bluuebunny/crossref_metadata_2025 huggingface dataset:
The α‐subunit of protein prenyltransferases is a member of the tetratricopeptide repeat family
Authors: Zhang Hong , Grishin Nick V
Published: February 2009

Lipidation catalyzed by protein prenyltransferases is essential for the biological function of a number of eukaryotic proteins, many of which are involved in signal transduction and vesicular traffic regulation. Sequence similarity searches reveal that the α‐subunit of protein prenyltransferases (PTα) is a member of the tetratricopeptide repeat (TPR) super‐family. This finding makes the three‐dimensional structure of the rat protein farnesyltransferase the first structural model of a TPR protein interacting with its protein partner. Structural comparison of the two TPR domains in protein farnesyltransferase and protein phosphatase 5 indicates that variation in TPR consensus residues may affect protein binding specificity through altering the overall shape of the TPR superhelix. A general approach to evolutionary analysis of proteins with repetitive sequence motifs has been developed and applied to the protein prenyltransferases and other TPR proteins. The results suggest that all members in PTα family originated from a common multirepeat ancestor, while the common ancestor of PTα and other members of TPR superfamily is likely to be a single repeat protein.
Escherichia coli maltose‐binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused
Authors: Kapust Rachel B. , Waugh David S.
Published: February 2009

Although it is usually possible to achieve a favorable yield of a recombinant protein in Escherichia coli, obtaining the protein in a soluble, biologically active form continues to be a major challenge. Sometimes this problem can be overcome by fusing an aggregation‐prone polypeptide to a highly soluble partner. To study this phenomenon in greater detail, we compared the ability of three soluble fusion partners—maltose‐binding protein (MBP), glutathione S‐transferase (GST), and thioredoxin (TRX)—to inhibit the aggregation of six diverse proteins that normally accumulate in an insoluble form. Remarkably, we found that MBP is a far more effective solubilizing agent than the other two fusion partners. Moreover, we demonstrated that in some cases fusion to MBP can promote the proper folding of the attached protein into its biologically active conformation. Thus, MBP seems to be capable of functioning as a general molecular chaperone in the context of a fusion protein. A model is proposed to explain how MBP promotes the solubility and influences the folding of its fusion partners.
Effects of N‐butyldeoxynojirimycin and the Lec3.2.8.1 mutant phenotype on N‐glycan processing in Chinese hamster ovary cells: Application to glycoprotein crystallization
Authors: Butters Terry D. , Sparks Lisa M. , Harlos Karl , Ikemizu Shinji , Stuart David I. , Jones E. Yvonne , Davis Simon J.
Published: February 2009

Heterologous gene expression in either (1) the glycosylation‐defective, mutant Chinese hamster ovary cell line, Lec3.2.8.1, or (2) the presence of the α‐glucosidase inhibitor, N‐butyldeoxynojirimycin facilitates the trimming of N‐linked glycans of glycoproteins to single N‐acetylglucosamine (GlcNAc) residues with endoglycosidase H (endo H). Both approaches are somewhat inefficient, however, with as little as 12% of the total protein being rendered fully endo H‐sensitive under these conditions. It is shown here that the combined effects of these approaches on the restriction of oligosaccharide processing are essentially additive, thereby allowing the production of glycoproteins that are essentially completely endo H‐sensitive. The preparation of a soluble chimeric form of CD58, the ligand of the human T‐cell surface recognition molecule CD2, illustrates the usefulness of the combined approach when expression levels are low or the deglycosylated protein is unstable at low pH. The endo H‐treated chimera produced crystals of space group P3121 or P3221, and unit cell dimensions a = b = 116.4 Å, c = 51.4 Å a = β = 90°, γ = 120°, that diffract to a maximum resolution of 1.8 Å.
Drug resistance mutations can affect dimer stability of HIV‐1 protease at neutral pH
Authors: Xie Dong , Gulnik Sergei , Gustchina Elena , Yu Betty , Shao Wei , Qoronfleh Walid , Nathan Anand , Erickson John W.
Published: February 2009

