bluuebunny/crossref_metadata_2025 huggingface dataset:
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

The 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

The 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

Heat‐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

The 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

Serpin 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

E47 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

The 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

The 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

More 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

Calbindin 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

Structurally 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

Using 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

The 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

Yeast 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

We 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.
Protein conformer selection by ligand binding observed with crystallography
Authors: Cao Yi , Musah Rabi A. , Wilcox Sheri K. , Goodin David B. , McRee Duncan E.
Published: July 2010

A large‐scale movement between “closed” and “open” conformations of a protein loop was observed directly with protein crystallography by trapping individual conformers through binding of an exogenous ligand and characterization with solution kinetics. The buried indole ring of Trp191 in cytochrome c peroxidase (CCP) was displaced by exogenous ligands, causing a conformational change of loop Pro190‐Asn195 and exposing Trp191 to the protein surface. Kinetic measurements are consistent with a two‐step binding mechanism in which the rate‐limiting step is a transition of the protein to the open state, which then binds the ligand. This large‐scale conformational change of a functionally important region of CCP is independent of ligand and indicates that about 4% of the wild‐type protein is in the open form in solution at any given time.
Prime region subsite specificity characterization of human cathepsin D: The dominant role of position 128
Authors: Beyer Brian M. , Dunn Ben M.
Published: July 2010

In order to contribute to our understanding of cathepsin D (CatD) active site specificity, two series of chromogenic octapeptides with systematic substitutions in positions P'2 and P'3 were synthesized. This panel was characterized with native human liver cathepsin D (nHuCatD) and yielded information concerning specificity trends within the S'2 and S'3 subsites. The pepstatin inhibited crystal structure of nHuCatD (Baldwin et al., 1993) was then utilized in conjunction with these subsite preference data to identify residues suspected of contributing to “prime” side subsite specificity. These residues were targeted for site‐directed mutagenesis using the re‐engineered recombinant model, “short” pseudocathepsin D (Beyer & Dunn, 1996). As a result of these analyses it was determined that prime region subsites do contribute to the unique specificity of human CatD. Furthermore, it was ascertained that the poly‐proline loop does not have an active role in S'3 subsite specificity. Lastly, it appears that Ile 128 has a dominant role on S'2 subsite specificity whereas Val130 does not.
NMR studies of internal dynamics of serine proteinase protein inhibitors: Binding region mobilities of intact and reactive‐site hydrolyzed Cucurbita maxima trypsin inhibitor (CMTI)‐III of the squash family and comparison with those of counterparts of CMTI‐V of the potato I family
Authors: Liu Jianhua , Gong Yuxi , Prakash Om , Krishnamoorthi Ramaswamy , Wen Lisa , Lee Insuk , Huang Jenq‐Kuen
Published: July 2010

Serine proteinase protein inhibitors follow the standard mechanism of inhibition (Laskowski M Jr, Kato I, 1980, Annu Rev Biochem 49593‐626), whereby an enzyme‐catalyzed equilibrium between intact (I) and reactive‐site hydrolyzed inhibitor (I*) is reached. The hydrolysis constant, Khyd is defined as [I*]/[I]. Here, we explore the role of internal dynamics in the resynthesis of the scissile bond by comparing the internal mobility data of intact and cleaved inhibitors belonging to two different families. The inhibitors studied are recombinant Cucurbita maxima trypsin inhibitor III (rCMTI‐III; Mr 3 kDa) of the squash family and rCMTI‐V (Mr ∼ 7 kDa) of the potato I family. These two inhibitors have different binding loop‐scaffold interactions and different Khyd values—2.4 (CMTI‐III) and 9 (CMTI‐V)—at 25°C. The reactive‐site peptide bond (P1‐P'1) is that between Arg5 and Ile6 in CMTI‐III, and that between Lys44 and Asp45 in CMTI‐V. The order parameters (S2) of backbone NHs of uniformly 15N‐labeled rCMTI‐III and rCMTI‐III* were determined from measurements of 15N spin‐lattice and spin‐spin relaxation rates, and {1H}‐15N steady‐state heteronuclear Overhauser effects, using the model‐free formalism, and compared with the data reported previously for rCMTI‐V and rCMTI‐V*. The backbones of rCMTI‐III (〈S2〉 = 0.71) and rCMTI‐III* (〈S2) = 0.63) are more flexible than those of rCMTI‐V (〈S2〉 = 0.83) and rCMTI‐V* (〈S2) = 0.85). The binding loop residues, P4‐P1, in the two proteins show the following average order parameters: 0.57 (rCMTI‐III) and 0.44 (rCMTI‐III*); 0.70 (rCMTI‐V) and 0.40 (rCMTI‐V*). The P1′‐P4′ residues, on the other hand, are associated with (S2) values of 0.56 (rCMTI‐III) and 0.47 (rCMTI‐III*); and 0.73 (rCMTI‐V) and 0.83 (rCMTI‐V*). The newly formed C‐terminal (Pn residues) gains a smaller magnitude of flexibility in rCMTI‐III* due to the Cys3‐Cys20 crosslink. In contrast, the newly formed N‐terminal (Pn′ residues) becomes more flexible only in rCMTI‐III*, most likely due to lack of an interaction between the P1′ residue and the scaffold in rCMTI‐III. Thus, diminished flexibility gain of the Pn residues and, surprisingly, increased flexibility of the Pn′ residues seem to facilitate the resynthesis of the P1‐P1′ bond, leading to a lower Khyd value.
The response of T4 lysozyme to large‐to‐small substitutions within the core and its relation to the hydrophobic effect
Authors: Xu Jian , Baase Walter A. , Baldwin Enoch , Matthews Brian W.
Published: July 2010

