bluuebunny/crossref_metadata_2025_split huggingface dataset:
p53 Family members p63 and p73 are SAM domain‐containing proteins
Authors: Thanos Christopher D. , Bowie James U. 
Published: February 2009

AbstractHomologs of the tumor suppressor p53, called p63 and p73, have been identified. The p63 and p73 family members possess a domain structure similar to p53, but contain variable C‐terminal extensions. We find that some of the C‐terminal extensions contain Sterile Alpha Motif (SAM) domains. SAM domains are protein modules that are involved in protein‐protein interactions. Consistent with this role, the C‐terminal SAM domains of the p63 and p73 may regulate function by recruiting other protein effectors.
Unusual folded conformation of nicotinamide adenine dinucleotide bound to flavin reductase P
Authors: Tanner John J. , Barbour Leonard J. , Barnes Charles L. , Tu Shiao‐Chun , Krause Kurt L. 
Published: February 2009

AbstractThe 2.1 Å resolution crystal structure of flavin reductase P with the inhibitor nicotinamide adenine dinucleotide (NAD) bound in the active site has been determined. NAD adopts a novel, folded conformation in which the nicotinamide and adenine rings stack in parallel with an inter‐ring distance of 3.6 Å. The pyrophosphate binds next to the flavin cofactor isoalloxazine, while the stacked nicotinamide/adenine moiety faces away from the flavin. The observed NAD conformation is quite different from the extended conformations observed in other enzyme/NAD (P) structures; however, it resembles the conformation proposed for NAD in solution. The flavin reductase P/NAD structure provides new information about the conformational diversity of NAD, which is important for understanding catalysis. This structure offers the first crystallographic evidence of a folded NAD with ring stacking, and it is the first enzyme structure containing an FMN cofactor interacting with NAD(P). Analysis of the structure suggests a possible dynamic mechanism underlying NADPH substrate specificity and product release that involves unfolding and folding of NADP(H).
Structural changes in the C‐terminus of Ca2+‐bound rat S100B(ββ) upon binding to a peptide derived from the C‐terminal regulatory domain of p53
Authors: Rustandi Richard R. , Baldisseri Donna M. , Drohat Alexander C. , Weber David J. 
Published: February 2009

AbstractS100B(ββ) is a dimeric Ca2+‐binding protein that interacts with p53, inhibits its phosphorylation by protein kinase C (PKC) and promotes disassembly of the p53 tetramer. Likewise, a 22 residue peptide derived from the C‐terminal regulatory domain of p53 has been shown to interact with S100B(ββ) in a Ca2+‐dependent manner and inhibits its phosphorylation by PKC. Hence, structural studies of Ca2+‐loaded S100B(ββ) bound to the p53 peptide were initiated to characterize this interaction. Analysis of nuclear Overhauser effect (NOE) correlations, amide proton exchange rates, 3JNH‐Hα coupling constants, and chemical shift index data show that, like apo‐ and Ca2+‐bound S100B(ββ), S100B remains a dimer in the p53 peptide complex, and each subunit has four helices (helix 1, Glu2–Arg20; helix 2, Lys29–Asn38; helix 3, Gln50–Asp61; helix 4, Phe70–Phe87), four loops (loop 1, Glu21–His25; loop 2, Glu39–Glu49; loop 3, Glu62–Gly66; loop 4, Phe88–Glu91), and two β‐strands (β‐strand 1, Lys26–Lys28; β‐strand 2, Glu67–Asp69), which forms a short antiparallel β‐sheet. However, in the presence of the p53 peptide helix 4 is longer by five residues than in apo‐ or Ca2+‐bound S100B(ββ). Furthermore, the amide proton exchange rates in helix 3 (K55, V56, E58, T59, L60, D61) are significantly slower than those of Ca2+‐bound S100B(ββ). Together, these observations plus inter‐molecular NOE correlations between the p53 peptide and S100B(ββ) support the notion that the p53 peptide binds in a region of S100B(ββ), which includes residues in helix 2, helix 3, loop 2, and the C‐terminal loop, and that binding of the p53 peptide interacts with and induces the extension of helix 4.
Predicting conformational switches in proteins
Authors: Young Malin , Kirshenbaum Kent , Dill Ken A. , Highsmith Stefan 
Published: February 2009

