bluuebunny/crossref_metadata_2025_split huggingface dataset:
Derivation of 3D coordinate templates for searching structural databases: Application to ser‐His‐Asp catalytic triads in the serine proteinases and lipases
Authors: Wallace Andrew C. , Laskowski Roman A. , Thornton Janet M. 
Published: July 2010

AbstractIt is well established that sequence templates (e.g., PROSITE) and databases are powerful tools for identifying biological function and tertiary structure for an unknown protein sequence. Here we describe a method for automatically deriving 3D templates from the protein structures deposited in the Brookhaven Protein Data Bank. As an example, we describe a template derived for the Ser‐His‐Asp catalytic triad found in the serine proteases and triacylglycerol lipases. We find that the resultant template provides a highly selective tool for automatically differentiating between catalytic and noncatalytic Ser‐His‐Asp associations. When applied to nonproteolytic proteins, the template picks out two “non‐esterase” catalytic triads that may be of biological relevance. This suggests that the development of databases of 3D templates, such as those that currently exist for protein sequence templates, will help identify the functions of new protein structures as they are determined and pinpoint their functionally important regions.
Intrinsic tryptophans of CRABPI as probes of structure and folding
Authors: Clark Patricia L. , Liu Zhi‐Ping , Zhang Jianhua , Gierasch Lila M. 
Published: July 2010

AbstractThe native state fluorescence and CD spectra of the predominantly β‐sheet cellular retinoic acid‐binding protein I (CRABPI) include contributions from its three tryptophan residues and are influenced by the positions of these residues in the three‐dimensional structure. Using a combination of spectroscopic approaches and single Trp‐mutants of CRABPI, we have deconvoluted these spectra and uncovered several features that have aided in our analysis of the development of structure in the folding pathway of CRABPI. The emission spectrum of native CRABPI is dominated by Trp 7. Trp 109 is fluorescence‐silent due to its interaction with the guanidino group of Arg 111. Although the far‐UV CD spectrum of CRABPI is largely determined by the protein's secondary structure, aromatic clustering around Trp 87 and the aromatic‐charge interaction between Arg 111 and Trp 109 give rise to a characteristic feature in the CD spectrum at 228 nm. The near‐UV CD bands of CRABPI arise largely from additive contributions of the three tryptophan residues. Trp 7 and Trp 87 give a negative CD band at 275 nm. The near‐UV CD band from Trp 109 is positive and shifted to longer wavelengths (to 302 nm) due to the charge‐aromatic interaction between Arg 111 and Trp 109. Our deconvolution of the equilibrium spectra have been used to interpret kinetic folding experiments monitored by stopped‐flow fluorescence. These dynamic experiments suggest the early evolution of a well‐populated, hydrophobically collapsed intermediate, which undergoes global rearrangement to form the fully folded structure. The results presented here suggest several additional strategies for dissecting the folding pathway of CRABPI.
Crystal structure of two new bifunctional nonsubstrate type thrombin inhibitors complexed with human α‐thrombin
Authors: Féthière J. , Tsuda Yuko , Coulombe René , Konishi Yasuo , Cygler Miroslaw 
Published: July 2010

AbstractThe crystal structures of two new thrombin inhibitors, P498 and P500, complexed with human α‐thrombin have been determined at 2.0 Å resolution and refined to crystallographic R‐factors of 0.170 and 0.169, respectively. These compounds, with picomolar binding constants, belong to a family of potent bifunctional inhibitors that bind thrombin at two remote sites: the active site and the fibrinogen recognition exosite (FRE). The inhibitors incorporate a nonsubstrate type active site binding fragment: Dansyl‐Arg‐(D)Pipecolic acid (Dns‐Arg‐(D)Pip), reminiscent of the active‐site directed inhibitors MD‐805 and MQPA, rendering them resistant to thrombin‐induced hydrolysis. The FRE binding fragment of these inhibitors corresponds to the hirudin55–65 sequence. They differ in the chemical nature of the nonpeptidyl linker bridging these two functional activities. In both cases, the active site binding fragment is well defined in the electron density. The DnsH1, ArgH2, and (D)PipH3 groups occupy the S3, S1, and S2 subsites of thrombin, respectively, in a way similar to that observed in the thrombin‐MQPA complexes. Binding in the active site of thrombin is characterized by numerous van der Waals contacts and ring‐ring system interactions. Unlike in the substrate‐like inhibitors, ArgH2 enters the S1 specificity pocket from the P2 position and adopts a bent conformation to make an hydrogen bond to the carboxylate of Asp189. In this noncanonical position, its carbonyl points away from the oxyanion hole, which is now occupied by well‐ordered solvent molecules. The linkers fit in the groove extending from the active site to the FRE. The C‐terminal fragments of both inhibitors bind in the same way as analogous FRE binding elements in previously described complexes.
Tumor marker disaccharide D‐Gal‐β1,3‐GalNAc complexed to heat‐labile enterotoxin from Escherichia coli
Authors: Akker Focco Van Den , Steensma Elles , Hol Wim G. J. 
Published: July 2010

