CB
LEU
B
128
10.511
−3.223
5.059
1.00
23.73


ATOM
1956
CG
LEU
B
128
11.749
−2.865
5.898
1.00
23.84


ATOM
1957
CD1
LEU
B
128
11.663
−3.458
7.290
1.00
23.49


ATOM
1958
CD2
LEU
B
128
13.010
−3.333
5.209
1.00
25.14


ATOM
1959
C
LEU
B
128
9.495
−0.971
5.567
1.00
23.52


ATOM
1960
O
LEU
B
128
9.653
−0.440
6.668
1.00
23.85


ATOM
1977
N
ALA
B
131
6.644
0.572
7.623
1.00
21.94


ATOM
1978
CA
ALA
B
131
6.866
0.327
9.054
1.00
21.30


ATOM
1979
CB
ALA
B
131
7.166
−1.143
9.295
1.00
20.73


ATOM
1980
C
ALA
B
131
7.991
1.213
9.620
1.00
21.62


ATOM
1981
O
ALA
B
131
7.889
1.731
10.736
1.00
21.42


ATOM
2042
N
MET
B
139
6.269
−6.221
14.687
1.00
31.52


ATOM
2043
CA
MET
B
139
5.752
−6.772
13.433
1.00
30.64


ATOM
2044
CB
MET
B
139
6.474
−8.062
13.065
1.00
30.37


ATOM
2045
CG
MET
B
139
7.683
−7.835
12.225
1.00
30.22


ATOM
2046
SD
MET
B
139
8.256
−9.376
11.518
1.00
30.12


ATOM
2047
CE
MET
B
139
9.770
−8.814
10.743
1.00
29.99


ATOM
2048
C
MET
B
139
4.250
−7.036
13.490
1.00
30.11


ATOM
2049
O
MET
B
139
3.747
−7.573
14.480
1.00
29.85


ATOM
2050
N
CYS
B
140
3.555
−6.639
12.421
1.00
29.34


ATOM
2051
CA
CYS
B
140
2.118
−6.876
12.238
1.00
28.75


ATOM
2052
CB
CYS
B
140
1.772
−8.366
12.411
1.00
28.65


ATOM
2053
SG
CYS
B
140
2.841
−9.467
11.455
1.00
29.16


ATOM
2054
C
CYS
B
140
1.239
−5.994
13.126
1.00
28.39


ATOM
2055
O
CYS
B
140
0.065
−6.302
13.357
1.00
28.43


ATOM
2065
N
PHE
B
142
0.389
−1.667
13.943
1.00
25.66


ATOM
2066
CA
PHE
B
142
0.537
−0.217
14.050
1.00
25.09


ATOM
2067
CB
PHE
B
142
0.094
0.503
12.767
1.00
24.60


ATOM
2068
CG
PHE
B
142
0.887
0.145
11.545
1.00
23.64


ATOM
2069
CD1
PHE
B
142
2.264
−0.020
11.606
1.00
21.72


ATOM
2070
CE1
PHE
B
142
2.995
−0.341
10.471
1.00
21.40


ATOM
2071
CZ
PHE
B
142
2.353
−0.493
9.244
1.00
21.81


ATOM
2072
CE2
PHE
B
142
0.971
−0.315
9.159
1.00
23.34


ATOM
2073
CD2
PHE
B
142
0.246
0.003
10.312
1.00
24.04


ATOM
2074
C
PHE
B
142
−0.358
0.260
15.188
1.00
25.17


ATOM
2075
O
PHE
B
142
−1.573
0.061
15.140
1.00
25.12


ATOM
2451
N
PHE
B
189
12.757
−4.136
14.489
1.00
27.50


ATOM
2452
CA
PHE
B
189
11.486
−4.532
13.896
1.00
27.79


ATOM
2453
CB
PHE
B
189
11.384
−3.922
12.498
1.00
27.46


ATOM
2454
CG
PHE
B
189
10.085
−4.177
11.802
1.00
26.05


ATOM
2455
CD1
PHE
B
189
8.886
−3.711
12.333
1.00
25.09


ATOM
2456
CE1
PHE
B
189
7.680
−3.937
11.675
1.00
24.46


ATOM
2457
CZ
PHE
B
189
7.668
−4.619
10.463
1.00
24.31


ATOM
2458
CE2
PHE
B
189
8.867
−5.087
9.917
1.00
25.07


ATOM
2459
CD2
PHE
B
189
10.064
−4.855
10.588
1.00
24.93


ATOM
2460
C
PHE
B
189
11.335
−6.053
13.847
1.00
28.94


ATOM
2461
O
PHE
B
189
10.249
−6.580
14.093
1.00
29.14


ATOM
2559
N
ILE
B
204
14.094
−15.322
10.184
1.00
29.32


ATOM
2560
CA
ILE
B
204
13.693
−13.913
10.146
1.00
28.78


ATOM
2561
CB
ILE
B
204
13.179
−13.406
11.518
1.00
28.99


ATOM
2562
CG1
ILE
B
204
11.731
−13.823
11.745
1.00
29.30


ATOM
2563
CD1
ILE
B
204
11.591
−15.090
12.517
1.00
31.74


ATOM
2564
CG2
ILE
B
204
13.236
−11.883
11.596
1.00
28.88


ATOM
2565
C
ILE
B
204
14.856
−13.037
9.677
1.00
28.15


ATOM
2566
O
ILE
B
204
14.692
−12.219
8.781
1.00
28.26


ATOM
2580
N
ALA
B
207
15.521
−13.559
5.951
1.00
25.24


ATOM
2581
CA
ALA
B
207
14.484
−12.882
5.189
1.00
25.15


ATOM
2582
CB
ALA
B
207
13.102
−13.349
5.634
1.00
25.21


ATOM
2583
C
ALA
B
207
14.607
−11.362
5.271
1.00
25.20


ATOM
2584
O
ALA
B
207
14.416
−10.670
4.265
1.00
25.68


ATOM
2585
N
LEU
B
208
14.945
−10.844
6.451
1.00
24.96


ATOM
2586
CA
LEU
B
208
15.113
−9.394
6.626
1.00
24.85


ATOM
2587
CB
LEU
B
208
15.102
−9.001
8.100
1.00
24.71


ATOM
2588
CG
LEU
B
208
13.746
−9.038
8.809
1.00
25.14


ATOM
2589
CD1
LEU
B
208
13.923
−8.622
10.264
1.00
25.27


ATOM
2590
CD2
LEU
B
208
12.710
−8.147
8.123
1.00
24.28


ATOM
2591
C
LEU
B
208
16.355
−8.848
5.923
1.00
24.52


ATOM
2592
O
LEU
B
208
16.313
−7.779
5.329
1.00
24.45


ATOM
2616
N
LEU
B
211
15.564
−8.585
2.472
1.00
25.74


ATOM
2617
CA
LEU
B
211
14.620
−7.487
2.359
1.00
25.81


ATOM
2618
CB
LEU
B
211
13.474
−7.660
3.359
1.00
25.74


ATOM
2619
CG
LEU
B
211
12.239
−6.840
3.033
1.00
25.44


ATOM
2620
CD1
LEU
B
211
11.726
−7.220
1.637
1.00
25.06


ATOM
2621
CD2
LEU
B
211
11.181
−7.070
4.099
1.00
26.22


ATOM
2622
C
LEU
B
211
15.257
−6.101
2.507
1.00
26.06


ATOM
2623
O
LEU
B
211
15.008
−5.230
1.689
1.00
26.06
















TABLE 20







Additional contacts that contribute to, but are not essential,


for contact between human Dcn3 and human Ube2f with hUbe2f Chain


F interactions with hDcn3P Chain A and hUbe2f Chain G interactions


with hDcn3P Chain B shown.















Distance



hUbe2f Atom

hDcn3 Atom
(Å)



















Lys
7F
CB
Glu
108A
CB
4.3






Glu
108A
CD
4.2






Glu
108A
OE2
4.14






Glu
108A
CG
4.31



Lys
7F
CD
Glu
108A
OE1
4.42






Cys
140A
CB
4.12






Cys
140A
SG
3.84



Lys
7F
CE
Glu
108A
CD
4.17






Glu
108A
OE1
3.79






Glu
108A
OE2
4.26



Lys
7F
O
Glu
108A
CD
4.41






Glu
108A
OE2
3.84






Glu
108A
CG
4.2



Leu
8F
CG
Glu
108A
CB
4.38



Leu
8F
CD1
Glu
108A
O
4.29






Glu
112A
CB
3.96






Glu
112A
CG
3.98



Leu
8F
CD2
Glu
108A
CB
4.03






Glu
108A
CG
3.88






Glu
108A
C
4.11






Glu
109A
N
4.18






Glu
109A
CA
4.26






Glu
109A
CG
4.16






Glu
108A
O
4.12



Lys
7G
CB
Glu
108B
CG
4.3






Glu
108B
CD
4.48






Glu
108B
CB
4.29



Lys
7G
CD
Glu
108B
OE1
4.02






Cys
140B
CB
4.12






Cys
140B
SG
4.04



Lys
7G
NZ
Glu
108B
OE1
4.21






Met
139B
O
3.95



Lys
7G
O
Glu
108B
CG
4.36



Leu
8G
CG
Glu
108B
CB
4.41



Leu
8G
CD1
Glu
112B
CG
3.84






Glu
112B
CB
3.89



Leu
8G
CD2
Glu
109B
CG
4.39






Glu
109B
CA
3.94






Glu
109B
N
3.88






Glu
108B
CG
4.02






Glu
108B
C
3.95






Glu
108B
O
4.01






Glu
108B
CB
4.12

















TABLE 21







Atomic coordinates of amino acid residues of hDcn3 Chain A that


contribute to, but are not required, for binding hUbe2F.


















Atom

Atom





Occu-
B-



No.

Name


X
Y
Z
pancy
factor




















ATOM
196
N
GLU
A
109
17.607
3.250
49.863
1.00
31.59


ATOM
197
CA
GLU
A
109
18.060
3.299
51.247
1.00
31.75


ATOM
198
CB
GLU
A
109
16.902
3.575
52.213
1.00
32.09


ATOM
199
CG
GLU
A
109
16.229
4.933
52.026
1.00
34.47


ATOM
200
CD
GLU
A
109
15.075
4.915
51.007
1.00
37.77


ATOM
201
OE1
GLU
A
109
14.960
3.957
50.200
1.00
37.79


ATOM
202
OE2
GLU
A
109
14.273
5.878
51.023
1.00
39.47


ATOM
203
C
GLU
A
109
18.775
1.997
51.598
1.00
31.35


ATOM
204
O
GLU
A
109
19.923
2.016
52.059
1.00
31.47


ATOM
284
N
VAL
A
120
32.343
−1.152
50.143
1.00
28.04


ATOM
285
CA
VAL
A
120
32.370
−0.816
48.732
1.00
27.63


ATOM
286
CB
VAL
A
120
31.772
−2.024
47.945
1.00
27.52


ATOM
287
CG1
VAL
A
120
30.862
−1.607
46.834
1.00
26.88


ATOM
288
CG2
VAL
A
120
32.884
−2.959
47.468
1.00
27.58


ATOM
289
C
VAL
A
120
31.644
0.511
48.420
1.00
27.54


ATOM
290
O
VAL
A
120
30.512
0.733
48.872
1.00
27.62


ATOM
291
N
ASP
A
121
32.307
1.388
47.662
1.00
26.97


ATOM
292
CA
ASP
A
121
31.673
2.594
47.134
1.00
26.54


ATOM
293
CB
ASP
A
121
32.683
3.431
46.341
1.00
26.32


ATOM
294
CG
ASP
A
121
32.118
4.781
45.877
1.00
26.75


ATOM
295
OD1
ASP
A
121
32.719
5.831
46.203
1.00
25.81


ATOM
296
OD2
ASP
A
121
31.087
4.801
45.167
1.00
27.68


ATOM
297
C
ASP
A
121
30.539
2.125
46.236
1.00
26.35


ATOM
298
O
ASP
A
121
30.760
1.257
45.389
1.00
26.75
















TABLE 22







Atomic coordinates of amino acid residues of hDcn3 Chain B that


contribute to, but are not required, for binding hUbe2F.


















Atom

Atom





Occu-
B-



No.