AbstractWe describe a new computational technique to predict conformationally switching elements in proteins from their amino acid sequences. The method, called ASP (Ambivalent Structure Predictor), analyzes results from a secondary structure prediction algorithm to identify regions of conformational ambivalence. ASP identifies ambivalent regions in 16 test protein sequences for which function involves substantial backbone rearrangements. In the test set, all sites previously described as conformational switches are correctly predicted to be structurally ambivalent regions. No such regions are predicted in three negative control protein sequences. ASP may be useful as a guide for experimental studies on protein function and motion in the absence of detailed three‐dimensional structural data.
X‐ray crystallographic analysis of the structural basis for the interactions of pokeweed antiviral protein with its active site inhibitor and ribosomal RNA substrate analogs
Authors: Kurinov I.V. , Myers D.E. , Uckun F.M. , Irvin J.D. 
Published: February 2009

AbstractThe pokeweed antiviral protein (PAP) belongs to a family of ribosome‐inactivating proteins (RIP), which depurinate ribosomal RNA through their site‐specific N‐glycosidase activity. We report low temperature, three‐dimensional structures of PAP co‐crystallized with adenyl‐guanosine (ApG) and adenyl‐cytosine‐cytosine (ApCpC). Crystal structures of 2.0–2.1 Å resolution revealed that both ApG or ApCpC nucleotides are cleaved by PAP, leaving only the adenine base clearly visible in the active site pocket of PAP. ApCpC does not resemble any known natural substrate for any ribosome‐inactivating proteins and its cleavage by PAP provides unprecedented evidence for a broad spectrum N‐glycosidase activity of PAP toward adenine‐containing single stranded RNA. We also report the analysis of a 2.1 Å crystal structure of PAP complexed with the RIP inhibitor pteoric acid. The pterin ring is strongly bound in the active site, forming four hydrogen bonds with active site residues and one hydrogen bond with the coordinated water molecule. The second 1808 rotation conformation of pterin ring can form only three hydrogen bonds in the active site and is less energetically favorable. The benzoate moiety is parallel to the protein surface of PAP and forms only one hydrogen bond with the guanido group of Arg135.
Change in oligomerization specificity of the p53 tetramerization domain by hydrophobic amino acid substitutions
Authors: Stavridi Elena S. , Caruso Loretta C. , Chehab Nabil H. , Halazonetis Thanos D. 
Published: February 2009

AbstractThe tumor suppressor function of the wild‐type p53 protein is transdominantly inhibited by tumor‐derived mutant p53 proteins. Such transdominant inhibition limits the prospects for gene therapy approaches that aim to introduce wild‐type p53 into cancer cells. The molecular mechanism for transdominant inhibition involves sequestration of wild‐type p53 subunits into inactive wild‐type/mutant hetero‐tetramers. Thus, p53 proteins, whose oligomerization specificity is altered so they cannot interact with tumor‐derived mutant p53, would escape transdominant inhibition. Aided by the known three‐dimensional structure of the p53 tetramerization domain and by trial and error we designed a novel domain with seven amino acid substitutions in the hydrophobic core. A full‐length p53 protein bearing this novel domain formed homo‐tetramers and had tumor suppressor function, but did not hetero‐oligomerize with tumor‐derived mutant p53 and resisted transdominant inhibition. Thus, hydrophobic core residues influence the oligomerization specificity of the p53 tetramerization domain.
Homology modeling and active‐site residues probing of the thermophilic Alicyclobacillus acidocaldarius esterase 2
Authors: Manco Giuseppe , Febbraio Ferdinando , Adinolfi Elena , Rossi Mosè 
Published: February 2009