The monomer‐dimer equilibrium for the human immunodeficiency virus type 1 (HIV‐1) protease has been investigated under physiological conditions. Dimer dissociation at pH 7.0 was correlated with a loss in β‐sheet structure and a lower degree of ANS binding. An autolysis‐resistant mutant, Q7K/L33I/L63I, was used to facilitate sedimentation equilibrium studies at neutral pH where the wild‐type enzyme is typically unstable in the absence of bound inhibitor. The dimer dissociation constant (KD) of the triple mutant was 5.8 μM at pH 7.0 and was below the limit of measurement (˜100 nM) at pH 4.5. Similar studies using the catalytically inactive D25N mutant yielded a KD value of 1.0 μMat pH 7.0. These values differ significantly from a previously reported value of 23 nM obtained indirectly from inhibitor binding measurements (Darke et al., 1994). We show that the discrepancy may result from the thermodynamic linkage between the monomer‐dimer and inhibitor binding equilibria. Under conditions where a significant degree of monomer is present, both substrates and competitive inhibitors will shift the equilibrium toward the dimer, resulting in apparent increases in dimer stability and decreases in ligand binding affinity. Sedimentation equilibrium studies were also carried out on several drug‐resistant HIV‐1 protease mutants: V82F, V82F/I84V, V82T/I84V, and L90M. All four mutants exhibited reduced dimer stability relative to the autolysis‐resistant mutant at pH 7.0. Our results indicate that reductions in drug affinity may be due to the combined effects of mutations on both dimer stability and inhibitor binding.
p53 Family members p63 and p73 are SAM domain‐containing proteins
Authors: Thanos Christopher D. , Bowie James U.
Published: February 2009

Homologs 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

The 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

S100B(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

We 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

The 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

The 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

The 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

Circular 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

Recent 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

The 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

Cellulases 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

The 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

Self‐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

Low 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

α‐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.
Crystal structure of an in vivo HIV‐1 protease mutant in complex with saquinavir: Insights into the mechanisms of drug resistance
Authors: Hong Lin , Zhang Xuenjun C. , Hartsuck Jean A. , Tang Jordan
Published: February 2009

Saquinavir is a widely used HIV‐1 protease inhibitor drug for AIDS therapy. Its effectiveness, however, has been hindered by the emergence of resistant mutations, a common problem for inhibitor drugs that target HIV‐1 viral enzymes. Three HIV‐1 protease mutant species, G48V, L90M, and G48V/L90M double mutant, are associated in vivo with saquinavir resistance by the enzyme (Jacobsen et al., 1996). Kinetic studies on these mutants demonstrate a 13.5‐, 3‐, and 419‐fold increase in Ki values, respectively, compared to the wild‐type enzyme (Ermolieff J, Lin X, Tang J, 1997, Biochemistry 36:12364‐12370). To gain an understanding of how these mutations modulate inhibitor binding, we have solved the HIV‐1 protease crystal structure of the G48V/L90M double mutant in complex with saquinavir at 2.6 Å resolution. This mutant complex is compared with that of the wild‐type enzyme bound to the same inhibitor (Krohn A, Redshaw S, Richie JC, Graves BJ, Hatada MH, 1991, J Med Chem 34:3340‐3342). Our analysis shows that to accommodate a valine side chain at position 48, the inhibitor moves away from the protease, resulting in the formation of larger gaps between the inhibitor P3 subsite and the flap region of the enzyme. Other subsites also demonstrate reduced inhibitor interaction due to an overall change of inhibitor conformation. The new methionine side chain at position 90 has van der Waals interactions with main‐chain atoms of the active site residues resulting in a decrease in the volume and the structural flexibility of S1/S1′ substrate binding pockets. Indirect interactions between the mutant methionine side chain and the substrate scissile bond or the isostere part of the inhibitor may differ from those of the wild‐type enzyme and therefore may facilitate catalysis by the resistant mutant.
The PA domain: A protease‐associated domain
Authors: Mahon Piers , Bateman Alex
Published: February 2009

