bluuebunny/crossref_metadata_2025_split huggingface dataset:
Free energies of protein decoys provide insight into determinants of protein stability
Authors: Vorobjev Yury N. , Hermans Jan 
Published: February 2009

AbstractWe have calculated the stability of decoy structures of several proteins (from the CASP3 models and the Park and Levitt decoy set) relative to the native structures. The calculations were performed with the force field–consistent ES/IS method, in which an implicit solvent (IS) model is used to calculate the average solvation free energy for snapshots from explicit simulations (ESs). The conformational free energy is obtained by adding the internal energy of the solute from the ESs and an entropic term estimated from the covariance positional fluctuation matrix. The set of atomic Born radii and the cavity‐surface free energy coefficient used in the implicit model has been optimized to be consistent with the all‐atom force field used in the ESs (cedar/gromos with simple point charge (SPC) water model). The decoys are found to have a consistently higher free energy than that of the native structure; the gap between the native structure and the best decoy varies between 10 and 15 kcal/mole, on the order of the free energy difference that typically separates the native state of a protein from the unfolded state. The correlation between the free energy and the extent to which the decoy structures differ from the native (as root mean square deviation) is very weak; hence, the free energy is not an accurate measure for ranking the structurally most native‐like structures from among a set of models. Analysis of the energy components shows that stability is attained as a result of three major driving forces: (1) minimum size of the protein‐water surface interface; (2) minimum total electrostatic energy, which includes solvent polarization; and (3) minimum protein packing energy. The detailed fit required to optimize the last term may underlie difficulties encountered in recovering the native fold from an approximate decoy or model structure.
The C‐terminal domain of biotin protein ligase from E. coli is required for catalytic activity
Authors: Chapman‐Smith Anne , Mulhern Terrence D. , Whelan Fiona , Cronan John E. , Wallace John C. 
Published: February 2009

AbstractBiotin protein ligase of Escherichia coli, the BirA protein, catalyses the covalent attachment of the biotin prosthetic group to a specific lysine of the biotin carboxyl carrier protein (BCCP) subunit of acetyl‐CoA carboxylase. BirA also functions to repress the biotin biosynthetic operon and synthesizes its own corepressor, biotinyl‐5′‐AMP, the catalytic intermediate in the biotinylation reaction. We have previously identified two charge substitution mutants in BCCP, E119K, and E147K that are poorly biotinylated by BirA. Here we used site‐directed mutagenesis to investigate residues in BirA that may interact with E119 or E147 in BCCP. None of the complementary charge substitution mutations at selected residues in BirA restored activity to wild‐type levels when assayed with our BCCP mutant substrates. However, a BirA variant, in which K277 of the C‐terminal domain was substituted with Glu, had significantly higher activity with E119K BCCP than did wild‐type BirA. No function has been identified previously for the BirA C‐terminal domain, which is distinct from the central domain thought to contain the ATP binding site and is known to contain the biotin binding site. Kinetic analysis of several purified mutant enzymes indicated that a single amino acid substitution within the C‐terminal domain (R317E) and located some distance from the presumptive ATP binding site resulted in a 25‐fold decrease in the affinity for ATP. Our data indicate that the C‐terminal domain of BirA is essential for the catalytic activity of the enzyme and contributes to the interaction with ATP and the protein substrate, the BCCP biotin domain.
Analysis of crystal structures of aspartic proteinases: On the role of amino acid residues adjacent to the catalytic site of pepsin‐like enzymes
Authors: Andreeva Natalia S. , Rumsh Lev D. 
Published: February 2009

AbstractTo elucidate the role of amino acid residues adjacent to the catalytic site of pepsin‐like enzymes, we analyzed and compared the crystal structures of these enzymes, their complexes with inhibitors, and zymogens in the active site area (a total of 82 structures). In addition to the water molecule (W1) located between the active carboxyls and playing a role of the nucleophile during catalytic reaction, another water molecule (W2) at the vicinity of the active groups was found to be completely conserved. This water molecule plays an essential role in formation of a chain of hydrogen‐bonded residues between the active site flap and the active carboxyls on ligand binding. These data suggest a new approach to understanding the role of residues around the catalytic site, which can assist the development of the catalytic reaction. The influence of groups adjacent to the active carboxyls is manifested by pepsin activity at pH 1.0. Some features of pepsin‐like enzymes and their mutants are discussed in the framework of the approach.
Characterization of the structure and dynamics of amyloidogenic variants of human lysozyme by NMR spectroscopy
Authors: Chamberlain Aaron K. , Receveur Veronique , Spencer Andrew , Redfield Christina , Dobson Christopher M. 
Published: February 2009