AbstractHeat‐labile enterotoxin (LT) is part of the cholera toxin (CT) family and consists of a catalytic A subunit and a B pentamer that serves to recognize the oligosaccharide part of the GM1 ganglioside receptor. We report here the crystal structure of heat‐labile enterotoxin in complex with the disaccharide portion of the Thomsen‐Friedenreich (T‐antigen) tumor marker. The toxin:carbohydrate complex is determined to 2.13 A resolution, yielding an R‐factor of 18.5%. The T‐antigen disaccharide, D‐Gal‐β1,3‐GalNAc‐Ser/Thr, is present in more than 85% of human carcinomas and monitoring its autoimmune response is used for the early detection of tumors. Insight into the molecular recognition of this tumor antigen by sugar binding proteins can benefit the development of a diagnostic tool for human carcinomas as well as a T‐antigen directed anticancer drug delivery system.
The crystal structure of the designed trimeric coiled coil coil‐VaLd: Implications for engineering crystals and supramolecular assemblies
Authors: Ogihara Nancy L. , Weiss Manfred S. , Eisenberg David , Degrado William F. 
Published: July 2010

AbstractThe three‐dimensional structure of the 29‐residue designed coiled coil having the amino acid sequence acetyl‐E VEALEKK VAALESK VQALEKK VEALEHG‐amide has been determined and refined to a crystallographic R‐factor of 21.4% for all data from 10‐Å to 2.1‐Å resolution. This molecule is called coil‐VaLd because it contains valine in the a heptad positions and leucine in the d heptad positions. In the trigonal crystal, three molecules, related by a crystallographic threefold axis, form a parallel three‐helix bundle. The bundles are stacked head‐to‐tail to form a continuous coiled coil along the c‐direction of the crystal. The contacts among the three helices within the coiled coil are mainly hydrophobic: four layers of valine residues alternate with four layers of leucine residues to form the core of the bundle. In contrast, mostly hydrophilic contacts mediate the interaction between trimers: here a total of two direct protein‐protein hydrogen bonds are found. Based on the structure, we propose a scheme for designing crystals of peptides containing continuous two‐, three‐, and four‐stranded coiled coils.
Importance of the release of strand 1C to the polymerization mechanism of inhibitory serpins
Authors: Chang Wun‐Shaing W. , Whisstock James , Hopkins Paul C.R. , Lesk Arthur M. , Carrell Robin W. , Wardell Mark R. 
Published: July 2010

AbstractSerpin polymerization is the underlying cause of several diseases, including thromboembolism, emphysema, liver cirrhosis, and angioedema. Understanding the structure of the polymers and the mechanism of polymerization is necessary to support rational design of therapeutic agents. Here we show that polymerization of antithrombin is sensitive to the addition of synthetic peptides that interact with the structure. A 12‐mer peptide (homologous to P14‐P3 of antithrombin reactive loop), representing the entire length of s4A, prevented polymerization totally. A 6‐mer peptide (homologous to P14‐P9 of antithrombin) not only allowed polymerization to occur, but induced it. This effect could be blocked by the addition of a 5‐mer peptide with the s1C sequence of antithrombin or by an unrelated peptide representing residues 26–31 of cholecystokinin. The s1C or cholecystokinin peptide alone was unable to form a complex with native antithrombin. Moreover, an active antitrypsin double mutant, Pro 361 → Cys, Ser 283 → Cys, was engineered for the purpose of forming a disulfide bond between s1C and s2C to prevent movement of s1C. This mutant was resistant to polymerization if the disulfide bridge was intact, but, under reducing conditions, it regained the potential to polymerize. We have also modeled long‐chain serpin polymers with acceptable stereochemistry using two previously proposed loop‐A‐sheet and loop‐C‐sheet polymerization mechanisms and have shown both to be sterically feasible, as are “mixed” linear polymers. We therefore conclude that the release of strand 1C must be an element of the mechanism of serpin polymerization.
Heteronuclear (1H, 13C, 15N) NMR assignments and secondary structure of the basic region‐helix‐loop‐helix domain of E47
Authors: Fairman Robert , Beran‐Steed Rita K. , Handel Tracy M. 
Published: July 2010