We have identified a similarity between the apical domain of the human transferrin receptor and several other protein families. This domain is found associated with two different families of peptidases. Therefore, we term it the PA domain for protease‐associated domain. The PA domain is found inserted within a loop of the peptidase domain of family M8/M33 zinc peptidases. The PA domain is also found in a vacuolar sorting receptor and a ring finger protein of unknown function that may be a cell surface receptor. The PA domain may mediate substrate determination of peptidases or form protein‐protein interactions.
New insight on β‐lactoglobulin binding sites by 1‐anilinonaphthalene‐8‐sulfonate fluorescence decay
Authors: Collini Maddalena , D'Alfonso Laura , Baldini Giancarlo
Published: February 2009

The fluorescence time decay parameters of the β‐lactoglobulin‐1‐anilinonaphthalene‐8‐sulfonate complex have been investigated under physical and chemical perturbations (2 < pH < 8 and added electrolyte 0 < NaCl < 0.5 M) to obtain new insight on the nature of the protein binding interactions. A double exponential decay of the bound probe lifetime has been confirmed by the presence of a longer component, 11 to 14.5 ns, and a shorter component, 2.5 to 3.5 ns. The two lifetimes are ascribed to different binding modes associated also with different exposure to the solvent; in particular, the longer component is attributed to binding inside the hydrophobic beta barrel, while a “surface” site is suggested for the shorter component. A detailed analysis of the lifetime fractional intensities correlates the binding constants with ionic strength and supports the presence of electrostatic effects at both sites. A Debye–Hückel approach, applied to extrapolate the electrostatic free energy contribution vs. pH at vanishing ionic strength, gives interesting clues on the effective charge felt by the ANS ligands in the proximity of each site. In particular, binding is found to parallel the aspartate and glutamate titrations between pH 3 and pH 4.5; the “surface” site mainly responds to the presence of these local titrating charges while the “internal” site more closely follows the overall protein net charge.
Role of a solvent‐exposed aromatic cluster in the folding of Escherichia coli CspA
Authors: Rodriguez Hector M. , Vu Dung M. , Gregoret Lydia M.
Published: February 2009

Escherichia coli CspA is a member of the cold shock protein family. All cold shock proteins studied to date fold rapidly by an apparent two‐state mechanism. CspA contains an unusual cluster of aromatic amino acids on its surface that is necessary for nucleic acid binding and also provides stability to CspA (Hillier et al., 1998). To elucidate the role this aromatic cluster plays in the determining the folding rate and pathway of CspA, we have studied the folding kinetics of mutants containing either leucine or serine substituted for Phe18, Phe20, and/or Phe31. The leucine substitutions are found to accelerate folding and the serine substitutions to decelerate folding. Because these residues exert effects on the free energy of the folding transition state, they may be necessary for nucleating folding. They are not responsible, however, for the very compact, native‐like transition state ensemble seen in the cold shock proteins, as the refolding rates of the mutants all show a similar, weak dependence of unfolding rate on denaturant concentration. Using mutant cycle analysis, we show that there is energetic coupling among the three residues between the unfolded and transition states, suggesting that the cooperative nature of these interactions helps to determine the unfolding rate. Overall, our results suggest that separate evolutionary pressures can act simultaneously on the same group of residues to maintain function, stability, and folding rate.
Engineering the substrate specificity of Escherichia coli asparaginase II. Selective reduction of glutaminase activity by amino acid replacements at position 248
Authors: Derst Christian , Henseling Johannes , Röhm Klaus‐Heinrich
Published: February 2009