To further examine the structural and thermodynamic basis of hydrophobic stabilization in proteins, all of the bulky non‐polar residues that are buried or largely buried within the core of T4 lysozyme were substituted with alanine. In 25 cases, including eight reported previously, it was possible to determine the crystal structures of the variants. The structures of four variants with double substitutions were also determined. In the majority of cases the “large‐to‐small” substitutions lead to internal cavities. In other cases declivities or channels open to the surface were formed. In some cases the structural changes were minimal (mainchain shifts ≤ 0.3 Å); in other cases mainchain atoms moved up to 2 Å. In the case of Ile 29 → Ala the structure collapsed to such a degree that the volume of the putative cavity was zero. Crystallographic analysis suggests that the occupancy of the engineered cavities by solvent is usually low. The mutants Val 149 → Ala (V149A) and Met 6 → Ala (M6A), however, are exceptions and have, respectively, one and two well‐ordered water molecules within the cavity. The Val 149 → Ala substitution allows the solvent molecule to hydrogen bond to polar atoms that are occluded in the wild‐type molecule. Similarly, the replacement of Met 6 with alanine allows the two solvent molecules to hydrogen bond to each other and to polar atoms on the protein. Except for Val 149 → Ala the loss of stability of all the cavity mutants can be rationalized as a combination of two terms. The first is a constant for a given class of substitution (e.g., − 2.1 kcal/mol for all Leu → Ala substitutions) and can be considered as the difference between the free energy of transfer of leucine and alanine from solvent to the core of the protein. The second term can be considered as the energy cost of forming the cavity and is consistent with a numerical value of 22 cal mol−1 Å−3, Physically, this term is due to the loss of van der Waal's interactions between the bulky sidechain that is removed and the atoms that form the wall of the cavity. The overall results are consistent with the prior rationalization of Leu → Ala mutants in T4 lysozyme by Eriksson et al. (Eriksson et al., 1992, Science 255:178‐183).
Fragment of GABAAreceptor containing key ligand‐binding residues overexpressed inEscherichia coli
Authors: Xue Hong , Chu Ruiai , Hang Jun , Lee Peggy , Zheng Hui
Published: July 2010

GABAAreceptor plays a major role in inhibitory synaptic transmission in the central nervous system and is the target of drugs such as the benzodiazepine tranquilizers. The polymeric membrane protein nature of GABAAreceptor has rendered structural elucidation of the receptor a formidable task, greatly hampering structure‐based drug design. We report here the first expression inEscherichia coliof a fragment of GABAAreceptor. This 131‐ residue fragment, spanning Cys166 to Leu296 of human GABAAreceptor α1 subunit, contains residues previously suggested to be involved in benzodiazepine binding. The overexpressed nonfusion recombinant protein was purified to near homogeneity and characterized by circular dichroism (CD), which showed that the recombinant protein has well defined secondary structures where β‐strands are dominant. The stability of the secondary structures was demonstrated by CD spectra at high pH and elevated temperature. Excluding part of the sequences from the carboxyl terminal of the fragment resulted in dramatic changes in the secondary structures comparable to the effects caused by SDS denaturation. Our results therefore suggest that the 131‐residue fragment harbors an integral structural domain of the receptor. The overexpression of the recombinant protein fragment thus opens the way to the biochemical and structural studies of a functionally important region of the receptor, and exemplifies an effective approach of expression and characterization that potentially may be extended to other members of the ligand gated channel receptor superfamily, to which the GABAAreceptor belongs.
Domain assignment for protein structures using a consensus approach: Characterization and analysis
Authors: Jones Susan , Stewart Michael , Michie Alex , Swindells Mark B. , Orengo Chirstine , Thornton Janet M.
Published: July 2010