AbstractE47 is an immunoglobulin enhancer DNA‐binding protein that contains a basic region‐helix‐loop‐helix (b/HLH) domain. This structural motif defines a class of transcription factors that are central to the developmental regulation of many tissues. Its function is to provide a dimerization interface through the formation of a parallel four‐helix bundle, resulting in the juxtaposition of two basic DNA‐recognition α‐helices that control sequence‐specific DNA‐binding. In order to gain insight into the biophysical nature of b/HLH domains, we have initiated structural studies of the E47 homodimer by NMR. Sequence‐specific resonance assignments have been obtained using a combination of heteronuclear double‐ and triple‐resonance NMR experiments. The secondary structure was deduced from characteristic patterns of NOEs, 13Cα/β chemical shifts, and measurements of 3JHNHα scalar couplings. Except for the basic region recognition helix, the secondary structural elements of the E47 homodimer are preserved in the absence DNA when compared with the co‐crystal structure of E47 bound to DNA (Ellenberger T, Fass D, Arnaud M, Harrison SC, 1994, Genes & Dev 8:970–980). As expected, the DNA‐binding helix is largely unstructured, but does show evidence of nascent helix formation. The HLH region of E47 is structured, but highly dynamic as judged by the rapid exchange of backbone hydrogen atoms and the relatively weak intensities of many of the NOEs defining the dimerization helices. This dynamic nature may be relevant to the ability of E47 both to homodimerize and to heterodimerize with MyoD, Id, and Tal1.
β‐Turn propensities as paradigms for the analysis of structural motifs to engineer protein stability
Authors: Ohage Ettore C. , Graml Werner , Walter Monika M. , Steipe Boris , Steinbacher Stefan 
Published: July 2010

AbstractThe thermodynamic stability of a protein provides an experimental metric for the relationship of protein sequence and native structure. We have investigated an approach based on an analysis of the structural database for stability engineering of an immunoglobulin variable domain. The most frequently occurring residues in specific positions of β‐turn motifs were predicted to increase the folding stability of mutants that were constructed by site‐directed mutagenesis. Even in positions in which different residues are conserved in immunoglobulin sequences, the predictions were confirmed. Frequently, mutants with increased β‐turn propensities display increased folding cooperativities, suggesting pronounced effects on the unfolded state independent of the expected effect on conformational entropy. We conclude that structural motifs with predominantly local interactions can serve as templates with which patterns of sequence preferences can be extracted from the database of protein structures. Such preferences can predict the stability effects of mutations for protein engineering and design.
Crystallization and preliminary crystallographic analysis of recombinant human p38 MAP kinase
Authors: Pav S. , White D.M. , Rogers S. , Crane K.M. , Cywin C.L. , Hopkins J. , Brown M.L. , Pargellis C.A. , Tong L. , Davidson W. 
Published: July 2010

AbstractThe recombinant human p38 MAP kinase has been expressed and purified from both Escherichia coli and SF9 cells, and has been crystallized in two forms by the hanging drop vapor diffusion method using PEG as precipitant. Both crystal forms belong to space group P212121. The cell parameters for crystal form 1 are a = 65.2 Å, b = 74.6 Å and c = 78.1 Å. Those for crystal form 2 are a = 58.3 Å, b = 68.3 Å and c = 87.9 Å. Diffraction data to 2.0 Å resolution have been collected on both forms.
SAM as a protein interaction domain involved in developmental regulation
Authors: Schultz Jörg , Bork Peer , Ponting Christopher P. , Hofmann Kay 
Published: July 2010

AbstractMore than 60 previously undetected SAM domain‐containing proteins have been identified using profile searching methods. Among these are over 40 EPH‐related receptor tyrosine kinases (RPTK), Drosophila bicaudal‐C, a p53 from Loligo forbesi, and diacylglycerol‐kinase isoform δ. This extended dataset suggests that SAM is an evolutionary conserved protein binding domain that is involved in the regulation of numerous developmental processes among diverse eukaryotes. A conserved tyrosine in the SAM sequences of the EPH related RPTKs is likely to mediate cell‐cell initiated signal transduction via the binding of SH2 containing proteins to phosphotyrosine.
A calbindin D9k mutant containing a novel structural extension: 1H nuclear magnetic resonance studies
Authors: Groves Patrick , Linse Sara , Thulin Eva , Forsen Sture 
Published: July 2010