The use of Escherichia coli asparaginase II as a drug for the treatment of acute lymphoblastic leukemia is complicated by the significant glutaminase side activity of the enzyme. To develop enzyme forms with reduced glutaminase activity, a number of variants with amino acid replacements in the vicinity of the substrate binding site were constructed and assayed for their kinetic and stability properties. We found that replacements of A sp248 affected glutamine turnover much more strongly than asparagine hydrolysis. In the wild‐type enzyme, N248 modulates substrate binding to a neighboring subunit by hydrogen bonding to side chains that directly interact with the substrate. In variant N248A, the loss of transition state stabilization caused by the mutation was 15 kJ mol−1 for l‐glutamine compared to 4 kJ mol−1 for l‐aspartic β‐hydroxamate and 7 kJ mol−1 for l‐asparagine. Smaller differences were seen with other N248 variants. Modeling studies suggested that the selective reduction of glutaminase activity is the result of small conformational changes that affect active‐site residues and catalytically relevant water molecules.
Dimer formation by a “monomeric” protein
Authors: Park Chiwook , Raines Ronald T.
Published: February 2009

Dimeric proteins can arise by the swapping of structural domains between monomers. The prevalence of this occurrence is unknown. Ribonuclease A (RNase A) is assumed to be a monomer near physiological conditions. Here, this hypothesis is tested and found to be imprecise. The two histidine residues (His12 and His119) in the active site of RNase A arise from two domains (S‐peptide and S‐protein) of the protein. The H12A and H119A variants have 105‐fold less ribonucleolytic activity than does the wild‐type enzyme. Incubating a 1:1 mixture of the H12A and H119A variants at pH 6.5 and 65°C results in a 103‐fold increase in ribonucleolytic activity. A large quantity of active dimer can be produced by lyophilizing a 1:1 mixture of the H12A and H119A variants from acetic acid. At pH 6.5 and 65°C, the ribonucleolytic activity of this dimer converges to that of the dimer formed by simply incubating the monomers, as expected for a monomer–dimer equilibrium. The equilibrium dissociation constant for the dimer is near 2 mM at both 65 and 37°C. This value of Kd is only 20‐fold greater than the concentration of RNase A in the cow pancreas, suggesting that RNase A dimers exist in vivo. The intrinsic ability of RNase A to form dimers under physiological conditions is consistent with a detailed model for the evolution of homodimeric proteins. Dimers of “monomeric” proteins could be more prevalent than is usually appreciated.
For the record: A logical sequence search for S100B target proteins
Authors: Mcclintock Kimberly A. , Shaw Gary S.
Published: February 2009

The EF‐hand calcium‐binding protein S100B has been shown to interact in vitro in a calcium‐sensitive manner with many substrates. These potential S100B target proteins have been screened for the preservation of a previously identified consensus sequence across species. The results were compared to known structural and in vitro properties of the proteins to rationalize choices for potential binding partners. Our approach uncovered four oligomeric proteins tubulin (α and β), glial fibrillary acidic protein (GFAP), desmin, and vimentin that have conserved regions matching the consensus sequence. In the type III intermediate filament proteins (GFAP, vimentin, and desmin), this region corresponds to a portion of a coiled‐coil (helix 2A), the structural element responsible for their assembly. In tubulin, the sequence matches correspond to regions of α and β tubulin found at the αβ tubulin interface. In both cases, these consensus sequence matches provide a logical explanation for in vitro observations that S100B is able to inhibit oligomerization of these proteins.
Reversible denaturation of oligomeric human chaperonin 10: Denatured state depends on chemical denaturant
Authors: Guidry Jesse J. , Moczygemba Charmaine K. , Wittung‐Stafshede Pernilla , Steede N. Kalaya , Landry Samuel J.
Published: February 2009