Name


X
Y
Z
pancy
factor




















ATOM
1798
N
GLU
B
109
−5.041
−8.978
11.450
1.00
29.25


ATOM
1799
CA
GLU
B
109
−6.225
−8.988
10.595
1.00
29.12


ATOM
1800
CB
GLU
B
109
−7.510
−9.163
11.425
1.00
29.53


ATOM
1801
CG
GLU
B
109
−7.538
−10.384
12.392
1.00
31.83


ATOM
1802
CD
GLU
B
109
−6.793
−10.146
13.734
1.00
34.71


ATOM
1803
OE1
GLU
B
109
−6.585
−8.973
14.138
1.00
34.75


ATOM
1804
OE2
GLU
B
109
−6.423
−11.148
14.392
1.00
36.82


ATOM
1805
C
GLU
B
109
−6.295
−7.707
9.753
1.00
28.56


ATOM
1806
O
GLU
B
109
−6.361
−7.773
8.523
1.00
28.30


ATOM
1886
N
VAL
B
120
0.008
−4.308
−2.141
1.00
21.20


ATOM
1887
CA
VAL
B
120
1.355
−4.805
−1.885
1.00
20.97


ATOM
1888
CB
VAL
B
120
2.209
−3.786
−1.090
1.00
21.14


ATOM
1889
CG1
VAL
B
120
2.557
−2.574
−1.957
1.00
21.30


ATOM
1890
CG2
VAL
B
120
1.518
−3.371
0.210
1.00
20.07


ATOM
1891
C
VAL
B
120
1.321
−6.131
−1.124
1.00
21.03


ATOM
1892
O
VAL
B
120
0.463
−6.332
−0.273
1.00
20.30


ATOM
1893
N
ASP
B
121
2.243
−7.032
−1.462
1.00
21.30


ATOM
1894
CA
ASP
B
121
2.501
−8.218
−0.657
1.00
21.87


ATOM
1895
CB
ASP
B
121
3.604
−9.058
−1.304
1.00
21.84


ATOM
1896
CG
ASP
B
121
3.835
−10.394
−0.597
1.00
22.12


ATOM
1897
OD1
ASP
B
121
4.155
−10.394
0.603
1.00
25.73


ATOM
1898
OD2
ASP
B
121
3.727
−11.446
−1.239
1.00
19.92


ATOM
1899
C
ASP
B
121
2.939
−7.731
0.719
1.00
22.56


ATOM
1900
O
ASP
B
121
3.844
−6.897
0.811
1.00
22.97









As will be apparent to those of ordinary skill in the art, the atomic structures presented herein are independent of their orientation, and the atomic co-ordinates identified herein merely represent one possible orientation of a particular E2 or E3 polypeptide. The atomic coordinates are a relative set of points that define a shape in three dimensions. Thus, it is possible that a different set of coordinates could define a similar or identical shape. Therefore, slight variations in the individual coordinates will have little effect on overall shape. It is apparent, therefore, that the atomic co-ordinates identified herein may be mathematically rotated, translated, scaled, or a combination thereof, without changing the relative positions of atoms or features of the respective structure. The variations in coordinates discussed may be generated because of mathematical manipulations of the structure coordinates. For example, the structure coordinates could be manipulated by crystallographic permutations of the structure coordinates, fractionalization of the structure coordinates, integer additions or subtractions to sets of the structure coordinates, inversion of the structure coordinates or any combination of the above.
As used herein, a “structural variant” comprises a three-dimensional molecular structure that is similar to another three-dimensional molecular structure. In some embodiments, the structural variant comprises a root mean square deviation (RMSD) from the back-bone atoms of the amino acids of listed in a particular table (e.g., Table 2, 3, 5, 6, 8, 9, 10, 11, 13, 14, 15, 16, 18, 19, 21, 22) of riot more than about 4 Å, including but not limited to about 3.5 Å, 3 Å, 2.5 Å, 2 Å, 1.9 Å, 1.8 Å, 1.7 Å, 1.6 Å, 1.5 Å, 1.4 Å, 1.3 Å, 1.2 Å, 1.1 Å, 1.0 Å, 0.9 Å, 0.8 Å, 0.7 Å, 0.6 Å, 0.5 Å, 0.4 Å, 0.3 Å, 0.2 Å, and 0.1 Å. In some of these embodiments, the structural variant comprises a root mean square deviation from the back-bone atoms of the amino acids listed in a particular table (e.g., Table 2, 3, 5, 6, 8, 9, 10, 11, 13, 14, 15, 16, 18, 19, 21, 22) of not more than about 2.0 Å. Modifications in the crystal structure due to mutations, additions, substitutions and/or deletions of amino acids, or other changes in any of the components that make up the crystal could also account for variations in the structure coordinates.
The novel E2-binding pocket of NEDD8 co-E3 proteins can be exploited to discover compounds that bind to the pocket and in some embodiments, inhibit the activity of the NEDD8 co-E3 protein. Such compounds can be identified using computational techniques or various in vitro or in vivo binding assays.
Computational techniques can be used to screen, identify, select and/or design compounds (i.e., small molecules, peptides) capable of associating with the E2-binding pocket of co-E3 proteins. Knowledge of the structure coordinates for the E2-binding pocket of co-E3 proteins permits the design and/or identification of synthetic compounds and/or other molecules which have a shape complementary to the conformation of the E2-binding pocket of co-E3 proteins. In particular, computational techniques can be used to identify or design compounds, such as inhibitors (also referred to herein as antagonists), that associate with a co-E3 protein E2-binding pocket. Potential inhibitors may bind to or interfere with all or a portion of the E2-binding pocket, and can be competitive, non-competitive, or uncompetitive inhibitors. Once identified and screened for biological activity, these inhibitors may be used therapeutically or prophylactically to inhibit co-E3 activity and, thus, prevent the onset and/or further progression of diseases or conditions associated with, for example, cell hyperproliferation (e.g., cancer, inflammatory disorders). Structure-activity data for inhibitors that bind to or interfere with the E2-binding pocket of co-E3 proteins can also be obtained computationally.
The design of compounds with particular structural relationships to part of a protein molecule are well established and described in the literature (see for example Cochran (2000) Chem. Biol. 7:85-94; Grzybowski et al. (2002) Acc. Chem. Res 35:261-269; Vejasquez-Campoy et al. (2001) Arch. Biochem. Biophys. 380:169-175; D'Aquino et al. (2000) Proteins: Struc. Func. Genet. Suppl. 4:93-107). Any of these “molecular modeling” methods for rational drug design can be used to find antagonists of the E2-binding pocket of co-E3 proteins. Most of these molecular modeling methods take into consideration the shape, charge distribution and the distribution of hydrophobic groups, ionic groups and hydrogen bonds in the binding site of the protein molecule. Using this information derived from the crystal structure of proteins and protein complexes, these methods suggest improvements to existing proposed compounds, construct new compounds on their own that are expected to have good binding affinity, screen through virtual compound libraries for such molecules, or otherwise support the interactive design of new drug compounds in silico. Programs such as GOLD (Jones et al. (1997) J. Mol. Biol. 267:727-748); FLEXX (Kramer et al. (1999) Structure, Functions, and Genetics 37:228-241); FLEXE (Rarey et al. (1996) JMB 261:470-489) DOCK (Kuntz (1992) Science 257:1078-1082); and AUTODOCK (Morris et al. (1998) J. Computational Chemistry 19:1639-1662) are virtual screening programs designed to calculate the binding position and conformation as well as the corresponding binding energy of an organic compound to a protein. These programs are specially trimmed to allow a great number of “dockings”, that is, calculations of the conformation with the highest binding energy of a compound to a binding site, per time unit. The binding energy is not always a real value, but can be statistically related to a real binding energy through a validation procedure.
In one embodiment, the method for identifying compounds that bind to the presently disclosed E2-binding pocket of NEDD8 co-E3 proteins comprises comparing the three-dimensional structure of a test compound and the three-dimensional molecular structure of the E2-binding pocket to determine if at least one atom of the test compound will interact with at least one atoms of the E2-binding pocket. As used herein, “interact” refers to both covalent and non-covalent interactions, including but not limited to hydrogen bonds, van der Waals interactions, hydrophobic interactions, and electrostatic interactions.
The term “test compound” refers to the molecule that is being assayed using the presently disclosed methods to determine if the test compound is capable of binding the E2-binding pocket of a NEDD8 co-E3 protein. The test compound can be naturally-occurring or non-naturally occurring and can be purified from a biological sample or synthetically derived. The structure of the test compound may be known or unknown. The test compound need not have a known biological activity or a known ability to interact with proteins. The compound may be solubilized in a solution, such as an aqueous solution or an organic solvent (e.g., dimethyl sulfoxide) prior to addition of the molecule to a sample comprising a NEDD8 co-E3 protein.
In some embodiments, the test compound is a member of a chemical library. A chemical library refers to a plurality of molecules. The components of the chemical library can be well-defined, containing known mixtures of molecules. For example, each molecule of a well-defined chemical library can be catalogued. Alternatively, the components of the library can be poorly defined, as is often the case with combinatorial libraries. Likewise, the structures of the molecules within the chemical library can be known or unknown.
In particular embodiments, the test compound is a member of a combinatorial chemical library. A combinatorial chemical library is a plurality of molecules or compounds which are formed by selectively combining a particular set of chemical building blocks. Combinatorial libraries can be constructed according to methods familiar to those skilled in the art. For example, see Rapoport et al., (1995) Immunology Today 16:43-49; Sepetov, N. F. et al., (1995) Proc. Natl. Acad. Sci. U.S.A. 92:5426-5430; Gallop, M. A. et al., (1994) J. Med. Chem. 9:1233-1251; Gordon, E. M. et al., (1994) J. Med. Chem. 37:1385-1401; Stankova, M. et al., (1994) Peptide Res. 7:292-298; Erb, E. et al., (1994) Proc. Natl. Acad. Sci. U.S.A. 91:11422-11426; DeWitt, S. H. et al., (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909-6913; Barbas, C. F. et al., (1992) Proc. Natl. Acad. Sci. U.S.A. 89:4457-4461; Brenner, S. et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89:5381-5383; Lam, K. S. et al., (1991) Nature 354:82-84; Devlin, J. J. et al., (1990) Science 245:404-406; Cwirla, S. E. et al., (1990) Proc. Natl. Acad. Sci, U.S.A. 87:6378-6382; Scott, J. K. et al., (1990) Science 249:386-390, and U.S. Pat. No. 5,463,564, each of which is herein incorporated by reference in its entirety.
To be a viable drug candidate, the compound identified or designed according to the method must be capable of structurally associating with at least part of the E2-binding pocket on co-E3 proteins, and must be able, sterically and energetically, to assume a conformation that allows it to associate with the E2-binding pocket of co-E3 proteins. Non-covalent molecular interactions important in this association include hydrogen bonding, van der Waals interactions, hydrophobic interactions, and electrostatic interactions. Conformational considerations include the overall three-dimensional structure and orientation of the compound in relation to the binding pocket, and the spacing between various functional groups of a compound that directly interact with the E2-binding pocket.
One embodiment of the method involves evaluating the potential association of a known compound with the E2-binding pocket of co-E3 proteins. The method of drug design thus includes computationally evaluating the potential of a selected compound to associate with any of the molecules or molecular complexes set forth above. This method includes the steps of (a) employing computational means to perform a fitting operation between the selected chemical entity and the E2-binding pocket; and (b) analyzing the results of said fitting operation to quantify the association between the compound and the binding pocket.
Specialized computer programs may also assist in the process of selecting compounds. Examples include GRID (Goodford (1985) J. Med. Chem. 28:849-57); available from Oxford University, Oxford, UK); MCSS (Miranker et al. (1991) Proteins: Struct. Funct. Gen. 11:29-34); available from Molecular Simulations, San Diego, Calif.); AUTODOCK (Goodsell et al. (1990) Proteins: Struct. Funct. Genet. 8:195-202; available from Scripps Research Institute, La Jolla, Calif.); and DOCK (Kuntz et al. (1982) J. Mol. Biol. 161:269-88); available from University of California, San Francisco, Calif.).
If these computational experiments suggest insufficient interaction and association between the test compound and the E2-binding pocket of co-E3 proteins, testing of the compound is obviated. However, if computer modeling indicates a strong interaction, the molecule may then be synthesized and tested for its ability to bind to or interfere with the E2-binding pocket of co-E3 proteins as disclosed elsewhere herein. In some cases, it may be advantageous to develop new inhibitors de novo, i.e. not on the basis and as a modification of a pre-existing compound. The term “de novo compound design” refers to a process whereby the binding pocket of the target macromolecule (e.g., the E2-binding pocket of co-E3 proteins) is determined; and its surfaces is used as a platform or basis for the rational design of compounds that will interact with those surfaces. The molecular modeling steps used in the methods may use the atomic coordinates of the E2-binding pocket disclosed herein and models or structures derived therefrom, to determine binding surfaces. Any such structure will preferably reveal van der Waals contacts, electrostatic interactions, and/or hydrogen bonding opportunities. Said binding surfaces will typically be used by grid-based techniques (e.g. GRID, CERIUS2, [Goodford (1985) J. Med. Chem. 28: 849-857]) and/or multiple copy simultaneous search (MCSS) techniques to map favorable interaction positions for functional groups. This preferably reveals positions in the E2-binding pocket of the co-E3 protein for interactions such as, but not limited to, those with protons, hydroxyl groups, amine groups, hydrophobic groups (e.g. methyl, ethyl, benzyl) and/or divalent cations.
Computer-based approaches to de novo compound design that can be used with the atomic co-ordinates of the E2-binding pocket of co-E3 proteins include LUDI(15 Bohm (1992) J. Comp. X ed Molec. Design 6: 61-78), SPROUT (Available from chem.leeds.ac. uk/ICAMS/SPROUT.html) and LEAPFROG (available from Tripos Inc (www.tripos.com)). Suitable in silico libraries include the Available Chemical Directory (MDL Inc), the Derwent World Drug Index (WDI), BioByteMasterFile, the National Cancer Institute database (NCl), and the Maybridge catalog. Compounds in these in silico libraries can also be screened for their ability to interact with the E2-binding pocket of co-E3 proteins by using their respective atomic coordinates in automated docking algorithms. An automated docking algorithm is one which permits the prediction of interactions of a number of compounds with a molecule having a given atomic structure. Suitable docking algorithms include: DOCK (Kuntz et al. (1982) J. Mol. Biol. 161: 269-288)), AUTODOCK (Goodsell et al. (1990) Proteins: Structure, Function and Genetics 8: 195-202), MOE-DOCK (Available from Chemical Computing Group Inc. (www.chemcomp.com/)) or FLEXX (Available from Tripos Inc (www.tripos.com)). Docking algorithms can also be used to verify interactions with ligands designed de novo.
Another approach encompassed by methods provided herein is the computational screening of small molecule databases for compounds that can bind in whole, or in part, to the E2-binding pocket of co-E3 proteins. In this screening, the quality of fit of such entities to the binding site may be judged either by shape complementarity or by estimated interaction energy (Meng et al. (1992) J. Comp. Chem. 13:505-24).
A compound that is identified or designed as a result of any of these methods can be obtained (or synthesized) and tested for its biological activity, e.g., inhibition of co-E3 activity. Binding assays to determine if a compound (e.g., an inhibitor) actually binds to or interferes with the E2-binding pocket on co-E3 enzymes can also be performed and are well known in the art and are described herein.
Further methods for identifying compounds that bind to an E2-binding pocket in a NEDD8 co-E3 protein disclosed herein comprise contacting a NEDD8 co-E3 protein with a test compound and determining if the test compound binds to the E2-binding pocket. The test compound may be contacted with the NEDD8 co-E3 protein in a cell-free system, in cells, or in vivo.
Binding assays to determine if the test compound binds the NEDD8 co-E3 protein may employ kinetic or thermodynamic methodology using a wide variety of techniques including, but not limited to, microcalorimetry, circular dichroism, capillary zone electrophoresis, nuclear magnetic resonance spectroscopy, fluorescence spectroscopy, and combinations thereof. One can determine if a test compound binds to the E2-binding pocket of a co-E3 enzyme using, for example, assays such as mutational analyses wherein residues that comprise the E2-binding pocket are mutated and the effect on test compound binding is assessed; and competitive binding assays, wherein a compound known to bind to the E2-binding pocket (for example, one of the peptides disclosed herein) is added to a binding reaction with the test compound to determine if the test compound is able to compete with the peptide for binding to the co-E3 protein. In some of these embodiments, the compound known to bind to the E2-binding pocket can be coupled with a detectable label, such as a radioisotope, fluorescent label, or enzymatic label, such that binding of the test compound to the E2-binding pocket can be determined by detecting a reduction in the labeled compound in a complex. For example, compounds known to bind to the E2-binding pocket (e.g., one of the peptides disclosed herein) can be labeled with 125I, 35S, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radio emmission or by scintillation counting. Alternatively, compounds known to bind to the E2-binding pocket can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
As another example, the compound known to bind to an E2-binding pocket can be coupled with a fluorescent label to allow for detection of binding using, for example, fluorescence polarization. In some embodiments, the NEDD8 co-E3 protein is also fluorescently labeled. The fluororescent molecules (i.e., fluorophores) on the compound and the NEDD8 co-E3 protein can have an overlapping excitation and emission spectra such as those generally used in fluorescence resonance energy transfer (FRET) technology assays, wherein the excitation of a first fluorescent molecule (donor fluorophore) at a first wavelength of light causes the first fluororescent molecule to emit light at a second wavelength, and wherein the second fluorescent molecule (acceptor fluorophore) is excited by the second wavelength of light if the two fluorescent molecules are in close enough proximity to one another, and subsequently, the second fluorescent molecule emits light at a third wavelength, which can be detected using any method or apparatus known in the art. Non-limiting examples of fluorophores that can be conjugated to antibodies include Cy3, Cy5, Cy5.5, Cy7, Alexa488, Alexa555, FITC, and rhodamine (TRITC). It is to be noted that the selection of the donor fluorophore depends on the excitation and emission spectra of the acceptor fluorophore and vice versa. Frequently used fluorophore pairs for FRET include but are not limited to, Cy3 and Cy5, Alexa488 and Alexa555, Alexa488 and Cy3, and FITC and rhodamine. In such an assay, the compound known to bind to an E2-binding pocket can be coupled to a donor fluorophore (in an alternative embodiment, to an acceptor fluorophore) and the NEDD8 co-E3 protein (e.g., at or near the E2-binding pocket) can be coupled to an acceptor fluorophore (in the alternative embodiment, to a donor fluorophore). If the two come in close contact with another, the acceptor fluorophore will emit light. One can determine if the test compound that is added to the assay binds to the E2-binding pocket of the NEDD8 co-E3 protein if the emission of the acceptor fluorophore is diminished due to competition with the tabled compound known to bind to the pocket.
In some assays, it may be desirable to immobilize either the NEDD8 co-E3 protein or a portion thereof or the compound known to bind to the E2-binding pocket to facilitate automation of the assay. For example, the NEDD8 co-E3 protein could be produced as a fusion protein that adds a domain that allows the NEDD8 co-E3 protein to be bound to a matrix. A non-limiting example of such a domain is glutathione-S-transferase, which can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione-derivatized microtitre plates. In other embodiments, the co-E3 protein or compounds can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated co-E3 proteins or compounds can be prepared from biotin-NHS(N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of a plate.
This invention further pertains to novel compounds identified by the above-described screening assays and uses thereof as described herein.
Further, the three-dimensional molecular structure of the test compound bound to the co-E3 protein can be determined using methods known in the art to determine if the test compound is binding within the presently disclosed E2-binding pocket of the NEDD8 co-E3 protein.
Methods known in the art and disclosed elsewhere herein can be used to determine if the test compound inhibits the co-E3 activity of NEDD8 co-E3 proteins (see Examples 1 and 4). Such binding assays can also be used to screen compounds or compound libraries for those that bind to the E2-binding pocket. Compounds that are found to bind can be further characterized for inhibition of co-E3 activity.
Compounds (e.g., small molecules and peptides) that bind to the E2-binding pocket of co-E3 proteins are provided herein. The term “compound” refers to any chemical molecule, including organic and inorganic molecules. In some embodiments, the compound is a small molecule. As used herein, the term “small molecule” refers to a chemical molecule that in some embodiments is a small organic compound, having a molecular weight of more than 100 and less than about 2,500 Daltons, including but not limited to less than 2000, or less than 1500 or less than 1000 or less than 500 D. The term “small molecule” does not encompass a peptide.
As used herein, the terms “peptide,” “polypeptide,” or “protein” are used interchangeably herein and are intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The terms “peptide” and “polypeptide” refer to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of “peptide” and “polypeptide”. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. Non-limiting examples of artificial amino acid residues include norleucine and selenomethionine. An amino acid residue is a molecule having a carboxyl group, an amino group, and a side chain and having the generic formula H2NCHRCOOH, where R is an organic substituent, forming the side chain. An amino acid residue, whether it is artificial or naturally occurring, is capable of forming a peptide bond with a naturally occurring amino acid residue.
An “isolated” or “purified” peptide is substantially or essentially free from components that normally accompany or interact with the peptide as found in its naturally occurring environment. Thus, an isolated or purified peptide is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. A peptide that is substantially free of cellular material includes preparations of peptide having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein. When the peptide is recombinantly produced, optimally culture medium represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-peptide-of-interest chemicals.
In some embodiments of the presently disclosed invention, peptides that bind to the E2-binding pocket of a NEDD8 co-E3 protein comprise the first 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or greater amino acid residues of a naturally-occurring NEDD8 E2 protein or a variant thereof. In particular embodiments, the peptide has the sequence set forth in SEQ ID NO: 7, 12, 13, 14, 15, or 16 or a fragment or variant thereof capable of binding to the E2-binding pocket, including but not limited to an amino acid sequence having at least 40%, at least about 50%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 7, 12, 13, 14, 15, or 16. In certain embodiments, the variant peptide may differ from SEQ ID NO: 7, 12, 13, 14, 15, or 16 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid residues or greater.
It was disclosed herein that N-terminal acetylation of yeast Ubc12 enhances its interaction with Dcn1 and that N-terminal acetylation of human Ubc12 is essential for its interaction with Dcn1. Acetylation of the nitrogen atom of the N-terminal methioinine of Ubc12 contributed to the binding of Ubc12 to Dcn1 by eliminating the N-terminal positive charge that otherwise would impede burial in the Dcn1 hydrophobic pocket, and the methyl group of the acetyl provides hydrophobic interactions with the hydrophobic pocket. Therefore, in some embodiments, the peptides of the invention that bind to the E2-binding pocket of a NEDD8 co-E3 protein are N-terminally acetylated; that is, the nitrogen atom in the amino-terminal amino acid residue is covalently bound to an acetyl group. An acetyl group has the formula