AbstractThe moderate thermophilic eubacterium Alicyclobacillus (formerly Bacillus) acidocaldarius expresses a thermostable carboxylesterase (esterase 2) belonging to the hormone‐sensitive lipase (HSL)‐like group of the esterase/lipase family. Based on secondary structures predictions and a secondary structure‐driven multiple sequence alignment with remote homologous protein of known three‐dimensional (3D) structure, we previously hypothesized for this enzyme the α/β‐hydrolase fold typical of several lipases and esterases and identified Ser155, Asp252, and His282 as the putative members of the catalytic triad. In this paper we report the construction of a 3D model for this enzyme based on the structure of mouse acetylcholinesterase complexed with fasciculin. The model reveals the topological organization of the fold corroborating our predictions. As regarding the active‐site residues, Ser155, Asp252, and His282 are located close to each other at hydrogen bond distances. Their catalytic role was here probed by biochemical and mutagenic studies. Moreover, on the basis of the secondary structure‐driven multiple sequence alignment and the 3D structural model, a residue supposed important for catalysis, Gly84, was mutated to Ser. The activity of the mutated enzyme was drastically reduced. We propose that Gly84 is part of a putative “oxyanion hole” involved in the stabilization of the transition state similar to the C group of the esterase/lipase family.
NMR studies of the low‐density lipoprotein receptor‐binding peptide of apolipoprotein E bound to dodecylphosphocholine micelles
Authors: Clayton Daniel , Brereton Ian M. , Kroon Paulus A. , Smith Ross 
Published: February 2009

AbstractCircular dichroism and NMR spectroscopy have been used to determine the structure of the low‐density lipoprotein (LDL) receptor‐binding peptide, comprising residues 130–152, of the human apolipoprotein E. This peptide has little persistent three‐dimensional structure in solution, but when bound to micelles of dodecylphosphocholine (DPC) it adopts a predominantly α‐helical structure. The three‐dimensional structure of the DPC‐bound peptide has been determined by using H‐NMR spectroscopy: the structure derived from NOE‐based distance constraints and restrained molecular dynamics is largely helical. The derived ϕ and ψ angle order parameters show that the helical structure is well defined but with some flexibility that causes the structures not to be superimposable over the full peptide length. Deuterium exchange experiments suggest that many peptide amide groups are readily accessible to the solvent, but those associated with hydrophobic residues exchange more slowly, and this helix is thus likely to be positioned on the surface of the DPC micelles. In this conformation the peptide has one hydrophobic face and two that are rich in basic amino acid side chains. The solvent‐exposed face of the peptide contains residues previously shown to be involved in binding to the LDL receptor.
Early events in the disulfide‐coupled folding of BPTI
Authors: Bulaj Grzegorz , Goldenberg David P. 
Published: February 2009

AbstractRecent studies of the refolding of reduced bovine pancreatic trypsin inhibitor (BPTI) have shown that a previously unidentified intermediate with a single disulfide is formed much more rapidly than any other one‐disulfide species. This intermediate contains a disulfide that is present in the native protein (between Cys14 and 38), but it is thermodynamically less stable than the other two intermediates with single native disulfides. To characterize the role of the [14–38] intermediate and the factors that favor its formation, detailed kinetic and mutational analyses of the early disulfide‐formation steps were carried out. The results of these studies indicate that the formation of [14–38] from the fully reduced protein is favored by both local electrostatic effects, which enhance the reactivities of the Cys14 and 38 thiols, and conformational tendencies that are diminished by the addition of urea and are enhanced at lower temperatures. At 25 °C and pH 7.3, approximately 35% of the reduced molecules were found to initially form the 14–38 disulfide, but the majority of these molecules then undergo intramolecular rearrangements to generate non‐native disulfides, and subsequently the more stable intermediates with native disulfides. Amino acid replacements, other than those involving Cys residues, were generally found to have only small effects on either the rate of forming [14–38] or its thermodynamic stability, even though many of the same substitutions greatly destabilized the native protein and other disulfide‐bonded intermediates. In addition, those replacements that did decrease the steady‐state concentration of [14–38] did not adversely affect further folding and disulfide formation. These results suggest that the weak and transient interactions that are often detected in unfolded proteins and early folding intermediates may, in some cases, not persist or promote subsequent folding steps.
Characterization of a soluble ternary complex formed between human interferon‐β‐1a and its receptor chains
Authors: Arduini Robert M. , Strauch Kathryn L. , Runkel LAURA A. , Carlson Melissa M. , Hronowski Xiaoping , Foley Susan F. , Young Carmen N. , Cheng Wenjie , Hochman Paula S. , Baker Darren P. 
Published: February 2009