A consensus approach for the assignment of structural domains in proteins is presented. The approach combines a number of previously published algorithms, and takes advantage of the elevated accuracy obtained when assignments from the individual algorithms are in agreement. The consensus approach is tested on a data set of 55 protein chains, for which domain assignments from four automated methods were known, and for which crystallographers assignments had been reported in the literature. Accuracy was found to increase in this test from 72% using individual algorithms to 100% when all four methods were in agreement. However a consensus prediction using all four methods was only possible for 52% of the dataset. The consensus approach (using three publicly available domain assignment algorithms (PUU, DETECTIVE, DOMAK)) was then used to make domain assignments for a data set of 787 protein chains from the Protein Data Bank. Analysis of the assignments showed 55.7% of assignments could be made automatically and of these, 13.5% were multi‐domain proteins. Of the remaining 44.3% that could not be assigned by the consensus procedure 90.4% had their domain boundaries assigned correctly by at least one of the algorithms. Once identified, these domains were analyzed for trends in their size and secondary structure class. In addition, the discontinuity of each domain along the protein chain was considered.
An interaction‐based analysis of calcium‐induced conformational changes in Ca2+ sensor proteins
Authors: Nelson Melanie R. , Chazin Walter J.
Published: July 2010

Calcium sensor proteins translate transient increases in intracellular calcium levels into metabolic or mechanical responses, by undergoing dramatic conformational changes upon Ca2+ binding. A detailed analysis of the calcium binding‐induced conformational changes in the representative calcium sensors calmodulin (CaM) and troponin C was performed to obtain insights into the underlying molecular basis for their response to the binding of calcium. Distance difference matrices, analysis of interresidue contacts, comparisons of interhelical angles, and inspection of structures using molecular graphics were used to make unbiased comparisons of the various structures. The calcium‐induced conformational changes in these proteins are dominated by reorganization of the packing of the four helices within each domain. Comparison of the closed and open conformations confirms that calcium binding causes opening within each of the EF‐hands. A secondary analysis of the conformation of the C‐terminal domin of CaM (CaM‐C) clearly shows that CaM‐C occupies a closed conformation in the absence of calcium that is distinct from the semi‐open conformation observed in the C‐terminal EF‐hand domains of myosin light chains. These studies provide insight into the structural basis for these changes and into the differential response to calcium binding of various members of the EF‐hand calcium‐binding protein family. Factors contributing to the stability of the Ca2+‐loaded open conformation are discussed, including a new hypothesis that critical hydrophobic interactions stabilize the open conformation in Ca2+ sensors, but are absent in “non‐sensor” proteins that remain closed upon Ca2+ binding. A role for methionine residues in stabilizing the open conformation is also proposed.
Structures of adamalysin II with peptidic inhibitors. Implications for the design of tumor necrosis factor α convertase inhibitors
Authors: Xavier Gomis‐Rüth F. , Meyer Edgar F. , Kress Lawrence F. , Politi Vincenzo
Published: July 2010

Crotalus adamanteus snake venom adamalysin II is the structural prototype of the adamalysin or ADAM family comprising proteolytic domains of snake venom metalloproteinases, multimodular mammalian reproductive tract proteins, and tumor necrosis factor α convertase, TACE, involved in the release of the inflammatory cytokine, TNFα. The structure of adamalysin II in noncovalent complex with two small‐molecule right‐hand side peptidomimetic inhibitors (Pol 647 and Pol 656) has been solved using X‐ray diffraction data up to 2.6 and 2.8 Å resolution. The inhibitors bind to the ‐side of the proteinase, inserting between two protein segments, establishing a mixed parallel‐antiparallel three‐stranded β‐sheet and coordinate the central zinc ion in a bidentate manner via their two C‐terminal oxygen atoms. The proteinase‐inhibitor complexes are described in detail and are compared with other known structures. An adamalysin‐based model of the active site of TACE reveals that these small molecules would probably fit into the active site cleft of this latter metalloproteinase, providing a starting model for the rational design of TACE inhibitors.
Trifluoroethanol effects on helix propensity and electrostatic interactions in the helical peptide from ribonuclease T1
Authors: Myers Jeffrey K. , Nick Pace C. , Martin Scholtz J.
Published: July 2010