Amino acid residues referred to herein with N-acetyl-preceding the name of the amino acid residue comprise an acetyl group covalently bound to the backbone nitrogen atom. In other embodiments, the nitrogen atom of the amino-terminal amino acid residue of the peptide is covalently bound to at least one —R group so that the nitrogen is not ionizable, wherein the —R group is any functional group that is not a hydrogen and is uncharged or non-ionizable under physiological conditions when bound to the backbone nitrogen atom. Non-limiting examples of appropriate —R groups include an alkyl (including, but not limited to, a C2 to a C20 chain), alkenyl, alkynyl, aryl, or heteroaryl, optionally substituted.

Alkyl refers to both straight, branched carbon chains and cyclic hydrocarbon groups; references to individual alkyl groups are specific for the straight chain (e.g. butyl=n-butyl). In one embodiment of alkyl, the number of carbons atoms is 1-20, in other embodiments of alkyl, the number of carbon atoms is 1-12, 1-10 or 1-8 carbon atoms. In yet another embodiment of alkyl, the number of carbon atoms is 1-4 carbon atoms. Other ranges of carbon numbers are also contemplated depending on the location of the alkyl moiety on the molecule;
Examples of C1-C10 alkyl include, but are not limited to, methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, 1-ethylpropyl, hexyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,1,2-trim ethylpropyl, 1,2,2-trimethylpropyl, 1-ethyl-1-methylpropyl, 1-ethyl-2-methylpropyl, heptyl, octyl, 2-ethylhexyl, nonyl and decyl and their isomers. C1-C4-alkyl means for example methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl, 2-methylpropyl or 1,1-dimethylethyl.
Cyclic alkyl groups, which are encompassed by alkyls, may be referred to as “cycloalkyl” and include those with 3 to 10 carbon atoms having single or multiple fused rings. Non-limiting examples of cycloalkyl groups include adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like.
The alkyl and cycloalkyl groups described herein can be unsubstituted or substituted with one or more moieties selected from the group consisting of alkyl, halo, haloalkyl, hydroxyl, carboxyl, acyl, acyloxy, amino, alkyl- or dialkylamino, amido, arylamino, alkoxy, aryloxy, nitro, cyano, azido, thiol, imino, sulfonic acid, sulfate, sulfonyl, sulfanyl, sulfinyl, sulfamonyl, ester, phosphonyl, phosphinyl, phosphoryl, phosphine, thioester, thioether, acid halide, anhydride, oxime, hydrozine, carbamate, phosphonic acid, phosphate, phosphonate, or any other viable functional group that does not inhibit the biological activity of the compounds of the invention, either unprotected, or protected as necessary, as known to those skilled in the art, for example, as taught in Greene, et al., Protective Groups in Organic Synthesis, John Wiley and Sons, Third Edition, 1999, hereby incorporated by reference.
Alkenyl refers to both straight and branched carbon chains which have at least one carbon-carbon double bond. In one embodiment of alkenyl, the number of double bonds is 1-3, in another embodiment of alkenyl, the number of double bonds is one. In one embodiment of alkenyl, the number of carbons atoms is 2-20, in other embodiments of alkenyl, the number of carbon atoms is 2-12, 2-10 or 2-8. In yet another embodiment of alkenyl, the number of carbon atoms is 2-4. Other ranges of carbon-carbon double bonds and carbon numbers are also contemplated depending on the location of the alkenyl moiety on the molecule;
“C2-C10-alkenyl” groups may include more than one double bond in the chain. Examples include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 1-methyl-ethenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-methyl-1-propenyl, 2-methyl-1-propenyl, 1-methyl-2-propenyl, 2-methyl-2-propenyl; 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-methyl-1-butenyl, 2-methyl-1-butenyl, 3-methyl-1-butenyl, 1-methyl-2-butenyl, 2-methyl-2-butenyl, 3-methyl-2-butenyl, 1-methyl-3-butenyl, 2-methyl-3-butenyl, 3-methyl-3-butenyl, 1,1-dimethyl-2-propenyl, 1,2-dimethyl-1-propenyl, 1,2-dimethyl-2-propenyl, 1-ethyl-1-propenyl, 1-ethyl-2-propenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-methyl-1-pentenyl, 2-methyl-1-pentenyl, 3-methyl-1-pentenyl, 4-methyl-1-pentenyl, 1-methyl-2-pentenyl, 2-methyl-2-pentenyl, 3-methyl-2-pentenyl, 4-methyl-2-pentenyl, 1-methyl-3-pentenyl, 2-methyl-3-pentenyl, 3-methyl-3-pentenyl, 4-methyl-3-pentenyl, 1-methyl-4-pentenyl, 2-methyl-4-pentenyl, 3-methyl-4-pentenyl, 4-methyl-4-pentenyl, 1,1-dimethyl-2-butenyl, 1,1-dimethyl-3-butenyl, 1,2-dimethyl-1-butenyl, 1,2-dimethyl-2-butenyl, 1,2-dimethyl-3-butenyl, 1,3-dimethyl-1-butenyl, 1,3-dimethyl-2-butenyl, 1,3-dimethyl-3-butenyl, 2,2-dimethyl-3-butenyl, 2,3-dimethyl-1-butenyl, 2,3-dimethyl-2-butenyl, 2,3-dimethyl-3-butenyl, 3,3-dimethyl-1-butenyl, 3,3-dimethyl-2-butenyl, 1-ethyl-1-butenyl, 1-ethyl-2-butenyl, 1-ethyl-3-butenyl, 2-ethyl-1-butenyl, 2-ethyl-2-butenyl, 2-ethyl-3-butenyl, 1,1,2-trimethyl-2-propenyl, 1-ethyl-1-methyl-2-propenyl, 1-ethyl-2-methyl-1-propenyl and 1-ethyl-2-methyl-2-propenyl.
Alkynyl refers to both straight and branched carbon chains which have at least one carbon-carbon triple bond. In one embodiment of alkynyl, the number of triple bonds is 1-3; in another embodiment of alkynyl, the number of triple bonds is one. In one embodiment of alkynyl, the number of carbons atoms is 2-20, in other embodiments of alkynyl, the number of carbon atoms is 2-12, 2-10 or 2-8. In yet another embodiment of alkynyl, the number of carbon atoms is 2-4. Other ranges of carbon-carbon double bonds and carbon numbers are also contemplated depending on the location of the alkenyl moiety on the molecule;
For example, the term “C2-C10-alkynyl” as used herein refers to a straight-chain or branched unsaturated hydrocarbon group having 2 to 10 carbon atoms and containing at least one triple bond, such as ethynyl, prop-1-yn-1-yl, prop-2-yn-1-yl, n-but-1-yn-1-yl, n-but-1-yn-3-yl, n-but-1-yn-4-yl, n-but-2-yn-1-yl, n-pent-1-yn-1-yl, n-pent-1-yn-3-yl, n-pent-1-yn-4-yl, n-pent-1-yn-5-yl, n-pent-2-yn-1-yl, n-pent-2-yn-4-yl, n-pent-2-yn-5-yl, 3-methylbut-1-yn-3-yl, 3-methylbut-1-yn-4-yl, n-hex-1-yn-1-yl, n-hex-1-yn-3-yl, n-hex-1-yn-4-yl, n-hex-1-yn-5-yl, n-hex-1-yn-6-yl, n-hex-2-yn-1-yl, n-hex-2-yn-4-yl, n-hex-2-yn-5-yl, n-hex-2-yn-6-yl, n-hex-3-yn-1-yl, n-hex-3-yn-2-yl, 3-methylpent-1-yn-1-yl, 3-methylpent-1-yn-3-yl, 3-methylpent-1-yn-4-yl, 3-methylpent-1-yn-5-yl, 4-methylpent-1-yn-1-yl, 4-methylpent-2-yn-4-yl or 4-methylpent-2-yn-5-yl and the like.
Aryl refers to a C6-C14 aromatic carbocyclic ring structure having a single ring or multiple fused rings. Aryl groups include, but are not limited to, phenyl, biphenyl, and naphthyl. In some embodiments aryl includes tetrahydronapthyl, phenylcyclopropyl and indanyl. Aryl groups may be unsubstituted or substituted by one or more moieties selected from halogen, cyano, nitro, hydroxy, mercapto, amino, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, haloalkyl, haloalkenyl, haloalkynyl, halocycloalkyl, halocycloalkenyl, alkoxy, alkenyloxy, alkynyloxy, haloalkoxy, haloalkenyloxy, haloalkynyloxy, cycloalkoxy, cycloalkenyloxy, halocycloalkoxy, halocycloalkenyloxy, alkylthio, haloalkylthio, arylthio, cycloalkylthio, halocycloalkylthio, alkylsulfinyl, alkenylsulfinyl, alkynyl-sulfinyl, haloalkylsulfinyl, haloalkenylsulfinyl, haloalkynylsulfinyl, alkylsulfonyl, alkenylsulfonyl, alkynylsulfonyl, haloalkyl-sulfonyl, haloalkenylsulfonyl, haloalkynylsulfonyl, alkylcarbonyl, haloalkylcarbonyl, alkylamino, alkenylamino, alkynylamino, di(alkyl)amino, di(alkenyl)-amino, di(alkynyl)amino, or SF5. In one embodiment of aryl, the moiety is phenyl, naphthyl, tetrahydronapthyl, phenylcyclopropyl and indanyl; in another embodiment of aryl, the moiety is phenyl. Arylo refers to an aryl substituted at two adjacent sites.
Heteroaryl refers to a monovalent aromatic group of from 1 to 15 carbon atoms, preferably from 1 to 10 carbon atoms, having one or more oxygen, nitrogen, and sulfur heteroatoms within the ring, preferably 1 to 4 heteroatoms, or 1 to 3 heteroatoms. The nitrogen and sulfur heteroatoms may optionally be oxidized. Such heteroaryl groups can have a single ring (e.g., pyridyl or furyl) or multiple fused rings provided that the point of attachment is through a heteroaryl ring atom. Preferred heteroaryls include pyridyl, piridazinyl, pyrimidinyl, triazinyl, pyrrolyl, quinolinyl, isoquinolinyl, quinazolinyl, quinoxalinnyl, furanyl, thienyl, furyl, imidazolyl, oxazolyl, isoxazolyl, isothiazolyl, pyrazolyl, benzofuranyl, and benzothienyl. Heteroaryl rings may be unsubstituted or substituted by one or more moieties as described for aryl above.
Exemplary monocyclic heterocyclic or heteroaryl groups also include, but are not limited to, pyrrolidinyl, oxetanyl, pyrazolinyl, imidazolinyl, imidazolidinyl, oxazolidinyl, isoxazolinyl, thiazolyl, thiadiazolyl, thiazolidinyl, isothiazolidinyl, tetrahydrofuryl, thienyl, oxadiazolyl, piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl, 2-oxoazepinyl, azepinyl, 4-piperidonyl, pyridinyl, pyrazinyl, pyridazinyl, tetrahydropyranyl, morpholinyl, thiamorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone, 1,3-dioxolane and tetrahydro-1,1-dioxothienyl, triazolyl, and the like.
In all embodiments, it is preferred that the R group is uncharged under physiological conditions.
Preferred hydrophobic moieties include substituted and unsubstituted alkyl, alkenyl, alkynyl, aryl and heteroaryl groups. More preferably, the hydrophobic moiety is a C4-C20 alkyl, alkenyl or alkynyl group. Most preferably, the hydrophobic moiety is a C6-C12 alkyl, alkenyl or alkynyl group.
In those embodiments wherein the nitrogen is bound to more than one —R group (e.g., 2 —R groups), the —R groups may be the same or distinct from one another. In particular embodiments, the —R group is not an acetyl group. In other embodiments, the —R group has Formula (I), wherein Formula (I) is:




wherein the —R2 group is any functional group that is uncharged under physiological conditions and is not a hydrogen. Non-limiting examples of appropriate —R2 groups include an alkyl (including, but not limited to, a C2 to a C20 chain), aryl, or heteroaryl. In certain embodiments, the —R group is a formyl group, wherein a formyl group has the formula





In particular embodiments, the amino-terminal methionine (at position 1) of the peptide that binds to the E2-binding pocket of a NEDD8 co-E3 protein (e.g., SEQ ID NO: 7, 12, 13, 14, 15, or 16) is substituted with an artificial amino acid residue having an unbranched hydrophobic side chain. As used herein, the term “hydrophobic” is a physical property of a molecule that is repelled from a mass of water and can be referred to as “nonpolar,” or “apolar,” all of which are terms that can be used interchangeably with “hydrophobic.” Non-limiting examples of an unbranched hydrophobic side chain include an alkyl (e.g., C2 to C20), an alkenyl, or alkynyl. In particular embodiments, peptides of the invention that bind to the E2-binding pocket of a NEDD8 co-E3 protein comprise a norleucine or selenomethionine at the first position of the peptide in place of a methioinine.
Analysis of the presently disclosed crystal structures revealed that in order to place the hydrophobic side chains of amino acid residues at positions 2 and 4 within the Ubc12 N-terminal extension on opposite sides of the amino-terminal acetylated methioinine to aid in binding, a helical structure is required. Thus, in certain embodiments, the peptide that binds to the E2-binding pocket of a NEDD8 co-E3 protein has an alpha-helical structure. As used herein, an “alpha helix” refers to the most abundant helical conformation found in globular proteins and the term is used in accordance with the standard meaning of the art. In an alpha helix, all amide protons point toward the N-terminus and all carbonyl oxygens point toward the C-terminus. Hydrogen bonds within an alpha helix also display a repeating pattern in which the backbone C═O of residue X (wherein X refers to any amino acid) hydrogen bonds to the backbone H—N of residue X+4. The alpha helix is a coiled structure characterized by 3.6 residues per turn, and translating along its axis 1.5 Å per amino acid. Thus the pitch is 3.6×1.5 or 5.4 Å. The screw sense of alpha helices is always right-handed.
In some of those embodiments wherein the peptide has an alpha helical structure, the peptide comprises a hydrophobic staple (such as those described in Bird et al. (2008) Methods Enzymol 446:369-386; Houston et al. (1995) J Pept Sci 1(4)274-282; Taylor (2002) Biopolymers 66(1):49-75; and Houston et al. (1996) Biochemistry 35(31):10041-50, each of which is herein incorporated by reference in its entirety), which is herein incorporated by reference in its entirety. As used herein, a “hydrophobic staple” refers to the presence of two amino acid residues having hydrophobic side chains that are covalently bound to one another. A hydrophobic staple is generated when the peptide comprises artificial amino acid residues comprising hydrophobic staple-forming amino acid residues at positions X and X+4 within the peptide, wherein X is any amino acid residue within any position of the peptide. One of skill in the art will appreciate that in order for a hydrophobic staple-forming amino acid residue to be present at positions X and X+4, X can not be any of the last four residues (i.e., the four most carboxy residues) within a peptide chain. In particular embodiments, the hydrophobic staple-forming amino acid residues are present at positions 4 and 8, 5 and 9, 6 and 10, or 7 and 11 of the peptide. Additional hydrophobic staple-forming amino acid residues can be present within the peptide as long as they are at least four amino acid residues away from another hydrophobic staple-forming amino acid residue.
As used herein a “hydrophobic staple-forming amino acid residue” is an amino acid residue as defined herein that comprises a side chain capable of forming a covalent bond with the side chain of another amino acid residue to form a hydrophobic staple. Non-limiting examples of hydrophobic staple-forming amino acid residues include (S)-2-(4-pentenyl)alanine, (R)-2(4-pentenyl)alanine, (S)-2-(3-butenyl)alanine, (S)-2-(7-octenyl)alanine, lysine, and glutamic acid.
Further, in particular embodiments, the carboxy terminus of the peptide that binds to the E2-binding pocket of a NEDD8 co-E3 protein is amidated, wherein the carbon atom of the backbone carboxyl group in the most carboxyl amino acid residue is covalently bound to an optionally substituted nitrogen. In other embodiments, the carbon atom of the carboxyl group in the most carboxyl amino acid residue is bound to at least one —R group, as defined elsewhere herein.
In particular embodiments, the peptide that binds to the E2-binding pocket has the sequence set forth in SEQ ID NO: 10, 11, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 87, 88, 89, 90, 91, 92, 93, or 94, or a fragment or variant thereof.
In some embodiments, the small molecule or peptide that binds to the E2-binding pocket of a NEDD8 co-E3 protein inhibits the co-E3 activity of the co-E3 protein. Therefore, the small molecule or peptide is capable of inhibiting the neddylation of at least one NEDD8 target proteins, including but not limited to, a cullin protein. Such a molecule is referred to herein as an antagonist or an inhibitor. In some embodiments, the co-E3 inhibitor inhibits the co-E3 activity of the co-E3 protein by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater. The ability of a small molecule or peptide to inhibit the activity of a co-E3 protein can be measured in vitro or in vivo or in cells in culture using any method known in the art, including those assays disclosed elsewhere herein (see, for example, Example 4). The compound may be assessed for its ability to inhibit co-E3 activity by measuring co-E3 activity directly or indirectly by assaying downstream effects of NEDD8 co-E3 proteins (e.g., inhibition of cullin-dependent ubiquitination and proteolysis). For example, activity may be assessed by detection of downstream protein substrate stabilization (e.g., stabilization of p27, of IκB, NFκB or p27 reporter assays).
In addition to NEDD8 co-E3 proteins, the amino terminal extension of NEDD8 E2 enzymes (e.g., Ubc12) also binds to NEDD8 E1 proteins (Huang et al. (2004) Nat Struct Mol Biol 11:927-935, which is herein incorporated by reference in its entirety). Therefore, in some embodiments, the NEDD8 co-E3 inhibitor that binds to the E2-binding pocket of the co-E3 protein also inhibits the activity of a NEDD8 E 1 protein. In order to generate an inhibitor molecule that is more specific for co-E3 proteins, in some embodiments, the phenylalanine corresponding to position 5 and leucine corresponding to position 7 of Ubc12 (SEQ ID NO: 2), which contribute to the bulk of the binding energy of Ubc12 to E1, are substituted with another amino acid residue, such as a hydrophobic staple-forming residue. Further, the addition of a hydrophobic staple to a peptide, locking it into an alpha helical conformation, prevents binding to NEDD8 E1 proteins (see Example 1).
Further, the peptide binding assays disclosed elsewhere herein (see Example 3 and Table 13) demonstrate that the assayed peptides exhibit binding specificity for particular co-E3 proteins (e.g., Dcn1, Dcn2, Dcn3, Dcn4, Dcn5). For example, SEQ ID NO: 8 (hUbc12Ac1-26) and SEQ ID NO: 87 (yUbc12Ac1-24) have greatest affinity for Den1 and 2, whereas SEQ ID NO: 88 (hUbe2fAcl-25) has greatest affinity for Dcn3 and is fairly specific for this particular co-E3 protein. The affinity of hUbc12Ac1-12 for Dcn3 and 5 can be enhanced by substituting amino acid residues at positions 6 and 10 with (S)-2-(3-butenyl)alanine and (S)-2-(7-octenyl)alanine, respectively, to form a hydrophobic staple. Similarly, the affinity of yUbc12Ac1-24 for Dcn1, 2, and 3 can be enhanced by substituting the first methionine with norleucine and amino acid residues at positions 6 and 10 with (S)-2-(4-pentenyl)alanine to form a hydrophobic staple. Interestingly, the affinity of yUbc12Ac1-24 for Dcn4 and Dcn5 is reduced with these modifications. Using this information, in combination with the disclosed crystal structures of the E2-binding pocket, one of skill in the art could generate a peptide that has enhanced specificity for one NEDD8 co-E3 protein over another.
The presently disclosed NEDD8 co-E3 inhibitors may be used to inhibit co-E3 activity and cellular growth and therefore, find use in treating disorders associated with unregulated cell growth. Further, as IκB, the inhibitor of the inflammatory mediator NFκB, is a target of the cullin-comprising SCF complex, the presently disclosed NEDD8 co-E3 inhibitors also find use in treating inflammatory disorders.
NEDD8 co-E3 proteins can be inhibited by the presently disclosed NEDD8 co-E3 inhibitors by contacting a NEDD8 co-E3 protein (e.g., Dcn1, Dcn2, Dcn3, Dcn4, Dcn5) with a NEDD8 co-E3 inhibitor, such as those described herein, in vivo or in vitro or in cells in culture.
In some embodiments, the NEDD8 co-E3 protein is contacted by a NEDD8 co-E3 inhibitor in a biological sample. As used herein, the phrase “biological sample” refers to a sample obtained from or comprising a cell, tissue, organ, or organism. Non-limiting examples of biological samples include cellular organelles, cells (e.g., mammalian cells, bacterial cells, cultured cells), a biological fluid, such as blood, plasma, serum, urine, bile, saliva, tears, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion), a transudate or exudate (e.g. fluid obtained from an abscess or other site of infection or inflammation), a fluid obtained from a joint (e.g. a normal joint or a joint affected by disease such as rheumatoid arthritis, osteoarthritis, gout or septic arthritis), or the like, and a lysate or extract of an organelle, cell, tissue, organ, or organism.
Biological samples may be obtained from any organ or tissue (normal or diseased, including a biopsy or autopsy specimen) or may comprise cells or a lysate or extract thereof (including primary cells, passaged or cultured primary cells, cell lines, cells conditioned by a specific medium or grown under a particular set of environmental conditions) or medium conditioned by cells. In some embodiments, the biological sample (e.g., cell, tissue, organism or extract thereof) after exposure to a hormone or other biological or pharmacological agent. If desired, the biological sample may be subjected to processing, such as lysis, extraction, subcellular fractionation, or other standard biochemical procedures known in the art to solubilize proteins. See, Deutscher (ed.) (1990) Methods in Enzymology 182:147-238, which is herein incorporated by reference in its entirety. For example, tissues or cells can be ground and homogenized in buffers appropriate for solubilizing proteins and retaining their native conformations, followed by clarification by centrifugation. Other methods known in the art can be used to process the biological sample to obtain the protein with co-E3 activity, including but not limited to osmotic lysis, detergent lysis, sonication, heat, and rapid decompression. In general, methods used to obtain the Nedd8 co-E3 protein are performed under non-denaturing conditions, allowing the majority of proteins to retain their native conformations.
The Nedd8 pathway is involved in the regulation of cell cycle progression and cell proliferation. Thus, the various inhibitors of the E2 binding pocket of the co-E3 enzymes provided herein may be useful for inhibiting cell growth and in the treatment of diseases or disorders that are associated with hyperproliferation (e.g., cancers, inflammatory disorders, pathogenic infections). As used herein, “cell growth” refers to cell proliferation, cell division, or progression through the cell cycle.
Methods for inhibiting cell growth comprise introducing into a cell a NEDD8 co-E3 inhibitor, such as those disclosed herein. As used herein, the terms “introduce” and “introducing” when referring to a compound refers to the presentation of the compound to a cell in such a manner that the compound gains access to the intracellular region of the cell. The compound can be introduced into the cell via any means known in the art.
Any method known in the art can be used to measure the growth rate of a cell, including, but not limited to, optical density (OD600), CO2 production, O2 consumption, assays that measure mitochondrial function, such as those utilizing tetrazolium salts (e.g., MTT, XTT), or other colorimetric reagents (e.g., the WST-1 reagent available from Roche), assays that measure or estimate DNA content, including, but not limited to fluoremetric assays such as those utilizing the fluorescent dye Hoechst 33258, assays that measure or estimate protein content, including, but not limited to, the sulforhodamine B (SRB) assay, manual or automated cell counts (with or without the Trypan Blue stain to distinguish live cells), and clonogenic assays with manual or automated colony counts. In some embodiments, the growth rate of a cell is inhibited by a NEDD co-E3 inhibitor by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater.
The NEDD8 co-E3 inhibitors disclosed herein can be used to treat disorders that are associated with hyperproliferation, such as cancers, inflammatory disorders, and pathogenic infections.
As used herein, “treatment” is an approach for obtaining beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, partial or complete restoration of eyesight (e.g., central vision, visual acuity), diminishment of extent of the disorder, stabilized (i.e., not worsening) state of the disorder (e.g., degeneration of cone photoreceptors), delaying or slowing of progression of the disorder, amelioration or palliation of the disorder, and prevention of, inhibition of, or reduction of risk of developing a retinal disorder. “Treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder (to prevent further degeneration) as well as those in which the disorder is to be prevented. “Palliating” a disorder means that the extent and/or undesirable clinical manifestations of the disorder are lessened and/or the time course of the progression is slowed or lengthened, as compared to a situation without treatment.
Methods for treating a disorder associated with hyperproliferation (e.g., cancer, inflammatory disorder, pathogenic infection) comprise administering a therapeutically effective amount of a NEDD co-E3 inhibitor, such as those disclosed herein, to a subject in need thereof.
By “therapeutically effective amount” is intended an amount that is useful in the treatment, prevention or diagnosis of a disease or condition. As used herein, a therapeutically effective amount of a NEDD8 co-E3 enzyme inhibitor is an amount which, when administered to a subject, is sufficient to achieve a desired effect, such as inhibiting NEDD8 co-E3 activity or cell growth in a subject being treated with that composition. The effective amount of a NEDD8 co-E3 protein inhibitor useful for inhibiting cell growth will depend on the subject being treated, the severity of the affliction, and the manner of administration of the NEDD8 co-E3 protein inhibitor.
By “subject” is intended an animal, including but not limited to, mammals, e.g., primates, humans, agricultural and domesticated animals such as, but not limited to, dogs, cats, cattle, horses, pigs, sheep, and the like. In some embodiments, the subject undergoing treatment with the pharmaceutical formulations of the invention is a human.
Given the involvement of NEDD8 in the regulation of cell cycle progression, NEDD8 co-E3 inhibitors, such as those disclosed herein, can be used to treat disorders that are associated with unregulated cell growth, such as cancer. Components of the Nedd8 pathway have been implicated in various types of cancer, including, but not limited to, gliomas, cancers of the lung, head and neck, esophagus, tongue, urinary tract, cervix, vulva, vagina and ovary (Broderick et al. (2010) Neoplasia 12(6):476-84; Sarkaria et al. (2006) Cancer Res 66(19): 9437-44; and Sarkaria et al. (2004) Ann Thorac Surg 78:1734-41, each of which are herein incorporated by reference in its entirety). In fact, human DCUN1D1 is also known as squamous cell carcinoma-related oncogene (SCCRO) due to its overexpression in various squamous cell carcinomas of mucosal origin, including, for example, oral tongue squamous cell carcinoma (Estilo et al. (2003) Clin Can Res 9:2300-06, which is herein incorporated by reference in its entirety).