Chaperonins cpn60/cpn10 (GroEL/GroES in Escherichia coli) assist folding of nonnative polypeptides. Folding of the chaperonins themselves is distinct in that it entails assembly of a sevenfold symmetrical structure. We have characterized denaturation and renaturation of the recombinant human chaperonin 10 (cpn10), which forms a heptamer. Denaturation induced by chemical denaturants urea and guanidine hydrochloride (GuHCl) as well as by heat was monitored by tyrosine fluorescence, far‐ultraviolet circular dichroism, and cross‐linking; all denaturation reactions were reversible. GuHCl‐induced denaturation was found to be cpn10 concentration dependent, in accord with a native heptamer to denatured monomer transition. In contrast, urea‐induced denaturation was not cpn10 concentration dependent, suggesting that under these conditions cpn10 heptamers denature without dissociation. There were no indications of equilibrium intermediates, such as folded monomers, in either denaturant. The different cpn10 denatured states observed in high [GuHCl] and high [urea] were supported by cross‐linking experiments. Thermal denaturation revealed that monomer and heptamer reactions display the same enthalpy change (per monomer), whereas the entropy‐increase is significantly larger for the heptamer. A thermodynamic cycle for oligomeric cpn10, combining chemical denaturation with the dissociation constant in absence of denaturant, shows that dissociated monomers are only marginally stable (3 kJ/mol). The thermodynamics for co‐chaperonin stability appears conserved; therefore, instability of the monomer could be necessary to specify the native heptameric structure.
Proline in α‐helical kink is required for folding kinetics but not for kinked structure, function, or stability of heat shock transcription factor
Authors: Hardy Jeanne A. , Nelson HILLARY C.M.
Published: February 2009

The DNA‐binding domain of the yeast heat shock transcription factor (HSF) contains a strictly conserved proline that is at the center of a kink. To define the role of this conserved proline‐centered kink, we replaced the proline with a number of other residues. These substitutions did not diminish the ability of the full‐length protein to support growth of yeast or to activate transcription, suggesting that the proline at the center of the kink is not conserved for function. The stability of the isolated mutant DNA‐binding domains was unaltered from the wild‐type, so the proline is not conserved to maintain the stability of the protein. The crystal structures of two of the mutant DNA‐binding domains revealed that the helices in the mutant proteins were still kinked after substitution of the proline, suggesting that the proline does not cause the α‐helical kink. So why are prolines conserved in this and the majority of other kinked α‐helices if not for structure, function, or stability? The mutant DNA‐binding domains are less soluble than wild‐type when overexpressed. In addition, the folding kinetics, as measured by stopped‐flow fluorescence, is faster for the mutant proteins. These two results support the premise that the presence of the proline is critical for the folding pathway of HSF's DNA‐binding domain. The finding may also be more general and explain why kinked helices maintain their prolines.
Homology modeling and molecular dynamics simulations of lymphotactin
Authors: Ma Buyong , Xiong Junjie , Lubkowski Jacek , Nussinov Ruth
Published: February 2009

We have modeled the structure of human lymphotactin (hLpnt), by homology modeling and molecular dynamics simulations. This chemokine is unique in having a single disulfide bond and a long C‐terminal tail. Because other structural classes of chemokines have two pairs of Cys residues, compared to one in Lpnt, and because it has been shown that both disulfide bonds are required for stability and function, the question arises how the Lpnt maintains its structural integrity. The initial structure of hLpnt was constructed by homology modeling. The first 63 residues in the monomer of hLpnt were modeled using the structure of the human CC chemokine, RANTES, whose sequence appeared most similar. The structure of the long C‐terminal tail, missing in RANTES, was taken from the human muscle fatty‐acid binding protein. In a Protein Data Bank search, this protein was found to contain a sequence that was most homologous to the long tail. Consequently, the modeled hLpnt C‐terminal tail consisted of both α‐helical and β‐motifs. The complete model of the hLpnt monomer consisted of two α‐helices located above the five‐stranded β‐sheet. Molecular dynamics simulations of the solvated initial model have indicated that the stability of the predicted fold is related to the geometry of Pro78. The five‐stranded β‐sheet appeared to be preserved only when Pro78 was modeled in the cis conformation. Simulations were also performed both for the C‐terminal truncated forms of the hLpnt that contained one or two (CC chemokine‐like) disulfide bonds, and for the chicken Lpnt (cLpnt). Our MD simulations indicated that the turn region (T30‐G34) in hLpnt is important for the interactions with the receptor, and that the long C‐terminal region stabilizes both the turn (T30‐G34) and the five‐stranded β‐sheet. The major conclusion from our theoretical studies is that the lack of one disulfide bond and the extension of the C‐terminus in hLptn are mutually complementary. It is very likely that removal of two Cys residues sufficiently destabilizes the structure of a chemokine molecule, particularly the core β‐sheet, to abolish its biological function. However, this situation is rectified by the long C‐terminal segment. The role of this long region is most likely to stabilize the first β‐turn region and α‐helix H1, explaining how this chemokine can function with a single disulfide bond.
The N‐terminal region of cystatin A (stefin A) binds to papain subsequent to the two hairpin loops of the inhibitor. Demonstration of two‐step binding by rapid‐kinetic studies of cystatin A labeled at the N‐terminus with a fluorescent reporter group
Authors: Estrada Sergio , Raub‐Segall Elke , Björk Ingemar , Olson Steven T.
Published: February 2009