AbstractCalbindin D9k is a small, well‐studied calcium‐binding protein consisting of two helix‐loop‐helix motifs called EF‐hands. The P43MG2 mutant is one of a series of mutants designed to sequentially lengthen the largely unstructured tether region between the two EF‐hands (F36‐S44). A lower calcium affinity for P43MG was expected on the basis of simple entropie arguments. However, this is not the case and P43MG (‐97 kJ‐mol−1) has a stronger calcium affinity than P43M (‐93 Kj‐mol−1), P43G (‐95 kJ‐mol−1) and even wild‐type protein (‐96 kj‐mol−1). An NMR study was initiated to probe the structural basis for these calcium‐binding results. The 1H NMR assignments and 3JHNHα values of the calcium‐free and calcium‐bound forms of P43MG calbindin D9k mutant are compared with those of P43G. These comparisons reveal that little structure is formed in the tether regions of P43MG(apo), P43G(apo) and P43G(Ca) but a helical turn (S38‐K41) appears to stabilize this part of the protein structure for P43MG(Ca). Several characteristic NOEs obtained from 2D and 3D NMR experiments support this novel helix. A similar, short helix exists in the crystal structure of calcium‐bound wild‐type calbindin D9k‐—but this is the first observation in solution for wild‐type calbindin D9k or any of its mutants.
Backbone and side‐chain dynamics of residues in a partially folded β‐sheet peptide from platelet factor‐4
Authors: Daragan Vladimir A. , Ilyina Elena E. , Fields Cynthia G. , Fields Gregg B. , Mayo Kevin H. 
Published: July 2010

AbstractStructurally characterizing partially folded states is problematic given the nature of these transient species. A peptide 20mer, T38AQLIATLKNGRKISLDLQA57 (P20), which has been shown to partially fold in a relatively stable turn/loop conformation (LKNGR) and transient β‐sheet structure, is a good model for studying backbone and side‐chain mobilities in a transiently folded peptide by using l3C‐NMR relaxation. Here, four residues in P20, A43, T44, G48, and 151, chosen for their positions in or near the loop conformation and for compositional variety, have been selectively 13C‐enriched. Proton‐coupled and decoupled 13C‐NMR relaxation experiments have been performed to obtain the temperature dependencies (278 K to 343 K) of auto‐ and cross‐correlation motional order parameters and correlation times. In order to differentiate sequence‐neighbor effects from folding effects, two shorter peptides derived from P20, IATLK (P5) and NGRKIS (P6), were similarly l3C‐enriched and investigated. For A43, T44, G48, and 151 residues in P20 relative to those in P5/P6, several observations are consistent with partial folding in P20: (1) CαH motional tendencies are all about the same, vary less with temperature, and are relatively more restricted, (2) G48 CαH2 ϕ(t),Ψ(t) rotations are more correlated, and (3) methyl group rotations are slower and yield lower activation energies consistent with formation of hydrophobic “pockets.” In addition, T44 and 151 CβH mobilities in P20 are more restricted at lower temperature than those of their CαH and display significantly greater sensitivity to temperature suggesting a larger enthalpic contribution to side‐chain mobility. Moreover, at higher temperatures, side‐chain methyls and methylenes in P20 are more motionally restricted than those in P5/P6, suggesting that some type of “folded” or “collapsed” structure remains in P20 for what normally would be considered an “unfolded” state.
The role of helix VIII in the lactose permease ofEscherichia coli: I. Cys‐scanning mutagenesis
Authors: Frillingos Stathis , Ujwal M.L. , Sun Jianzhong , Kaback H. Ronald 
Published: July 2010

AbstractUsing a functional lactose permease mutant devoid of Cys residues (C‐less permease), each amino acid residue in transmembrane domain VIII and flanking hydrophilic loops (from Gin 256 to Lys 289) was replaced individually with Cys. Of the 34 single‐Cys mutants, 26 accumulate lactose to >70% of the steady state observed with C‐less permease, and an additional 7 mutants (Gly 262 → Cys, Gly 268 → Cys, Asn 272 → Cys, Pro 280 → Cys, Asn 284 → Cys, Gly 287 → Cys, and Gly 288 → Cys) exhibit lower but significant levels of accumulation (30‐50% of C‐less). As expected (Ujwal ML, Sahin‐Toth M, Persson B, Kaback HR, 1994,Mol Membr Biol1:9–16), Cys replacement for Glu 269 abolishes lactose transport. Immunoblot analysis reveals that the mutants are inserted into the membrane at concentrations comparable to C‐less permease, with the exceptions of mutants Pro 280 → Cys, Gly 287 → Cys, and Lys 289 → Cys, which are expressed at reduced levels. The transport activity of the mutants is inhibited byN‐ethylmaleimide (NEM) in a highly specific manner. Most of the mutants are insensitive, but Cys replacements render the permease sensitive to inactivation by NEM at positions that cluster in manner indicating that they are on one face of an α‐helix (Gly 262 → Cys, Val 264 → Cys, Thr 265 → Cys, Gly 268 → Cys, Asn 272 → Cys, Ala 273 → Cys, Met 276 → Cys, Phe 277 → Cys, and Ala 279 → Cys). The results indicate that transmembrane domainVIIIis in α‐helical conformation and demonstrate that, although only a single residue in this region of the permease is essential for activity (Glu 269), one face of the helix plays an important role in the transport mechanism. More direct evidence for the latter conclusion is provided in the companion paper (Frillingos S, Kaback HR, 1997,Protein Sci6:438–443) by using site‐directed sulfhydryl modification of the Cys‐replacement mutants in situ.
Importance of van der Waals contact between Glu 35 and Trp 109 to the catalytic action of human lysozyme
Authors: Muraki Michiro , Goda Shuichiro , Nagahora Hitoshi , Harata Kazuaki 
Published: July 2010