The term “cancer” refers to the condition in a subject that is characterized by unregulated cell growth, wherein the cancerous cells are capable of local invasion and/or metastasis to noncontiguous sites. As used herein, “cancer cells,” “cancerous cells,” or “tumor cells” refer to the cells that are characterized by this unregulated cell growth and invasive property. The term “cancer” encompasses all types of cancers, including, but not limited to, all forms of carcinomas, melanomas, sarcomas, gliomas, lymphomas and leukemias, including without limitation, lung cancer, head and neck cancer, esophageal cancer, tongue cancer, cancers of the urinary tract, cervical cancer, vulval cancer, vaginal cancer, ovarian cancer. In particular embodiments, the cancer that is being treated with the presently disclosed NEDD8 co-E3 inhibitors is a squamous cell carcinoma, such as an oral tongue squamous cell carcinoma.
The neddylation of cullin proteins promotes the ubiquitination of IκB and subsequent activation of the inflammatory mediator NFκB. Therefore, NEDD8 co-E3 inhibitors can be used to treat inflammatory disorders.
As used herein, an “inflammatory disorder” is a condition characterized by inflammation and tissue destruction, or a combination thereof. An “inflammatory disorder” includes any inflammatory immune-mediated process where the initiating event or target of the immune response involves non-self antigen(s), including, for example, alloantigens, xenoantigens, viral antigens, bacterial antigens, unknown antigens, or allergens.
Further, for purposes of the present invention, the term “inflammatory disorder(s)” includes “autoimmune disease(s).” As used herein, the term “autoimmunity” is generally understood to encompass inflammatory immune-mediated processes involving “self” antigens. In autoimmune diseases, self antigen(s) trigger host immune responses. The term “inflammatory disorder” also refers to inflammation associated with tissue transplant rejection, such as graft versus host disease. “Transplant rejection” or “graft rejection” refers to any host-mounted immune response against a graft including but not limited to HLA antigens, blood group antigens, and the like.
Non-limiting examples of inflammatory disorders that can be treated with the NEDD8 co-E3 inhibitors disclosed herein include systemic lupus erythematosus (SLE), CREST syndrome, inflammatory myositis, Sjogren's syndrome, mixed connective tissue disease, multiple sclerosis, inflammatory bowel disease, acute respiratory distress syndrome, pulmonary inflammation, idiopathic pulmonary fibrosis, osteoporosis, delayed type hypersensitivity, asthma, primary biliary cirrhosis, and idiopathic thrombocytopenic purpura, discoid lupus, lupus nephritis, sarcoidosis, inflammatory arthritis, including juvenile arthritis, rheumatoid arthritis, psoriatic arthritis, Reiter's syndrome, ankylosing spondylitis, and gouty arthritis, rejection of an organ or tissue transplant, hyperacute, acute, or chronic rejection and/or graft versus host disease, multiple sclerosis, hyper IgE syndrome, polyarteritis nodosa, primary biliary cirrhosis, inflammatory bowel disease, Crohn's disease, celiac's disease (gluten-sensitive enteropathy), autoimmune hepatitis, pernicious anemia, autoimmune hemolytic anemia, psoriasis, scleroderma, myasthenia gravis, autoimmune thrombocytopenic purpura, autoimmune thyroiditis, Grave's disease, Hasimoto's thyroiditis, immune complex disease, chronic fatigue immune dysfunction syndrome (CFIDS), polymyositis and dermatomyositis, cryoglobulinemia, thrombolysis, cardiomyopathy, pemphigus vulgaris, pulmonary interstitial fibrosis, Type I and Type II diabetes mellitus, type 1,2, 3, and 4 delayed-type hypersensitivity, allergy or allergic disorders, unwanted/unintended immune responses to therapeutic proteins (see for example, U.S. Patent Application No. US 2002/0119151 and Koren, et al. (2002) Curr. Pharm. Biotechnol. 3:349-60), asthma, Churg-Strauss syndrome (allergic granulomatosis), atopic dermatitis, allergic and irritant contact dermatitis, urtecaria, IgE-mediated allergy, atherosclerosis, vasculitis, idiopathic inflammatory myopathies, hemolytic disease, Alzheimer's disease, chronic inflammatory demyelinating polyneuropathy, and the like.
Given the conservation of the NEDD8 signaling pathway in all eukaryotes, including fungal species, the presently disclosed NEDD8 co-E3 inhibitors inhibit the growth of pathogens and can be used to treat pathogenic infections, such as fungal infections. Further, viruses and bacterial pathogens routinely hijack the cullin-RING ligase machinery for ubiquitination and degradation of specific host factors. For example, HIV-1 Vif targets a RNA-editing enzyme (APOBEC3G) for Cu15-mediated degradation in a manner that reportedly depends on the NEDD8 pathway (see, for example, Barry and Fruh (2006) Sci STKE 335:21; Ribert and Cossart (2010) Cell 143:694-702). Therfore, the growth of viruses and bacterial pathogens that target host proteins for cullin RING ligase-dependent degradation can also be inhibited by the presently disclosed NEDD8 co-E3 inhibitors and these inhibitors find further use in treating infections of these viral and bacterial pathogens. Non-limiting examples of pathogenic organisms whose growth can be attenuated by the NEDD8 co-E3 inhibitors disclosed herein include fungal species within the genus Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis, Stachybotrys, and Paracoccidioides, including but not limited to, Candida albicans, Aspergillus fumigatus, Aspergillus flavus, Cryptococcus neoformans, Cryptococcus gattii, Histoplasma capsulatum, Pneumocystis jirovecii, Stachybotrys chartarum, and Paracoccidioides brasiliensis (see, for example, San-Blas and Calderone (2008) Pathogenic Fungi Insights in Molecular Biology. Caister Academic Press. 264 pages; and Chen et al. (2010) Curr Opin Pharmacol 10(5):522-30, each of which is herein incorporated by reference in its entirety); viral pathogens such as human immunodeficiency virus (HIV) and other lentiviruses; and bacterial pathogens that target host proteins for cullin RING ligase-dependent degradation.
When administration is for the purpose of treatment, administration may be for either a prophylactic (i.e., preventative) or therapeutic purpose. When provided prophylactically, the substance is provided in advance of any symptom. The prophylactic administration of the substance serves to prevent or attenuate any subsequent symptom. When provided therapeutically, the substance is provided at (or shortly after) the onset of a symptom. The therapeutic administration of the substance serves to attenuate any actual symptom.
It will be understood by one of skill in the art that the treatment modalities described herein may be used alone or in conjunction with other therapeutic modalities (i.e., as adjuvant therapy), including, but not limited to, surgical therapy, radiotherapy, chemotherapy (e.g., with any chemotherapeutic agent well known in the art) or immunotherapy.
The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a NEDD8 co-E3 inhibitor can include a single treatment or, preferably, can include a series of treatments. It will also be appreciated that the effective dosage of a NEDD8 co-E3 inhibitor used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein.
It is understood that appropriate doses of such active compounds depends upon a number of factors within the knowledge of the ordinarily skilled physician, veterinarian, or researcher. The dose(s) of the active compounds will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable. Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of an active agent depend upon the potency of the active agent with respect to the expression or activity to be modulated. Such appropriate doses may be determined using the assays described herein. When one or more of these molecules is to be administered to an animal (e.g., a human) in order to reduce the activity of the NEDD8 co-E3 protein, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.
Therapeutically effective amounts of a NEDD8 co-E3 inhibitor can be determined by animal studies. When animal assays are used, a dosage is administered to provide a target tissue concentration similar to that which has been shown to be effective in the animal assays. It is recognized that the method of treatment may comprise a single administration of a therapeutically effective amount or multiple administrations of a therapeutically effective amount of the NEDD8 co-E3 inhibitor.
The inhibitors of the E2 binding pocket of the co-E3 enzyme disclosed herein can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the compound (e.g., peptide, small molecule) and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. In addition, it may be desirable to administer a therapeutically effective amount of the pharmaceutical composition locally to an area in need of treatment (e.g., to an area of the body where inhibiting a TR cell function is desired). This can be achieved by, for example, local or regional infusion or perfusion during surgery, topical application, injection, catheter, suppository, or implant (for example, implants formed from porous, non-porous, or gelatinous materials, including membranes, such as sialastic membranes or fibers), and the like. In one embodiment, administration can be by direct injection at the site (or former site) of a cancer that is to be treated. In another embodiment, the therapeutically effective amount of the pharmaceutical composition is delivered in a vesicle, such as liposomes (see, e.g., Langer (1990) Science 249:1527-33; and Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez Berestein and Fidler (eds.), Liss, N.Y., pp. 353-65, 1989).
In another embodiment, the peptide NEDD8 co-E3 inhibitors can be formulated with a cell-penetrating peptide, including but not limited to the HIV tat protein, that is either covalently bound to the peptide or otherwise associated therewith (see, for example, Okuyama et al. (2007) Nature Methods 4:153-159).
In yet another embodiment, the therapeutically effective amount of the pharmaceutical composition can be delivered in a controlled release system. In one example, a pump can be used (see, e.g., Langer (1990) Science 249:1527-33; Sefton (1987) Crit. Rev. Biomed. Eng. 14:201-40; Buchwald et al. (1980) Surgery 88:507-16; Saudek et al. (1989) N. Engl. J. Med. 321:574-79). In another example, polymeric materials can be used (see, e.g., Levy et al. (1985) Science 228:190-92; During et al. (1989) Ann. Neurol. 25:351-56; Howard et al. (1989) J. Neurosurg. 71:105-12). Other controlled release systems, such as those discussed by Langer (1990) Science 249:1527-33, can also be used.
Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor® EL (BASF; Parsippany, N.J.), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride, in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth, or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated with each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “a peptide” is understood to represent one or more peptides. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
Throughout this specification and the claims, the words “comprise,” “comprises,” and “comprising” are used in a non-exclusive sense, except where the context requires otherwise.
As used herein, the term “about,” when referring to a value is meant to encompass variations of, in some embodiments ±50%, in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.
Further, when an amount, concentration, or other value or parameter is given as either a range, preferred range, or a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. Where a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range. It is not intended that the scope of the presently disclosed subject matter be limited to the specific values recited when defining a range.
The following examples are offered by way of illustration and not by way of limitation.
EXPERIMENTAL
Example 1
Activation of Ubiquitin-Like Protein Ligation by Acetylation-Dependent E2-E3 Interaction
Exogenously expressed C-terminally tagged and/or untagged human or yeast Ubc12 were purified from 293T cells, yeast, and/or SF9 cells (hereafter human proteins denoted with “h”, and yeast with “y”). Endogenous hUbc12 was co-immunoprecipitated with TAPhNedd8 expressed in 293T cells. Tandem mass spectrometry (LC-MS/MS) identified exogenous yeast and human, and endogenous human Ubc12 as retaining the amino-terminal (N-terminal) methionine (Met) and being N-terminally acetylated (data not shown). N-terminal acetylation has been well-characterized in budding yeast: N-terminal Acetyltransferase (Nat) enzyme specificities depend upon a combination of: (1) whether or not the N-terminal Met is removed by methionine-aminopeptidase; and (2) the second residue in a protein's sequence (Polevoda and Sherman (2003) J Mol Biol 325(4):595-622). yUbc12's N-terminal sequence, Met-Leu, is predicted to retain the Met and be acetylated by NatC, a heterotrimeric enzyme consisting of Mak3p, Mak10p and Mak31p (Polevoda and Sherman (2001) J Biol Chem 276(23):20154-20159). Indeed, deletion of the yeast Mak3 gene prevents yUbc12 N-terminal acetylation. Furthermore, coexpression with NatC subunits is sufficient for yUbc12 N-terminal acetylation in bacteria (FIG. 1A). Thus, yeast NatC performs yUbc12 N-terminal acetylation.
To address the question of whether Ubc12 N-terminal acetylation influences Nedd8 ligation to cullins such as Cul1, levels of yNedd8 ̃yCul1 (aka Rub1p ̃Cdc53p, but here human nomenclature used for simplification) complexes in yeast were examined with gene deletions for subunits of the major N-terminal Acetyltransferases, NatA, NatB, and NatC. Only yeast deleted for genes encoding NatC subunits displayed decreased levels of yNedd8 ̃yCul1 (FIG. 1B), indicating a correlation between yUbc12 N-terminal acetylation and yNedd8 modification of yCul1 in vivo. Furthermore, loss of NatC activity was synthetically lethal in combination with the cdc34-2 temperature-sensitive allele (data not shown)—a hallmark for Nedd8 pathway components due to roles in yCul1/SCF-regulated cell division (Kurz et al. (2008) Mol Cell 29:33; Lammer et al. (1998) Genes Dev 12:914).
In vitro, Ubc12 N-terminal acetylation strikingly and specifically dictates Dcn1P-mediated Nedd8 transfer to Cul1, as revealed by assays with three forms of Ubc12: Ubc12AcMet is N-terminally acetylated; Ubc12Met is identical to Ubc12AcMet sequence but lacks N-terminal acetylation; and yUbc12GSMet is not acetylated and has Gly-Ser N-terminal of Met (Scott et al. (2010) Mol Cell 39(5):784-795). Although yDcn1P enhances yNedd8 transfer to yCul1 even from yUbc12Met or yUbc12GSMet, consistent with the partial defect observed in NatC null yeast (FIG. 1B), yDcn1P E3 activity is substantially magnified for yUbc12AcMet (FIG. 2A). For human proteins, hUbc12 N-terminal acetylation is absolutely required to observe any hDcn1P mediated potentiation of neddylation (FIG. 2B). With apparently saturating concentrations of hDcn1P, hNedd8 transfer to hCul1 from hUbc12AcMet is complete in less than 20 seconds under conditions where we estimate the reaction would take  ̃8 minutes either in the absence of hDcn1P, or with hDcn1P but without Ubc12 N-terminal acetylation (FIGS. 2B and 3C). The effect of Ubc12 N-terminal acetylation is specific for Dcn1P, because Rbx1-mediated transfer of Nedd8 to Cul1 is independent of the state of Ubc12's N-terminus (FIGS. 2A and 2B). Nonetheless, even in the presence of Dcn1P and Ubc12 N-terminal acetylation, Cul1 neddylation still requires Rbx1's RING E3 activity and is blocked by an inhibitor, CAND1 (FIG. 3).
To gain insights into mechanistic roles of Ubc12 N-terminal acetylation, isothermal titration calorimetry (ITC) was used to quantify interactions with peptides corresponding to the N-termini of yeast or human Ubc12. Strikingly, N-terminal acetylation increases affinity for Dcn1P by roughly two orders-of-magnitude (Table 23). Ubc12's N-terminal Met is also required for Dcn1P-binding. For comparison, N-terminal acetylation has little effect on Ubc12 peptide binding to E1, although the role of the Met differs between yeast and human, likely due to idiosynchratic Nedd8 E1-E2 interactions in yeast (FIG. 4) (Huang et al. (2009) Mol Cell 33(4):483-495).