AbstractThe structures and dynamics of the native states of two mutational variants of human lysozyme, I56T and D67H, both associated with non‐neuropathic systemic amyloidosis, have been investigated by NMR spectroscopy. The 1H and 15N main‐chain amide chemical shifts of the I56T variant are very similar to those of the wild‐type protein, but those of the D67H variant are greatly altered for 28 residues in the β‐domain. This finding is consistent with the X‐ray crystallographic analysis, which shows that the structure of this variant is significantly altered from that of the wild‐type protein in this region. The 1H–15N heteronuclear NOE values show that, with the exception of V121, every residue in the wild‐type and I56T proteins is located in tightly packed structures characteristic of the native states of most proteins. In contrast, D67H has a region of substantially increased mobility as shown by a dramatic decrease in heteronuclear NOE values of residues near the site of mutation. Despite this unusual flexibility, the D67H variant has no greater propensity to form amyloid fibrils in vivo or in vitro than has I56T. This finding indicates that it is the increased ability of the variants to access partially folded conformations, rather than intrinsic changes in their native state properties, that is the origin of their amyloidogenicity.
Competing protein:protein interactions are proposed to control the biological switch of the E coli biotin repressor
Authors: Weaver Larry H. , Kwon Keehwan , Beckett Dorothy , Matthews Brian W. 
Published: February 2009

AbstractA model is suggested for the complex between the biotin repressor of Escherichia coli, BirA, and BCCP, the biotin carboxyl carrier protein to which BirA transfers biotin. The model is consistent with prior physical and biochemical studies. Measurement of transfer rates for variants of BirA with single‐site mutations in the proposed BirA:BCCP interface region also provides support. The unique feature of the proposed interaction between BirA and BCCP is that it uses the same β‐sheet region on the surface of BirA that the protein uses for homodimerization into a form competent to bind DNA. The resulting mutually exclusive protein:protein interfaces explain the novel feature of the BirA regulatory system, namely, that transcription of the genes involved in biotin synthesis is not determined by the level of biotin, per se, but by the level of unmodified BCCP. The model also provides a role for the C‐terminal domain of BirA that is structurally similar to an SH3 domain.
Identification of residues critical for metallo‐β‐lactamase function by codon randomization and selection
Authors: Materon Isabel C. , Palzkill Timothy 
Published: February 2009

AbstractIMP‐1 β‐lactamase is a zinc metallo‐enzyme encoded by the transferable blaIMP‐1 gene, which confers resistance to virtually all β‐lactam antibiotics including carbapenems. To understand how IMP‐1 recognizes and hydrolyzes β‐lactam antibiotics it is important to determine which amino acid residues are critical for catalysis and which residues control substrate specificity. We randomized 27 individual codons in the blaIMP‐1 gene to create libraries that contain all possible amino acid substitutions at residue positions in and near the active site of IMP‐1. Mutants from the random libraries were selected for the ability to confer ampicillin resistance to Escherichia coli. Of the positions randomized, >50% do not tolerate amino acid substitutions, suggesting they are essential for IMP‐1 function. The remaining positions tolerate amino acid substitutions and may influence the substrate specificity of the enzyme. Interestingly, kinetic studies for one of the functional mutants, Asn233Ala, indicate that an alanine substitution at this position significantly increases catalytic efficiency as compared with the wild‐type enzyme.
Allosteric inhibition of glycogen phosphorylase a by the potential antidiabetic drug 3‐isopropyl 4‐(2‐chlorophenyl)‐1,4‐dihydro‐1‐ethyl‐2‐methyl‐pyridine‐3,5,6‐tricarboxylate
Authors: Oikonomakos Nikos G. , Tsitsanou Katerina E. , Zographos Spyros E. , Skamnaki Vicky T. , Goldmann Siegfried , Bischoff Hilmar 
Published: February 2009