Trifluoroethanol (TFE) is often used to increase the helicity of peptides to make them usable as models of helices in proteins. We have measured helix propensities for all 20 amino acids in water and two concentrations of trifluoroethanol, 15 and 40% (v/v) using, as a model system, a peptide derived from the sequence of the α‐helix of ribonuclease T1. There are three main conclusions from our studies. (1) TFE alters electrostatic interactions in the ribonuclease T1 helical peptide such that the dependence of the helical content on pH is lost in 40% TFE. (2) Helix propensities measured in 15% TFE correlate well with propensities measured in water, however, the correlation with propensities measured in 40% TFE is significantly worse. (3) Propensities measured in alanine‐based peptides and the ribonuclease T1 peptide in TFE show very poor agreement, revealing that TFE greatly increases the effect of sequence context.
The early folding kinetics of apomyoglobin
Authors: Pappu Rohit V. , Weaver David L.
Published: July 2010

The folding pathway of apomyoglobin has been experimentally shown to have early kinetic intermediates involving the A, B, G, and H helices. The earliest detected kinetic events occur on a ns to μis time scale. We show that the early folding kinetics of apomyoglobin may be understood as the association of nascent helices through a network of diffusioncollision‐coalescence steps G + H GH + A AGH + B ABGH obtained by solving the diffusion‐collision model in a chemical kinetics approximation. Our reproduction of the experimental results indicates that the model is a useful way to analyze folding data. One prediction from our fit is that the nascent A and H helices should be relatively more helix‐like before coalescence than the other apomyoglobin helices.
High level, context dependent misincorporation of lysine for arginine in Saccharomyces cerevisiae al homeodomain expressed in Escherichia coli
Authors: Forman Michael D. , Stack Robert F. , Masters Paul S. , Hauer Charles R. , Baxter Susan M.
Published: July 2010

The Saccharomyces cerevisiae al homeodomain is expressed as a soluble protein in Escherichia coli when cultured in minimal medium. Nuclear magnetic resonance (NMR) spectra of previously prepared al homeodomain samples contained a subset of doubled and broadened resonances. Mass spectroscopic and NMR analysis demonstrates that the heterogeneity is largely due to a lysine misincorporation at the arginine (Arg) 115 site. Arg 115 is coded by the 5′‐AGA‐3′ sequence, which is quite rare in E. coli genes. Lower level mistranslation at three other rare arginine codons also occurs. The percentage of lysine for arginine misincorporation in al homeodomain production is dependent on media composition. The dnaY gene, which encodes the rare 5′‐AGA‐3′ tRNAARG, was co‐expressed in E. coli with the al‐encoding plasmid to produce a homogeneous recombinant al homeodomain. Co‐expression of the dnaY gene completely blocks mistranslation of arginine to lysine during al overexpression in minimal media, and homogeneous protein is produced.
A LexA mutant repressor with a relaxed inter‐domain linker
Authors: Oertel‐Buchheit Pascale , Reinbolt Joseph , Johan Matthias , Granger‐Schnarr Michèle , Schnarr Manfred
Published: July 2010

The LexA protein is part of a large family of prokaryotic transcriptional repressors that contain an amino‐terminal DNA binding domain and a carboxy‐terminal dimerization domain. These domains are separated by a linker or hinge region, which is generally considered to be rather flexible and unconstrained. So far, no structure of any of the full‐length repressors is available. Here we show that a mutant LexA repressor harboring several point mutations in the hinge region gets sensitive to trypsin and Glu‐C cleavage over a segment of at least 20 amino acids, whereas the LexA wild‐type hinge region is resistant to these proteases. These data are not compatible with the hypothesis of an fully flexible and/or unstructured inter‐domain linker and suggest that the LexA hinge region is, in fact, constrained by contacts with the carboxy‐terminal domain and/or a fairly stable local structure of the linker region.
Crystal structures of the psychrophilic α‐amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor
Authors: Aghajari Nushin , Haser Richard , Feller Georges , Gerday Charles
Published: July 2010