TABLE 23







Thermodynamic parameters determined by isothermal


titration calorimetry (ITC) for binding between the indicated


Ubc12 peptides and Dcn1P and E1.
















ΔS





Kd
ΔH
(cal/mol/


Protein
Titrant
(μM)
(cal/mol)
deg)
N















yDcn1P
yUbc121-24
>40
3,777
33.4
1.16


(44)
(10)


yDcn1P
Acetyl-yUbc121-24
0.444
4,145
43.6
0.99


(44)


yDcn1P
Acetyl-yUbc122-24
>37
3,761
33.4
0.92


hDcn1P
hUbc121-26
>130
1,259
22.2
0.75


hDcn1P
Acetyl-hUbc121-26
1.11
−6,107
5.84
0.92


hDcn1P
Acetyl-hUbc122-26
>100
1,719
24.1
0.52


yE1
yUbc121-24
0.070
−8,571
2.65
1.05


yE1
Acetyl-yUbc121-24
0.016
−11,260
−3.8
0.99


yE1
Acetyl-yUbc122-24
1.58
−9,625
−7.21
0.92


hE1
hUbc121-26
0.585
−8,881
−1.99
1.29


hE1
Acetyl-hUbc121-26
0.943
−9,425
−4.79
1.14


hE1
Acetyl-hUbc122-26
0.390
−8,899
−1.24
1.04





Kd: dissociation constant; ΔH: change in enthalpy; ΔS: change in entropy; N: stoichiometry, number of molecules of titrant the protein binds






To understand how N-acetyl-methionine mediates interactions, the crystal structures of yeast and human Dcn1P bound to Ubc12 peptides were determined (Table 24). The structure with human proteins is also in complex with a domain of the Nedd8 target, Cul1. hDcn1P-hCul1 interactions superimpose with the prior structure of yeast counterparts (Scott et al. (2010) Mol Cell 39(5):784-796), and are not discussed in detail here. Overall, Dcn1P superimposes with prior structures, forming a helical domain containing 2 EFhand-like folds (Kurz et al. (2008) Mol Cell 29(1):23-25; Scott et al. (2010) Mol Cell 39(5):784-796; and Yang et al. (2007) J Biol Chem 282(34):24490-24494). The Ubc12 N-terminal peptides are α-helical, as in full-length yUbc12GSMet (Scott et al. (2010) Mol Cell 39(5):784-796). A Dcn1P groove at the junction between the two EF-hand-like subdomains cradles Ubc12's helix, culminating in Ubc12's N-acetyl-methionine filling a conserved, hydrophobic crevasse in Dcn1P (FIGS. 5 and 6).







TABLE 24







Data collection and refinement statistics for three crystal structures.














hCut1WHB:hDcn1P:Acetyl-




yDcn1P:Acetyl-
hCul1WHB:hDcn1P:Acetyl-
hUbc121-12(5:9
hDcn3P:Acetyl-



yUbc121-24
hUbc121-15
Staple)
hUbe2f1-25















DATA COLLECTION






Space group
C2221
C2
C2221
P21


Cell dimensions


a, b, c (Å)
72.906, 98.421, 143.923
135.431, 65.454, 64.182
131.790, 190.210, 67.506
83.464, 44.579, 101.216


α, β, γ (°)
90.0, 90.0, 90.0
90.0, 104.73, 90.0
90.0, 90.0, 90.0
90.000, 103.305, 90.000













Resolution (Å)
40.0-2.3
(2.38-2.3)
50.0-1.5
(1.55-1.5)
50.0-2.0
50.0-2.4















Rmerge
0.123
(0.370)
0.090
(0.413)
0.078
(0.439)
0.110
(0.474)














|/σ|
13.5
(2.6)
27.2
(2.6)
24.9
(2.5)
1.857















Completeness (%)
89.9
(65.8)
96.2
(88.0)
96.7
(77.2)
91.6
(70.0)


Redundancy
5.4
(3.4)
3.7
(3.3)
6.6
(3.5)
3.6
(3.2)











REFINEMENT






Resolution (Å)
35-2.3
50.0-1.5
50.0-2.0
40.0-2.4


No. reflections
19967
78739
53080
27355


Rwork/Rfree
21.3/26.9
21.0/23.7
19.89/22.77
21.52/23.93


No. atoms
3788
5365
5131
3437












Protein
3651
4776
4646













Water
137
589
485
135


B-factors


Protein
27.06
19.56
17.2
34.381


Water
33.3
30.75
26.0
37.21











R.m.s deviations















Bond lengths (Å)
0.009
0.006
0.008
0.009


Bond angles (°)
1.021
0.914
0.992
1.297


RAMACHANDRAN


PLOT STATISTICS (%)


Preferred regions
97.1
97.8
97.5
94.62


Allowed regions
2.9
2.2
2.5
4.1


Disallowed regions
0
0
0
1.28





Data for highest resolution shell is shown in parentheses. Rwork = Σ|F0-Fc|/ΣF0•Rfree is the cross-validation of R-factor, with 5-10% of the total reflections omitted in model refinement.






N-acetyl-methionine recognition consists of three major components (FIG. 5). First, the methyl portion of the acetyl group fits snuggly in a hydrophobic pocket consisting of the α-carbon from yDcn1's Glu105 (hDcn1's Ala98) and side-chains from yDcn1's Leu107, Leu 110, and Leu193 (hDcn1's Ile100, Val102, Leu103, and Leu184). Second, the amide makes a hydrogen bond with the carbonyl oxygen from yDcn1's Leu104 (hDcn1's Pro97). Third, the Met side-chain packs in an extensive hydrophobic channel formed by side-chains from yDcn1's Leu93, Ile97, Leu104, Thr109, Leu112, Ala113, Tyr118, Leu121, and Leu173 (hDcn1's Ile86, Cys90, Pro97, Val102, Ile105, Ala106, Cys115, Phe117, Leu121, and Phe164).
Four additional structural elements, two each from Dcn1P and Ubc12, secure Ubc12's N-acetyl-methionine in place. First, yDcn1's Tyr190 (hDcn1's Tyr181) clamps between Ubc12's N-acetyl-Met1 and yDcn1-Leu2/hDcn1-Ile2 to press the N-acetyl-Met into the hydrophobic crevasse in Dcn1P. Second, the loop between Dcn1P's E- and F-á-helices acts as an additional clamp, closing down on top of Ubc12's N-acetyl-methionine. Notably, in 7 prior structures of yDcn1P (Kurz et al. (2008) Mol Cell 29(1):23-35; Scott et al. (2010) Mol Cell 39(5):784-796; and Yang et al. (2007) J Biol Chem 282(34):24490-24494), none of which are bound to Ubc12, both of these elements are repositioned and partially occupy and occlude access to the hydrophobic crevasse (data not shown). It seems likely that yDcn1P is sufficiently flexible to initially engage Ubc12's acetylated N-terminus, and subsequently clamps down around the hydrophobic yUbc12 N-acetyl-methionine. Such conformational flexibility may allow structural rearrangement accounting for yDcn1P's ability to mediate low-level activation of yUbc12 even in the absence of N-terminal acetylation.
Ubc12's N-acetyl-methionine is further sealed into the hydrophobic crevasse by interactions involving hydrophobic residues at positions 2 and 4 in Ubc12 (FIGS. 5C and 5D). This requires the helical structure, which places these side-chains on opposite sides of the N-acetyl-methionine. On one side, yUbc12's Leu2 (hUbc12's Ile2) buries the acetyl moiety via interacting with the aliphatic portion of yDcn1's Glu105 and Gln189 side-chains, Tyr190, and Leu193. The interaction is more extensive for the human proteins, with interactions with hDcn1's Ala98, Met177, Ala180, Tyr181, and Leu184. On the other side, Ubc12's Leu4 side-chain seals the Met1 side-chain into place via interactions with yDcn1's Ile90, Val94, Leu104, and Leu121 (hDcn1's Ile83, Ile86, Gln87, Pro97, and Cys115). Furthermore, there are numerous additional, species-specific interactions between other downstream residues from both faces of the Ubc12 helix and surrounding residues from the Dcn1P groove. For example, yUbc12's Lys3 fits in an anionic pocket formed by side-chains from yDcn1P's Glu122 and Asp 176, and carbonyls from Ile174 and Leu175. Other interactions include the following: yUbc12's Arg5 forms a salt-bridge with yDcn1P's Glu105; yUbc12's Leu7 makes hydrophobic contacts with yDcn1P's Ile90 and Leu121; and yUbc12's Lys11 fits in an anionic pocket formed by side-chains from yDcn1P's Asp89 and Asp91. Examples of interactions between hUbc12 and hDcn1 include the following: hUbc12's Lys3 makes a salt-bridge with hDcn1P's Asn167; hUbc12's Leu7 makes hydrophobic contacts with hDcn1P's Ile83 and Cys115; hUbc12's Lys8 forms a hydrogen bond with hDcn1P's Gln87; and hUbc12's Lys11 forms a salt-bridge with hDcn1P's Asp84.
Although yUbc12Met's N-terminal extension is helical in a prior structure (Scott et al. (2010) Mol Cell 39(5):784-796), experiments were performed to confirm a role for the helix with human proteins because hUbc12Met's N-terminal region is extended in complex with E1 (Huang et al, (2004) Nat Struct Mol Biol 11(10):927-935; Huang et al. (2007) Nature 445(7126):394-398). Specifically, Phe5 and Gln9 were replaced with (S)-2-(4′-pentenyl) alanine and these side-chains were covalently linked to test the effect of “stapling” an acetylated Ubc12 peptide into a helix (Bird et al. (2008) Methods Enzymol 446:369-386). The stapled helical peptide no longer binds E1 (data not shown), consistent with prior structures (Huang et al. (2004) Nat Struct Mol Biol 11(10):927-935; and Huang et al. (2007) Nature 445(7126):394-398). Moreover, a 2.0 Å resolution structure with the stapled peptide superimposes with the unstapled hUbc12AcMet complex with hDcn1P, confirming that the staple is completely solvent-exposed (data not shown). The helical staple improves binding to hDcn1P 14-fold (Table 25). This is largely due to decreasing the entropic cost of binding, supporting the notion that locking the flexible hUbc12 N-terminal region into a helical conformation is important for the hDcn1P interaction.







TABLE 25







Thermodynamic parameters determined by isothermal titration


calorimetry (ITC) for binding between hDcn1P or HE1 to


control or hydrocarbon-stapled Ubc12 peptides.
