AbstractThe effect of the potential antidiabetic drug (‐)(S)‐3‐isopropyl 4‐(2‐chlorophenyl)‐1,4‐dihydro‐1‐ethyl‐2‐methyl‐pyridine‐3,5,6‐tricarboxylate (W1807) on the catalytic and structural properties of glycogen phosphorylase a has been studied. Glycogen phosphorylase (GP) is an allosteric enzyme whose activity is primarily controlled by reversible phosphorylation of Ser14 of the dephosphorylated enzyme (GPb, less active, predominantly T‐state) to form the phosphorylated enzyme (GPa, more active, predominantly R‐state). Upon conversion of GPb to GPa, the N‐terminal tail (residues 5‐22), which carries the Ser14(P), changes its conformation into a distorted 310 helix and its contacts from intrasubunit to intersubunit. This alteration causes a series of tertiary and quaternary conformational changes that lead to activation of the enzyme through opening access to the catalytic site. As part of a screening process to identify compounds that might contribute to the regulation of glycogen metabolism in the noninsulin dependent diabetes diseased state, W1807 has been found as the most potent inhibitor of GPb (Ki = 1.6 nM) that binds at the allosteric site of T‐state GPb and produces further conformational changes, characteristic of a T′‐like state. Kinetics show W1807 is a potent competitive inhibitor of GPa (‐AMP) (Ki = 10.8 nM) and of GPa (+1 mM AMP) (Ki = 19.4 μM) with respect to glucose 1‐phosphate and acts in synergism with glucose. To elucidate the structural features that contribute to the binding, the structures of GPa in the T‐state conformation in complex with glucose and in complex with both glucose and W1807 have been determined at 100 K to 2.0 Å and 2.1 Å resolution, and refined to crystallographic R‐values of 0.179 (Rfree = 0.230) and 0.189 (Rfree = 0.263), respectively. W1807 binds tightly at the allosteric site and induces substantial conformational changes both in the vicinity of the allosteric site and the subunit interface. A disordering of the N‐terminal tail occurs, while the loop of chain containing residues 192‐196 and residues 439‐499 shift to accommodate the ligand. Structural comparisons show that the T‐state GPa‐glucose‐W1807 structure is overall more similar to the T‐state GPb‐W1807 complex structure than to the GPa‐glucose complex structure, indicating that W1807 is able to transform GPa to the T′‐like state already observed with GPb. The structures provide a rational for the potency of the inhibitor and explain GPa allosteric inhibition of activity upon W1807 binding.
Probing the nature of interactions in SH2 binding interfaces–evidence from electrospray ionization mass spectrometry
Authors: Chung Evonne W. , Henriques Denise A. , Renzoni Debora , Morton Craig J. , Mulhern Terrence D. , Pitkeathly Maureen C. , Ladbury John E. , Robinson Carol V. 
Published: February 2009

AbstractWe have adopted nanoflow electrospray ionization mass spectrometry (ESI‐MS) and isothermal titration calorimetry (ITC) to probe the mechanism of peptide recognition by the SH2 domain from the Src family tyrosine kinase protein, Fyn. This domain is involved in the mediation of intracellular signal transduction pathways by interaction with proteins containing phosphorylated tyrosine (Y*) residues. The binding of tyrosyl phosphopeptides can mimic these interactions. Specificity in these interactions has been attributed to the interaction of the Y* and residues proximal and C‐terminal to it. Previous studies have established that for specific binding with Fyn, the recognition sequence consists of pTyr‐Glu‐Glu‐Ile. The specific interactions involve the binding of Y* with the ionic, and the Y* + 3 Ile residue with the hydrophobic binding pockets on the surface of the Fyn SH2 domain. In this work, a variation in the Y* + 3 residue of this high‐affinity sequence was observed to result in changes in the relative binding affinities as determined in solution (ITC) and in the gas phase (nanoflow ESI‐MS). X‐ray analysis shows that a feature of the Src family SH2 domains is the involvement of water molecules in the peptide binding site. Under the nanoflow ESI conditions, water molecules appear to be maintained in the Fyn SH2‐ligand complex. Compelling evidence for these molecules being incorporated in the SH2‐peptide interface is provided by the prevalence of the peaks assigned to water‐bound over the water‐free complex at high‐energy conditions. Thus, the stability of water protein‐ligand complex appears to be intimately linked to the presence of water.
Crystal structure of penicillin G acylase from the bro1 mutant strain of providencia rettgeri
Authors: Mcdonough Michael A. , Klei Herbert E. , Kelly Judith A. 
Published: February 2009