AbstractThe extracellular portions of the chains that comprise the human type I interferon receptor, IFNAR1 and IFNAR2, have been expressed and purified as recombinant soluble His‐tagged proteins, and their interactions with each other and with human interferon‐β‐1a (IFN‐β‐1a) were studied by gel filtration and by cross‐linking. By gel filtration, no stable binary complexes between IFN‐β‐1a and IFNAR1, or between IFNAR1 and IFNAR2 were detected. However, a stable binary complex formed between IFN‐β‐1a and IFNAR2. Analysis of binary complex formation using various molar excesses of IFN‐β‐1a and IFNAR2 indicated that the complex had a 1:1 stoichiometry, and reducing SDS‐PAGE of the binary complex treated with the cross‐linking reagent dissucinimidyl glutarate (DSG) indicated that the major cross‐linked species had an apparent Mr consistent with the sum of its two individual components. Gel filtration of a mixture of IFNAR1 and the IFN‐β‐1a/IFNAR2 complex indicated that the three proteins formed a stable ternary complex. Analysis of ternary complex formation using various molar excesses of IFNAR1 and the IFN‐β‐1a/IFNAR2 complex indicated that the ternary complex had a 1:1:1 stoichiometry, and reducing SDS‐PAGE of the ternary complex treated with DSG indicated that the major cross‐linked species had an apparent Mr consistent with the sum of its three individual components. We conclude that the ternary complex forms by the sequential association of IFN‐β‐1a with IFNAR2, followed by the association of IFNAR1 with the preformed binary complex. The ability to produce the IFN‐β‐1a/IFNAR2 and IFN‐β‐1a/IFNAR1/IFNAR2 complexes make them attractive candidates for X‐ray crystallography studies aimed at determining the molecular interactions between IFN‐β‐1a and its receptor.
A comparative study of the unfolding of the endoglucanase Cel45 from Humicola insolens in denaturant and surfactant
Authors: Otzen Daniel E. , Christiansen Lars , Schülein Martin 
Published: February 2009

AbstractCellulases are increasingly being used for industrial purposes, particularly in washing powders, yet little is known of the factors governing the stability of proteins in detergent solutions. We present a comparative analysis of the behavior of the cellulase Cel45 from Humicola insolens in the presence of the denaturant guanidinium chloride and the anionic detergent C12‐LAS. Although Cel45 unfolds in GdmCl according to a simple two‐state model under equilibrium conditions, it accumulates a transient intermediate during refolding. The four disulfide bonds do not contribute detectably to the stability of the native state. Cel45 is unfolded by very low concentrations of C12‐LAS (1–4 mM). An analysis of 16 mutants of Cel45 shows a very weak correlation between unfolding rates in denaturant and detergent; mutants that have the same unfolding rate in GdmCl (within a factor of 1.5) vary 1, 000‐fold in their unfolding rates in C12‐LAS. The data support a simple model for unfolding by detergent, in which the introduction of positive charges or removal of negative charges greatly increases detergent sensitivity, while interactions with the hydrophobic detergent tail contribute to a smaller extent. This implies that different detergent‐mediated unfolding pathways exist, whose accessibilities depend on individual residues. Double‐mutant cycles reveal that mutations in two proximal residues lead to repulsion and a destabilization greater than the sum of the individual mutations as measured by GdmCl denaturation, but they also reduce the affinity for LAS and therefore actually stabilize the protein relative to wild‐type. Ligands that interact strongly with the denatured state may therefore alter the unfolding process.
Histidine placement in de novo–designed heme proteins
Authors: Gibney Brian R. , Dutton P. Leslie 
Published: February 2009

AbstractThe effects of histidine residue placement in a de novo‐designed four‐α‐helix bundle are investigated by placement of histidine residues at coiled coil heptad a positions in two distinct heptads and at each position within a single heptad repeat of our prototype heme protein maquette, [H10H24]2 [{Ac‐CGGGELWKL·HEELLKK·FEELLKL·HEERLKK·L‐CONH2}2]2 composed of a generic (α‐SS‐α)2 peptide architecture. The heme to peptide stoichiometry of variants of [H10H24]2 with either or both histidines on each helix replaced with noncoordinating alanine residues ([H10A24]2, [A10H24]2, and [A10A24]2) demonstrates the obligate requirement of histidine for biologically significant heme affinity. Variants of [A10A24]2, [{Ac‐CGGGELWKL·AEELLKK·FEELLKL·AEERLKK·L‐CONH2}2]2, containing a single histidine per helix in positions 9 to 15 were evaluated to verify the design based on molecular modeling. The bis‐histidine site formed between heptad positions a at 10 and 10′ bound ferric hemes with the highest affinity, Kd1 and Kd2 values of 15 and 800 nM, respectively. Placement of histidine at position 11 (heptad position b) resulted in a protein that bound a single heme with moderate affinity, Kd1 of 9.5 μM, whereas the other peptides had no measurable apparent affinity for ferric heme with Kd1 values >200 μM. The bis‐histidine ligation of heme to [H10A24]2 and [H11A24]2 was confirmed by electron paramagnetic resonance spectroscopy. The protein design rules derived from this study, together with the narrow tolerances revealed, are applicable for improving future heme protein designs, for analyzing the results of randomized heme protein combinatorial libraries, as well as for implementation in automated protein design.
Nucleotide‐dependent oligomerization of C1pB from Escherichia coli
Authors: Zolkiewski Michal , Kessel Martin , Ginsburg Ann , Maurizi Michael R. 
Published: February 2009