ΔS





Kd
ΔH
(cal/mol/


Protein
Titrant
(μM)
(cal/mol)
deg)
N















hDcn1P
Acetyl-hUbc121-12
2.15
−4,992
8.79
0.99


hDcn1P
Acetyl-hUbc121-12
0.15
−3,386
19.3
1.18



(5:9Staple)


hE1
Acetyl-hUbc121-12
0.63
−11,490
−11.0
0.78


hE1
Acetyl-hUbc121-12
NB
NB
NB
NB



(5:9Staple)









The structures suggest two primary mechanisms by which Ubc12's N-terminal acetylation dictates binding to Dcn1P. First, the acetyl group interacts directly with Dcn1P. Second, acetylation eliminates an N-terminal positive charge, which would impede burial in the Dcn1P hydrophobic crevasse. To test these concepts, Dcn1P binding to Ubc12 peptides that were N-terminally formylated was examined by ITC (Table 26). The formylated peptides lack the methyl portion of an acetyl, but retain the amide and are uncharged. Indeed, the formylated peptides bind better than those with a free N-terminus. Furthermore, the Kds were decreased  ̃9- and  ̃17-fold compared with the acetylated human and yeast peptides, respectively, highlighting the importance of the acetyl methyl for mediating Ubc12-Dcn1P interactions.







TABLE 26







Thermodynamic parameters determined by isothermal


titration calorimetry (ITC) for binding between N-terminally


formylated Ubc12 peptides and Dcn1P.
















ΔS





Kd
ΔH
(cal/mol/


Protein
Titrant
(μM)
(cal/mol)
deg)
N















yDcn1P
Formyl-yUbc121-24
7.46
2,824
33.4
0.90


hDcn1P
Formyl-hUbcU126
9.71
−4,893
5.79
1.00









The ultimate test of understanding the structural role of Ubc12 N-terminal acetylation would be to design Dcn1P mutants that potentiate neddylation with unacetylated Ubc12. Given the structural malleability of yDcn1P, it was reasoned that mutations alleviating repulsion of yUbc12's N-terminal charge might enhance the low-level yDcn1P E3 activity toward yUbc12Met. The structure indicated that aspartate substitutions for yDcn1P Leu110 or Leu173 would approach yUbc12Met's N-terminus to balance the positive charge. Also, an Ala replacement for the Tyr190 “clamp” would not force a charged yUbc12Met's N-terminus directly into the hydrophobic pocket. Indeed, the three Dcn1P mutants showed enhanced activity specifically toward unacetylated yUbc12 in potentiating yNedd8 transfer to yCul1 (FIG. 7A). To address whether a mutation could compensate for loss of NatC-mediated yUbc12 acetylation in vivo, HA-tagged versions of either wild-type or mutant yDcn1 were expressed in strains deleted for either Dcn1, or both Dcn1 and the NatC subunit Mak10. Although Leu110Asp or Leu173Asp mutants were not expressed at levels comparable to the wild-type, strains expressing the Tyr190Ala mutant were obtained. Consistent with the in vitro results, relative to wild-type Dcn1, the Tyr190Ala mutant partially rescued the defect in yCul1 ̃yNedd8 conjugate formation in the absence of NatC activity (FIG. 7B).
Methods
Constructs, Protein Preparation, Antibodies, and Peptides
Constructs for expression and purification of hUbc12, yUbc12GSMet (also referred to as Ubc12), yDcn1P, hNedd8, yNedd8 (also referred to as Rub1), hCul1ctd-hRbx1, yCul1C+-yRbx1 (also referred to as Cdc53C+-Hrt1), yCul1C+-yRbx1R (also referred to as Cdc53C+-Hrt1R), hE1 for Nedd8 (also referred to as APPBP1-UBA3 and NAE1-UBA3), yE1 for yNedd8 (also referred to as Ula1Uba3), and hCAND1 were expressed in E. coli and purified as described previously (Walden et al. (2003) Nature 422(6929):330-334; Duda et al. (2008) Cell 134(6):995-1006; Scott et al. (2010) Mol Cell 39(5):784-796). hCul1-hRbx1 was expressed in insect cells and purified as described previously (Duda et al. (2008) Cell 134(6):995-1006).
hCul1ctd-hRbx1R contains a stop mutation introduced after residue 36 in hRbx1 to delete the RING domain, and was expressed and purified as previously described for hCul1ctdhRbx1 (Duda et al. (2008) Cell 134(6):995-1006). Expression constructs generated for this study were prepared by standard molecular biology techniques and coding sequences were entirely verified. Mutant versions used in this study were generated by QuickChange (Stratagene). hCul1WHB corresponds to residues 702 to the C-terminal residue 776 of hCul1 (SEQ ID NO: 79). hDcn1P corresponds to residues 62 to the C-terminal residue 259 of hDcn1 (aka SCCRO or DCUN1D1 (Sarkaria et al. (2006) Cancer Res 66(19):9437-9444); SEQ ID NO: 33).
hCul1WHB, hDcn1P, and hDcn1FL were expressed as GST fusions in BL21(DE3)Gold E. coli and purified by glutathione affinity chromatography. After TEV protease treatment to release GST, proteins were further purified by ion exchange and gel filtration chromatography in 25 mM Tris, 125 mM NaCl, 5 mM DTT, pH 7.6 (Buffer A), concentrated (Amicon Ultra), aliquotted, flash-frozen and stored at −80° C.
For expression and purification of hUbc12Met and yUbc12Met proteins, His-SUMO-1 fusions were generated in pRSF-Duet, in which the Met start codon of hUbc12 or yUbc12 was directly fused to follow the GlyGly SUMO-1 C-terminus. His-SUMO-1 fusions were expressed in BL21(DE3)Gold E. coli and purified by Ni+2 affinity chromatography. After treatment with the SUMO protease SENP2, hUbc12Met and yUbc12Met were further purified by gel filtration and passed back over a Ni+2 affinity resin to remove any residual His-SUMO-1 that co-purified over sizing.
For purification of hUbc12AcMet, hUbc12 was expressed untagged in insect cells. The Ubc12 coding sequence was cloned into pFastbac1, and baculoviruses were prepared using the Bac-to-Bac system (Invitrogen). Insect cell expression of hUbc12AcMet was performed at 24° C. for 1 day, followed by incubation at 18° C. for 2 days, with lysates initially purified over gravity S-Sepharose with a step gradient elution. Fractions containing hUbc12AcMet were diluted and reapplied to a Source-S column via FPLC, and eluted with a linear salt gradient. Fractions were pooled and concentrated (Amicon Ultra), and further purified by gel filtration chromatography in Buffer A.
For expression and purification of yUbc12AcMet, the yUbc12 ORF was fused to a C-terminal hexahistidine tag and cloned into pRS426-Gal1 (Mumberg et al. (1994) Nucleic Acids Res 22(25):5767-5768). Proteins expressed from this vector are referred to as “yUbc12-His6”, but subsequently the N-terminally acetylated version is referred to as “yUbc12ACMet”. BY4742, or mak3 (i.e., strain Y15470) yeast cells harboring pRS426-Gal1-yUbc12-His6 under control of the galactose-inducible promoter were grown at 30° C. in SD-Ura 2% Raffinose 0.1% Glucose media to mid-log phase (OD600=0.4-0.6). Cells were pelleted and resuspended at a density of 0.1 OD/ml in pre-warmed SD-Ura 2% Galactose media and induced at 30° C. for 14-16 hours. Cell pellets were resuspended in Phosphate-Buffer Saline containing Aprotonin, Leupeptin, PMSF, and protease inhibitor tablets (Sigma). Cells were lysed by three passages through a cell disruptor at 15,000 psi and protein subesquently purifed by Ni+2 affinity chromatography. Ni+2 column eluates were further purified over gel filtration chromatography in Buffer A.
N-terminal acetylation of bacterially expressed yUbc12 was achieved by co-expression of Mak3, Mak10, Mak31, and yUbc12-His6, followed by Ni+2 affinity and gel filtration chromatography. Briefly, untagged Mak10 and Mak3 were cloned into site 1 and 2, respectively, of pET-Duet (EMD Biosciences), and untagged Mak31 and yUbc12-His6 were cloned into site 1 and 2, respectively, of pRSF-Duet (EMD Biosciences).
For wild type and mutant rescue of dcn1 yeast cells, yDcn1-HA was built by overlap PCR from yeast genomic DNA and cloned into the CEN/Leu vector pRS315. The resultant clone contains approximately 600 nucleotides 5′ and 3′ of the start and stop codon, respectively, in order to preserve the natural regulatory elements of genomic yDcn1.
Antibodies against Cdc53 (sc-50444) and HA tag (H6908) were obtained from Santa Cruz Biotechnology and Sigma, respectively, and used according to the manufacturer's instructions. Peptides were synthesized and purified by reversed-phase HPLC by the Hartwell Center for Bioinformatics and Biotechnology at St. Jude. All peptides were C-terminally amidated. For helical hydrocarbon stapling, residues 5 and 9 of Ac-hUbc121-12 were substituted with (S)—N-Fmoc-2-(4-pentenyl)alanine, and the staple generated as described (Schafineister et al. (2000) J Am Chem Soc 122:5891-5892). Sequences of peptides used in this study are provided in Table 27.







TABLE 27







Sequences of peptides used in Example 1.









SEQ ID NO:
Peptide name
Sequence





15
yUbc121-24
MLKLRQLQKKKQKENENSSSIQPN-NH2


 


87
Acetyl-yUbc121-24
Ac-MLKLRQLQKKKQKENENSSSIQPN-


 


89
Acetyl-yUbc122-24
Ac-LKLRQLQKKKQKENENSSSIQPN-


 


7
hUbc121-26
MIKLFSLKQQKKEEESAGGTKGSSKK-


 


8
Acetyl-hUbc121-26
Ac-


 


9
Acetyl-hUbc122-26
Ac-IKLFSLKQQKKEEESAGGTKGSSKK-


 


90
Acetyl-hUbc121-12
Ac-MIKLFSLKQQKK-NH2


 


20
Acetyl-hUbc121-12(5:9Staple)
Ac-MIKLXSLKXQKK-NH2




X = 2-(4-pentenyl)alanine-


 


91
Formyl-yUbc121-24
Formyl-




MLKLRQLQKKKQKENENSSSIQPN-NH2


 


10
Formyl-hUbc121-26
Formyl-




MIKLFSLKQQKKEEESAGGTKGSSKK-


 


92
Acetyl-hUbc121-15
Ac-MIKLFSLKQQKKEEE-NH2









Cell Line Generation and Sample Preparation

293T cells with stable expression of C-terminally Flag-HA tagged hUbc12 were generated using lentiviral-mediated transduction. Puromycin-resistant cells were grown in DMEM supplemented with L-glutamine, 10% FBS, and pen/strep. TAP-Nedd8 cells were generated as described previously (Bennett et al. (2010) Cell 143(6):951-965). Cells were harvested and lysed in buffer containing 50 mM Tris pH 7.8, 150 mM NaCl, 0.5% NP40, and protease inhibitors (Roche). Cell lysates were then immunoprecipitated with HA conjugated resin. Complexes were washed, eluted with HA peptide, precipitated with TCA, and digested with GIuC (NEB).
Yeast Strains Used in this Study
Yeast strains for expression and purification of C-terminally His6-tagged yUbc12 (“yUbc12AcMet”) and for monitoring the yNedd8 modification status of yCul1 were obtained from the EUROScarf collection (Brachmann et al. (1998) Yeast 14(2):115-132). YBS001 was constructed by PCR-mediated gene disruption using pUG72 as a template for generation of a loxP-His3-loxP Dcn1 deletion cassette. Correct integration was confirmed by PCR screening of purified genomic DNA utilizing two primers that anneal internal to the His3 cassette and 5′ and 3′ primers flanking the intended disruption gene. Marker rescue was achieved as described with pSH47 (a kind gift from Randy Schekman's lab). After counter-selection on 5-FOA, YBS001 was subsequently used to generate YBS002 and correct integration verified as described above. Yeast strains used in this study are described in Table 28.







TABLE 28







Yeast strains used in Example 1.