AbstractPenicillin G acylase is an important enzyme in the commercial production of semisynthetic penicillins used to combat bacterial infections. Mutant strains of Providencia rettgeri were generated from wild‐type cultures subjected to nutritional selective pressure. One such mutant, Bro1, was able to use 6‐bromohexanamide as its sole nitrogen source. Penicillin acylase from the Bro1 strain exhibited an altered substrate specificity consistent with the ability of the mutant to process 6‐bromohexanamide. The X‐ray structure determination of this enzyme was undertaken to understand its altered specificity and to help in the design of site‐directed mutants with desired specificities. In this paper, the structure of the Bro1 penicillin G acylase has been solved at 2.5 Å resolution by molecular replacement. The R‐factor after refinement is 0.154 and R‐free is 0.165. Of the 758 residues in the Bro1 penicillin acylase heterodimer (α‐subunit, 205; β‐subunit, 553), all but the eight C‐terminal residues of the α‐subunit have been modeled based on a partial Bro1 sequence and the complete wild‐type P. rettgeri sequence. A tightly bound calcium ion coordinated by one residue from the α‐subunit and five residues from the β‐subunit has been identified. This enzyme belongs to the superfamily of Ntn hydrolases and uses Oγ of Serβ1 as the characteristic N‐terminal nucleophile. A mutation of the wild‐type Metα140 to Leu in the Bro1 acylase hydrophobic specificity pocket is evident from the electron density and is consistent with the observed specificity change for Bro1 acylase. The electron density for the N‐terminal Gln of the α‐subunit is best modeled by the cyclized pyroglutamate form. Examination of aligned penicillin acylase and cephalosporin acylase primary sequences, in conjunction with the P. rettgeri and Escherichia coli penicillin acylase crystal structures, suggests several mutations that could potentially allow penicillin acylase to accept charged β‐lactam R‐groups and to function as a cephalosporin acylase and thus be used in the manufacture of semi‐synthetic cephalosporins.
Three‐dimensional structures of enzyme‐substrate complexes of the hydroxynitrile lyase from hevea brasiliensis
Authors: Zuegg J. , Gruber K. , Gugganig M. , Wagner U. G. , Kratky C. 
Published: February 2009

AbstractThe 3D structures of complexes between the hydroxynitrile lyase from Hevea brasiliensis (Hb‐HNL) and several substrate and/or inhibitor molecules, including trichloracetaldehyde, hexafluoracetone, acetone, and rhodanide, were determined by X‐ray crystallography. The complex with trichloracetaldehyde showed a covalent linkage between the protein and the inhibitor, which had apparently resulted from nucleophilic attack of the catalytic Ser80‐Oγ. All other complexes showed the substrate or inhibitor molecule merely hydrogen bonded to the protein. In addition, the native crystal structure of Hb‐HNL was redetermined at cryo‐temperature and at room temperature, eliminating previous uncertainties concerning residual electron density within the active site, and leading to the observation of two conserved water molecules. One of them was found to be conserved in all complex structures and appears to have mainly structural significance. The other water molecule is conserved in all structures except for the complex with rhodanide; it is hydrogen bonded to the imidazole of the catalytic His235 and appears to affect the Hb‐HNL catalyzed reaction. The observed 3D structural data suggest implications for the enzyme mechanism. It appears that the enzyme‐catalyzed cyanohydrin formation is unlikely to proceed via a hemiacetal or hemiketal intermediate covalently attached to the enzyme, despite the observation of such an intermediate for the complex with trichloracetaldehyde. Instead, the data are consistent with a mechanism where the incoming substrate is activated by hydrogen bonding with its carbonyl oxygen to the Ser80 and Thr11 hydroxy groups. A hydrogen cyanide molecule subsequently replaces a water molecule and is deprotonated presumably by the His235 base. Deprotonation is facilitated by the proximity of the positive charge of the Lys236 side chain.
Active site specificity of plasmepsin II
Authors: Westling Jennifer , Cipullo Patty , Hung Su‐Hwi , Saft Howard , Dame John B. , Dunn Ben M. 
Published: February 2009