Alteromonas haloplanctis is a bacterium that flourishes in Antarctic sea‐water and it is considered as an extreme psychrophile. We have determined the crystal structures of the α‐amylase (AHA) secreted by this bacterium, in its native state to 2.0 Å resolution as well as in complex with Tris to 1.85 Å resolution. The structure of AHA, which is the first experimentally determined three‐dimensional structure of a psychrophilic enzyme, resembles those of other known α‐amylases of various origins with a surprisingly greatest similarity to mammalian α‐amylases. AHA contains a chlorideion which activates the hydrolytic cleavage of substrate α‐l,4‐glycosidic bonds. The chloride binding site is situated ∼5 Å from the active site which is characterized by a triad of acid residues (Asp 174, Glu 200, Asp 264). These are all involved in firm binding of the Tris moiety. A reaction mechanism for substrate hydrolysis is proposed on the basis of the Tris inhibitor binding and the chloride activation.A trio of residues (Ser 303, His 337, Glu 19) having a striking spatial resemblance with serine‐protease like catalytic triads was found ∼22 Å from the active site. We found that this triad is equally present in other chloride dependent α‐amylases, and suggest that it could be responsible for autoproteolytic events observed in solution for this cold adapted α‐amylase.
Solvation studies of DMP323 and A76928 bound to HIV protease: Analysis of water sites using grand canonical Monte Carlo simulations
Authors: Marrone Tami J. , McCammon J. Andrew , Resat Haluk , Hodge C. Nicholas , Chang Chong‐Hwan
Published: July 2010

We examine the water solvation of the complex of the inhibitors DMP323 and A76928 bound to HIV‐1 protease through grand canonical Monte Carlo simulations, and demonstrate the ability of this method to reproduce crystal waters and effectively predict water positions not seen in the DMP323 or A76928 structures. The simulation method is useful for identifying structurally important waters that may not be resolved in the crystal structures. It can also be used to identify water positions around a putative drug candidate docked into a binding pocket. Knowledge of these water positions may be useful in designing drugs to utilize them as bridging groups or displace them in the binding pocket. In addition, the method should be useful in finding water sites in homology models of enzymes for which crystal structures are unavailable.
Solution structure of Compstatin, a potent complement inhibitor
Authors: Morikis Dimitrios , Assa‐Munt Nuria , Sahu Arvind , Lambris John D.
Published: July 2010

The third component of complement, C3, plays a central role in activation of the classical, alternative, and lectin pathways of complement activation. Recently, we have identified a 13‐residue cyclic peptide (named Compstatin) that specifically binds to C3 and inhibits complement activation. To investigate the topology and the contribution of each critical residue to the binding of Compstatin to C3, we have now determined the solution structure using 2D NMR techniques; we have also synthesized substitution analogues and used these to study the structure‐function relationships involved. Finally, we have generated an ensemble of a family of solution structures of the peptide with a hybrid distance geometry‐restrained simulated‐annealing methodology, using distance, dihedral angle, and 3JNH‐Hα‐coupling constant restraints. The Compstatin structure contained a type I β‐turn comprising the segment Gln5‐Asp6‐Trp7‐Gly8. Preference for packing of the hydrophobic side chains of Val3, Val4, and Trp7 was observed. The generated structure was also analyzed for consistency using NMR parameters such as NOE connectivity patterns, 3JNH‐Hα‐coupling constants, and chemical shifts. Analysis of Ala substitution analogues suggested that Val3, Gln5, Asp6, Trp7, and Gly8 contribute significantly to the inhibitory activity of the peptide. Substitution of Gly8 caused a 100‐fold decrease in inhibitory potency. In contrast, substitution of Val4, His9, His10, and Arg11 resulted in minimal change in the activity. These findings indicate that specific side‐chain interactions and the β‐turn are critical for preservation of the conformational stability of Compstatin and they might be significant for maintaining the functional activity of Compstatin.
LIF receptor‐gp130 interaction investigated by homology modeling: Implications for LIF binding
Authors: Smith David K. , Treutlein Herbert R.
Published: July 2010

Leukemia inhibitory factor (LIF), a member of the gpl30 family of helical cytokines, is involved in the hemopoietic and neural systems. The LIF signal transducing complex contains two receptor molecules, the LIF receptor (LIFR) and gp130. The extracellular region of the LIFR is unique in that it includes three membrane‐proximal fibronectin type III domains and two cytokine binding domains (CBDs) separated by an immunoglobulin‐like domain. Although some mutagenesis data on LIF are available, it is not yet known which regions of LIFR or gp130 bind LIF. Nor is it known whether LIFR contacts gp130 in a manner similar to the growth hormone receptor dimer and, if so, through which of its CBDs. To attempt to elucidate these matters and to investigate the receptor complex, models of the CBDs of LIFR and the CBD of gp130 were constructed. Analyses of the electrostatic isopotential surfaces of the CBD models suggest that gp130 and the membrane‐proximal CBD of LIFR hetero‐dimerize and bind LIF through contacts similar to those seen in the growth hormone receptor dimer. This work further demonstrates the utility of electrostatic analyses of homology models and suggests a strategy for biochemical investigations of the LIF‐receptor complex.