AbstractSelf‐association of ClpB (a mixture of 95‐ and 80‐kDa subunits) has been studied with gel filtration chromatography, analytical ultracentrifugation, and electron microscopy. Monomeric ClpB predominates at low protein concentration (0.07 mg0mL), while an oligomeric form is highly populated at >4 mg/mL. The oligomer formation is enhanced in the presence of 2 mM ATP or adenosine 5′‐O‐thiotriphosphate (ATPγS). In contrast, 2 mMADP inhibits full oligomerization of ClpB. The apparent size of the ATP‐ or ATPγS‐induced oligomer, as determined by gel filtration, sedimentation velocity and electron microscopy image averaging, and the molecular weight, as determined by sedimentation equilibrium, are consistent with those of a ClpB hexamer. These results indicate that the oligomerization reactions of ClpB are similar to those of other Hsp100 proteins.
A simple in vivo assay for increased protein solubility
Authors: Maxwell Karen L. , Mittermaier Anthony K. , Forman‐Kay Julie D. , Davidson Alan R. 
Published: February 2009

AbstractLow solubility is a major stumbling block in the detailed structural and functional characterization of many proteins and isolated protein domains. The production of some proteins in a soluble form may only be possible through alteration of their sequences by mutagenesis. The feasibility of this approach has been demonstrated in a number of cases where amino acid substitutions were shown to increase protein solubility without altering structure or function. However, identifying residues to mutagenize to increase solubility is difficult, especially in the absence of structural knowledge. For this reason, we have developed a method by which soluble mutants of an insoluble protein can be easily distinguished in vivo in Escherichia coli. This method is based on our observation that cells expressing fusions of an insoluble protein to chloramphenicol acetyltransferase (CAT) exhibit decreased resistance to chloramphenicol compared to fusions with soluble proteins. We found that a soluble mutant of an insoluble protein fused to CAT could be selected by plating on high levels of chloramphenicol.
Structure of a rat α1‐macroglobulin receptor‐binding domain dimer
Authors: Xiao Tsan , Decamp Dianne L. , Sprang Stephen R. 
Published: February 2009

Abstractα‐Macroglobulin inhibits a broad spectrum of proteinases by forming macromolecular cages inside which proteinases are cross‐linked and trapped. Upon formation of a complex with proteinase, α‐macroglobulin undergoes a large conformational change that results in the exposure of its receptor‐binding domain (RBD). Engagement of this domain by α‐macroglobulin receptor permits clearance of the α‐macroglobulin: proteinase complex from circulation. The crystal structure of rat α1‐macroglobulin RBD has been determined at 2.3 Å resolution. The RBD is composed of a nine‐stranded β‐sandwich and a single α‐helix that has been implicated as part of the receptor binding site and that lies on the surface of the β‐sandwich. The crystallographic asymmetric unit contains a dimer of RBDs related by approximate twofold symmetry such that the putative receptor recognition sites of the two monomers are contiguous. By gel filtration and ultracentrifugation, it is shown that RBD dimers form in solution with a dissociation constant of ∼50 μM. The structure of the RBD dimer might mimic a conformation of transformed α‐macroglobulin in which the proposed receptor binding residues are exposed on one face of the dimer. A pair of phenylalanine residues replaces a cystine that is conserved in other members of the macroglobulin family. These residues participate in a network of aromatic side‐chain interactions that appears to stabilize the dimer interface.