AbstractThe importance of van der Waals contact between Glu 35 and Tip 109 to the active‐site structure and the catalytic properties of human lysozyme (HL) has been investigated by site‐directed mutagenesis. The X‐ray analysis of mutant HLs revealed that both the replacement of Glu 35 by Asp or Ala, and the replacement of Trp 109 by Phe or Ala resulted in a significant but localized change in the active‐site cleft geometry. A prominent movement of the backbone structure was detected in the region of residues 110 to 120 and in the region of residues 100 to 115 for the mutations concerning Glu 35 and Trp 109, respectively. Accompanied by the displacement of the main‐chain atoms with a maximal deviation of Ca atom position ranging from 0.7 Å to 1.0 Å, the mutant HLs showed a remarkable change in the catalytic properties against Micrococcus luteus cell substrate as compared with native HL. Although the replacement of Glu 35 by Ala completely abolished the lytic activity, HL‐Asp 35 mutant retained a weak but a certain lytic activity, showing the possible involvement of the side‐chain carboxylate group of Asp 35 in the catalytic action. The kinetic consequence derived from the replacement of Trp 109 by Phe or Ala together with the result of the structural change suggested that the structural detail of the cleft lobe composed of the residues 100 to 115 centered at Ala 108 was responsible for the turnover in the reaction of HL against the bacterial cell wall substrate. The results revealed that the van der Waals contact between Glu 35 and Trp 109 was an essential determinant in the catalytic action of HL.
Crystallization and preliminary structural analysis of catalase A from Saccharomyces cerevisiae
Authors: Berthet Serge , Bravo Jeronimo , Mate Maria J. , Fita Ignacio , Nykyri Leena M. , Koller Franz , Berthet‐Colominas Carmen , Alzari Pedro M. 
Published: July 2010

AbstractYeast peroxisomal catalase A, obtained at high yields by over expression of the C‐terminally modified gene from a 2μ‐plasmid, has been crystallized in a form suitable for high resolution X‐ray diffraction studies. Brownish crystals with bipyrimidal morphology and reaching ca. 0.8 mm in size were produced by the hanging drop method using ammonium sulphate as precipitant. These crystals diffract better than 2.0 Å resolution and belong to the hexagonal space group P6122 with unit cell parameters a = b = 184.3 Å and c = 305.5 Å. An X‐ray data set with 76% completeness at 3.2 Å resolution was collected in a rotating anode generator using mirrors to improve the collimation of the beam. An initial solution was obtained by molecular replacement only when using a beef liver catalase tetramer model in which fragments with no sequence homology had been omitted, about 150 residues per subunit. In the structure found a single molecule of catalase A (a tetramer with accurate 222 molecular symmetry) is located in the asymmetric unit of the crystal with an estimated solvent content of about 61%. The preliminary analysis of the structure confirms the absence of a carboxy terminal domain as the one found in the catalase from Penicillium vitalae, the only other fungal catalase structure available. The NADPH binding site appears to be involved in crystal contacts, suggesting that heterogeneity in the occupancy of the nucleotide can be a major difficulty during crystallization.
Architecture of helix bundle membrane proteins: An analysis of cytochrome c oxidase from bovine mitochondria
Authors: Wallin Erik , Tsukihara Tomitake , Yoshikawa Shinya , Heijne Gunnar Von , Elofsson Arne 
Published: July 2010

AbstractWe have analyzed the structure of mitochondrial cytochrome c oxidase in terms of general characteristics thought to be important for describing the architecture of helix bundle membrane proteins. Many aspects of the structure are similar to what has previously been found for the photosynthetic reaction center and bacteriorhodopsin. Our results lead to a considerably more precise general picture of membrane protein architecture than has hitherto been possible to obtain.