Strain




name or


proccession

Refer-


Number
Genotype
ence





BY4742
MAT his3- 1 leu2- 0 lys2- 0 ura3- 0
*


Y14085
Same as B Y4742 but also dcn1::kanMX4
*


Y15214
Same as B Y4742 but also ubc12::kanMX4
*


Y13914
Same as B Y4742 but also csn5::kanMX4
*


Y10976
Same as B Y4742 but also ard1::kanMX4
*


Y13736
Same as B Y4742 but also nat1::kanMX4
*


Y15546
Same as B Y4742 but also nat3::kanMX4
*


Y15470
Same as B Y4742 but also mak3::kanMX4
*


Y10294
Same as B Y4742 but also mak10::kanMX4
*


Y13501
Same as B Y4742 but also mak31::kanMX4
*


YBS001
Same as B Y4742 but also dcn1::loxP
This




study


YBS002
Same as B Y4742 but also
This



mak10::loxP-His3-IoxP
study





* Brachmann et al (1998) Yeast 14: 115-132






Yeast Extract Preparation for Western Blotting

For FIG. 1, cells were inoculated into YPD media and grown overnight at 30° C. After OD600 measurements, cells were subcultured into fresh YPD media at 0.2 OD/ml and grown at 30° C. until OD600=0.9-1.0. Cells were pelleted from 2 ml of media and extracts made by the NaOH/BME/TCA method as described (Knop et al. (1999) Yeast 15(10B):963-972). For western blots from cell extracts in FIG. 7, cells were treated as described above except they were grown in SD-Leu 2% glucose for plasmid maintenance. Cell extracts corresponding to approximately 0.3 OD's of cells were run on 10% NuPAGE gels (Invitrogen). After western transfer, nitrocellulose membranes were probed with anti-Cdc53 (referred to here as yCul1) or anti-HA antibodies as indicated and visualized by enhanced chemiluminescence.
Mass Spectrometry
Peptides generated from GluC digestion of sample proteins were desalted offline using C18 stage tips. Peptides were eluted from the stage tip, dried down using a speed vac, and resuspended in 10 μl 5% formic acid, 5% acetonitrile. Peptide mixtures were separated by in line reverse phase using an 18 cm×150 μm (ID) column packed with C18 (MAGIC C18 5 μm particle, 200 angstrom pore size) using a 50-minute 8%-26% acetonitrile gradient. MS/MS data was generated using an LTQ-Velos mass spectrometer (Thermo), a data-dependent top 10 instrument method, and zoom scan for MS1 assignments. Data was acquired using CID with the normalized collision energy set to 35% with activation times of 10 ms. MS/MS triggering thresholds were set to 2000 and a 30s dynamic exclusion was used with an exclusion list size of 500. All of the mass spec data was validated using an LTQ-Orbitrap (Thermo) instrument to obtain high mass accuracy MS1 assignments for all Ubc12 peptides. Resultant MS/MS spectra were searched using Sequest against a concatenated forward and reverse human IPI database (v3.6), or a Saccharomyces cerevisiae database as needed. Methionine oxidation (+15.99), acetylation (+42.01), and the combined modification (+58.09) were set as dynamic modifications.
For intact mass analyses, protein was desalted using a reverse phase C8 Zip Tip and eluted in 50% acetonitrile, 2% formic acid. The eluent was ionized by static nanospray on Waters LCT Premier XE mass spectrometer using positive mode to obtain MaxEnt LC-TOF spectra.
Isothermal Titration calorimetry (ITC)
Protein samples were buffer matched by desalting over a NAP-5 column (GE Healthcare) into 50 mM Hepes, 125 mM NaCl, 1 mM BME, pH 7.0 (ITC buffer). Peptides were dissolved by weight to a final concentration of 10 mM in ITC buffer and diluted further in ITC buffer as required for the experiments. Measurements were performed using a MicroCal ITC200. hDcn1P or yDcn1P were placed into the sample cell at a final concentration of 400 μM at 18° C. The peptide ligands (4 mM) were constantly injected (1.5 μl). The interval time between each injection was 3 minutes and the duration of each injection was 3 seconds. For titrations involving hE1 and yE1, proteins were placed into the sample cell at a final concentration of 25 μM at 22° C. Peptide ligands (250 μM) were constantly injected (2.5 μl). The interval time between each injection was three minutes and the duration of each injection was 5 seconds. For hCul1ctd-Rbx1:hDcn1P binding, proteins were buffer matched by desalting over a NAP-5 column in 25 mM Tris, 0.1 M NaCl, 1 mM BME, pH 7.6. hCul1ctd-Rbx1 was placed into the sample cell at a final concentration of 55 μM at 22° C. hDcn1P (550 μM) was constantly injected (2.5 μl). The interval time between each injection was 3 minutes and the duration of each injection was 5 seconds. Obtained spectra were evaluated using Origin (V 7.0) to determine heats of binding and Kd values.
Crystallography
Crystals were grown by the hanging-drop vapor-diffusion method. Crystals of yDcn1P-Acetyl-yUbc121-24 grew as plate clusters at room temperature in 19-22% PEG3350, 0.1 M Bis-Tris Propane, 0.2 M sodium/potassium tartrate, pH 8.5. Single crystals were obtained by streak seeding into 16% PEG 3350, 0.1 M Bis-Tris Propane, 0.2 M sodium/potassium tartrate, pH 7.9. The crystals were harvested from mother liquor supplemented with 25% glycerol prior to flash-freezing in liquid nitrogen. Reflection data were collected at NECAT ID-24-E at the Advanced Photon Source. The crystals belong to space group C2221 with two yDcn1P-Acetyl-yUbc121-24 complexes in the asymmetric unit.
Crystals of hCul1WHB-hDcn1P-Acetyl-hUbc121-15 were grown at 4° C. in 27% PEG1500, 0.1 M MIB buffer (Qiagen), pH 4.0. The crystals grew as multiple clusters. Single crystals of the complex were obtained by streak-seeding into 17% PEG1500, 0.1 M MIB pH 4.0. The crystals were soaked in step gradients for 1-5 minutes, with sequential soaks containing well solution supplemented with 10%, 20%, and 30% ethylene glycol prior to flash-freezing in liquid nitrogen. Reflection data were collected at beamline 8.2.2 at the Advanced Light Source. Even after seeding, reflections from data for both the yDcn1P-Acetyl-yUbc121-24 and hCul1WHB-hDcn1P-Acetyl-hUbc121-15 complexes were streaky.
Crystals of hCul1WHB-hDcn1P-AcetylhUbc12Ac1-12(5,9 Staple) contained a selenomethionine version of the peptide used for convenience in crystallography that binds hDcn1P with essentially the same Kd as the methionine-containing peptide (not shown), and were grown at 4° C. in 21% PEG3350, 0.2 M KCl. The crystals were soaked in step gradients for 1-5 minutes, with sequential soaks containing well solution supplemented with 10%, 20%, and 30% of a 50:50 mixture of glycerol:ethylene glycol prior to flash-freezing in liquid nitrogen. Reflection data were collected at SERCAT 22-ID beamline at the Advanced Photon Source. All reflection data were processed with HKL2000 (Otwinowski and Minor (1997) Methods in Enzymology, Macromolecular Crystallography, part A 276:307-326).
Phases for all structures were obtained by molecular replacement using PHASER (Storoni et al. (2004) Acta Crystallogr D Biol Crystallogr 60(Pt 3):432-438) using the following search models: (1) for yDcn1P-Acetyl-yUbc121-24 structure, 2 copies of yDcn1P (3); (2) for hCul1WHB-hDcn1P-Acetyl-hUbc121-15 structure, 2 copies each of (a) a model of hDcn1P generated by Modeller (Eswar et al. (2006) Curr Protoc Bioinformatics Chapter 5, Unit 5 6) and (b) residues 707-776 from a prior structure of hCul1 (SEQ ID NO: 79) (Zheng et al. (2002) Nature 416(6882):703-709); (3) for hCul1wHB-hDcn1P-Acetyl-hUbc12Ac1-12(5,9 Staple) structure, 2 copies of hCul1WHB-hDcn1P from the hCul1WHB-hDcn1P-Acetyl-hUbc121-15 structure. In all cases, the peptides were built manually, and general manual rebuilding was performed with COOT (Emsley et al. (2004) Acta Crystallogr D Biol Crystallogr 60(Pt12Pt1):2126-2132). Refinement was performed using Phenix (Adams et al. (2010) Acta Crystallogr D Biol Crystallogr 66(Pt2):213-221) and Refmac (Murshudov et al. (1997) Acta Crystallogr D Biol Crystallogr D53:240-255). Details of Refinement are provided in Table 24.
Biochemical Assays
Dcn1P-mediated co-E3 activity was monitored using pulse-chase assays to exclusively monitor effects of acetylation on Nedd8 transfer to Cul1 without sensing earlier steps in the reaction. For the “pulse”, 10 μM of the indicated versions of yUbc12 were charged with [32P]-yNedd8 (also referred to as [32P]-Rub1) as described previously (Scott et al. (2010) Mol Cell 39(5):784-796), resulting in formation of a thiolester-linked yUbc12-[32P]-yNedd8 intermediate. 10 μM of the indicated versions of hUbc12 were charged similarly with [32P]-hNedd8, for 15 minutes at room temperature using 0.1 μM hE1, 15 μM [32P]-hNedd8, in 50 mM Hepes, 100 mM NaCl, 1.25 mM ATP, 2.5 mM MgCl2, pH 7.5. Formation of a yUbc12-[32P]-yNedd8 or hUbc12-[32P]-hNedd8 intermediate was quenched with 50 mM EDTA on ice for 5 minutes. Other than in FIG. 3, where additional details are provided, chase reactions with hUbc12 and yUbc12 variants involved dilution of the Ubc12-[32P]-Nedd8 thioester conjugate to 40 nM in 50 mM BisTris, 100 mM NaCl, 50 mM EDTA, 0.5 mg/ml BSA, pH 6.7. Chase reactions were initiated at 0° C. by the addition of hCul1ctd-hRbx1, yCul1C+-yRbx1, hDcn1P-hCul1ctd-hRbx1, or yDcn1P-yCul1C+-yRbx1 at a final concentration of 125 nM. Aliquots were removed at the indicated times and quenched with 2×SDS-PAGE sample buffer. Reaction products were heated at 70° C. for 1.5 minutes and separated on 4-12% NuPAGE gels (Invitrogen). Dried gels were exposed, as indicated in the figure legends, to a Storm (GE) Phosphorimager screen or film (Kodak).
Example 2
DCUND1D(1-5) Family Members Stimulate Ubc12AcMet and Ube2fAcMet Nedd8 Transfer to Cul1-5 Family Members
Pulse-chase assays similar to those described in Example 1 were used to determine if the DCUN1D family members, DCUN1D1, DCUN1D2, DCUN1D3, DCUN1D4, and DCUN1D5 could stimulate Nedd8 transfer to cullin family members, Cul1, Cul2, Cul3, Cul4, and Cul5. Results are shown in FIGS. 8A and 8B.
Example 3
Peptide Binding Assays
The affinity of various peptides for hDcn1P, hDcn2P, hDcn3P, hDcn4P, and hDcn5P was tested using isothermal titration calorimetry as described in Example 1 and is provided in Table 29.







TABLE 29







Binding Kd (in μM) of peptides for hDcn1P, hDcn2P, hDcn3P, hDcn4P, and hDcn5P.













SEQ ID








NO*:
Peptide
hDcn1P
hDcn2P
hDcn3P
hDcn4P
hDcn5P
















8
hUbc12Ac1−26
1.5
0.735
10.8
10
8.6


7
hUbc121−26
>200
—
—
—
—


9
hUbc12Ac−2−26
>150
—
—
—
—


10
hUbc12Formyl−1−26
9.7
—
—
—
—


11
hUbc12Ac1−Ac3−26
4.6
—
—
—
—


12
hUbc12Ac1−12
2.1
—
6.3
13.1
>30


17
hUbc12Ac1−12
5.6
—
—
—
—



(NorLeu)







93
hUbc121−12
>400
—
—
—
—



(NorLeu)







18
hUbc12Ac1−12
0.440
—
—
—
—



(NorLeu) 5, 9







19
hUbc12Ac1−12
0.8/1.2**
—
—
—
—



(NorLeu) 6, 10







20
hUbc12Ac1−12
0.153
—
—
—
—



(Met) 5, 9







21
hUbc12Ac1−12
0.0018/0.187** 
—
—
—
—



(Met) SS 6, 10







22
hUbc12Ac1−12
0.34/1 × 10{circumflex over ( )}−29**
—
—
—
—



(Met) RR 6, 10







23
hUbc12Ac1−12
0.182
—
—
—
—



(SeMet) 5, 9







24
hUbc12Ac1−12
0.00093/0.361** 
—
—
—
—



(SeMet) 6, 10







25
hUbc12Ac1−12
0.129
—
—
—
—



(Met) Oct:But








6, 10







26
hUbc12Ac1−12
0.120
—
1.52
—
0.781



(Met) But:Oct








6, 10







27
hUbc12Ac1−12
1.06
—
—
—
—



(Met) KE 5, 9







94
hUbc12Ac1−12
0.483
—
—
—
—



(Met) KE 6, 10







88
hUbe2fAc1−25
>500
>400
2
>40
>30


14
hUbe2f1−25
—
—
>400
—
—


87
yUbc12Ac1−24
0.311
0.204
3.4
*1.2/13
2.4


88
yUbc12Ac1−12
0.628
0.793
—
—
—



(NorLeu)







29
yUbc12Ac1−12
0.418
0.515
—
—
—



(NorLeu) 5, 9







30
yUbc12Ac1−12
0.068
0.075
1.76
4.3
4.78



(NorLeu) 6, 10







31
yUbc12Ac1−12
0.038/0.037**
—
—
—
—



(Met) 6, 10





*Each of the peptides had an amidated carboxy-terminus.


**The binding isotherm from the titration best fit a two-site model for binding, but the stoichiometry of binding is additive to approximately one.






Example 4
Peptide Inhibition
Pulse chase assays were used to monitor the transfer of Nedd8 from Ubc12AcMet ̃Nedd8 to Cul1CTD-Rbx1 in the absence or presence of DCUN1D1P and the indicated amounts of the peptide inhibitor Acetyl-yUbc121-12(6:10Staple). Results are provided in FIGS. 9A and 9B.
All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. This application hereby incorporates by reference in its entirety U.S. Provisional Application No. 61/486,972, filed May 17, 2011.
Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

## Claims

1. A method for identifying compounds that bind to an E2-binding pocket in a NEDD8 co-E3 protein, said method comprising performing a competitive binding assay using a peptide selected from the group consisting of:
a peptide having the sequence set forth in SEQ ID NO: 7, 12, 13, 14, 15, or 16,
wherein the nitrogen atom of the amino terminus of said peptide is covalently bound to at least one —R group, wherein said —R group is a substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, heteroaryl or has Formula (I):




 wherein said —R2 group is a hydrophobic moiety or hydrogen, wherein the methionine corresponding to position 1 of SEQ ID NO: 7, 12, 13, 14, 15, or 16 can be substituted with another amino acid residue having an unbranched hydrophobic side chain,
wherein said peptide can comprise a hydrophobic staple, and
wherein said peptide binds to an E2-binding pocket of Dcn1, DCUN1D1, DCUN1D2, DCUN1D3, DCUN1D4, or DCUN1D5.

2. The method of claim 1, wherein said —R group is a substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, or heteroaryl.

3. The method of claim 1, wherein said —R group is a formyl group.

4. The method of claim 1, wherein said —R group is not an acetyl group.

5. The method of claim 1, wherein said —R group has Formula (I):



wherein said —R2 group is a hydrophobic moiety.

6. The method of claim 5, wherein said —R2 group is alkyl or aryl.

7. The method of claim 1, wherein said unbranched hydrophobic side chain is an alkyl, alkenyl, or alkynyl.

8. The method of claim 1, wherein the methionine corresponding to position 1 in SEQ ID NO: 7, 12, 13, 14, 15, or 16 is substituted with a selenomethionine or a norleucine.

9. The method of claim 1, wherein said peptide comprises a hydrophobic staple.

10. The method of claim 9, wherein amino acid residues at positions X and X+4 are substituted with hydrophobic staple-forming amino acid residues, wherein X is not the first amino acid residue of the peptide.

11. The method of claim 10, wherein said hydrophobic staple-forming amino acid residues are selected from the group consisting of: (S)-2-(4-pentenyl)alanine, (R)-2(4-pentenyl)alanine, (S)-2-(3-butenyl)alanine, (S)-2-(7-octenyl)alanine, lysine, and glutamic acid.

12. The method of claim 1, wherein the carboxy terminus of said peptide is amidated.

13. The method of claim 1, wherein said peptide inhibits co-E3 activity of said Dcn1, DCUN1D1, DCUN1D2, DCUN1D3, DCUN1D4, or DCUN1D5.

14. The method of claim 13, wherein said peptide inhibits the neddylation of a cullin.

15. The method of claim 1, wherein said method further comprises assaying the ability of said compound to inhibit the co-E3 activity of said Dcn1, DCUN1D1, DCUN1D2, DCUN1D3, DCUN1D4, or DCUN1D5.