The three‐dimensional structures of cystatins, and other evidence, suggest that the flexible N‐terminal region of these inhibitors may bind to target proteinases independent of the two rigid hairpin loops forming the remainder of the inhibitory surface. In an attempt to demonstrate such two‐step binding, which could not be identified in previous kinetics studies, we introduced a cysteine residue before the N‐terminus of cystatin A and labeled this residue with fluorescent probes. Binding of AANS‐ and AEDANS‐labeled cystatin A to papain resulted in ∼4‐fold and 1.2‐fold increases of probe fluorescence, respectively, reflecting the interaction of the N‐terminal region with the enzyme. Observed pseudo‐first‐order rate constants, measured by the loss of papain activity in the presence of a fluorogenic substrate, for the reaction of the enzyme with excess AANS‐cystatin A increased linearly with the concentration of the latter. In contrast, pseudo‐first‐order rate constants, obtained from measurements of the change of probe fluorescence with either excess enzyme or labeled inhibitor, showed an identical hyperbolic dependence on the concentration of the reactant in excess. This dependence demonstrates that the binding occurs in two steps, and implies that the labeled N‐terminal region of cystatin A interacts with the proteinase in the second step, subsequent to the hairpin loops. The comparable affinities and dissociation rate constants for the binding of labeled and unlabeled cystatin A to papain indicate that the label did not appreciably perturb the interaction, and that unlabeled cystatin therefore also binds in a similar two‐step manner. Such independent binding of the N‐terminal regions of cystatins to target proteinases after the hairpin loops may be characteristic of most cystatin–proteinase reactions.
Phosphoryl transfer by a concerted reaction mechanism in UMP/CMP‐kinase
Authors: Hutter Michael C. , Helms Volkhard
Published: February 2009

The reaction mechanism of phosphoryl transfer catalyzed by UMP/CMP‐kinase from Dictyostelium discoideum was investigated by semiempirical AM1 molecular orbital computations of an active site model system derived from crystal structures that contain a transition state analog or a bisubstrate inhibitor. The computational results suggest that the nucleoside monophosphate must be protonated for the forward reaction while it is unprotonated in the presence of aluminium fluoride, a popular transition state analog for phosphoryl transfer reactions. Furthermore, a compactification of the active site model system during the reaction and for the corresponding complex containing AlF3 was observed. For the active site residues that are part of the LID domain, conformational flexibility during the reaction proved to be crucial. On the basis of the calculations, a concerted phosphoryl transfer mechanism is suggested that involves the synchronous shift of a proton from the monophosphate to the transferred PO3‐group. The proposed mechanism is thus analogous to the phosphoryl transfer mechanism in cAMP‐dependent protein kinase that phosphorylates the hydroxyl groups of serine residues.