AbstractMembers of the aspartic proteinase family of enzymes have very similar three‐dimensional structures and catalytic mechanisms. Each, however, has unique substrate specificity. These distinctions arise from variations in amino acid residues that line the active site subsites and interact with the side chains of the amino acids of the peptides that bind to the active site. To understand the unique binding preferences of plasmepsin II, an enzyme of the aspartic proteinase class from the malaria parasite, Plasmodium falciparum, chromogenic octapeptides having systematic substitutions at various positions in the sequence were analyzed. This enabled the design of new, improved substrates for this enzyme (Lys‐Pro‐Ile‐Leu‐Phe*Nph‐Ala/Glu‐Leu‐Lys, where * indicates the cleavage point). Additionally, the crystal structure of plasmepsin II was analyzed to explain the binding characteristics. Specific amino acids (Met13, Ser77, and Ile287) that were suspected of contributing to active site binding and specificity were chosen for site‐directed mutagenesis experiments. The Met13Glu and Ile287Glu single mutants and the Met13Glu/Ile287Glu double mutant gain the ability to cleave substrates containing Lys residues.
Protein and ligand adaptation in a retinoic acid binding protein
Authors: Pattanayek Rekha , Newcomer Marcia E. 
Published: February 2009

AbstractA retinoic acid binding protein isolated from the lumen of the rat epididymis (ERABP) is a member of the lipocalin superfamily. ERABP binds both the all‐trans and 9‐cis isomers of retinoic acid, as well as the synthetic retinoid (E)‐4‐[2‐(5,6,7,8)‐tetrahydro‐5,5,8,8‐tetramethyl‐2 napthalenyl‐1 propenyl]‐benzoic acid (TTNPB), a structural analog of all‐trans retinoic acid. The structure of ERABP with a mixture of all‐trans and 9‐cis retinoic acid has previously been reported. To elucidate any structural differences in the protein when bound to the all‐trans and 9‐cis isomers, the structures of all‐trans retinoic acid‐ERABP and 9‐cis retinoic acid ERABP were determined. Our results indicate that the all‐trans isomer of retinoic acid adopts an 8‐cis structure in the binding cavity with no concomitant conformational change in the protein. The structure of TTNPB‐ERABP is also reported herein. To accommodate this all‐trans analog, which cannot readily adopt a cis‐like structure, alternative positioning of critical binding site side chains is required. Consequently, both protein and ligand adaption are observed in the formation of the various holo‐proteins.
Backbone dynamics of the human cc chemokine eotaxin: Fast motions, slow motions, and implications for receptor binding
Authors: Crump Matthew P. , Spyracopoulos Leo , Lavigne Pierre , Kim Key‐Sun , Clark‐Lewis Ian , Sykes Brian D. 
Published: February 2009

AbstractEotaxin is a member of the chemokine family of about 40 proteins that induce cell migration. Eotaxin binds the CC chemokine receptor CCR3 that is highly expressed by eosinophils, and it is considered important in the pathology of chronic respiratory disorders such as asthma. The high resolution structure of eotaxin is known. The 74 amino acid protein has two disulfide bridges and shows a typical chemokine fold comprised of a core of three antiparallel β‐strands and an overlying α‐helix. In this paper, we report the backbone dynamics of eotaxin determined through 15N‐T1, T2, and [H]‐15N nuclear Overhauser effect heteronuclear multidimensional NMR experiments. This is the first extensive study of the dynamics of a chemokine derived from 600, 500, and 300 MHz NMR field strengths. From the T1, T2, and NOE relaxation data, parameters that describe the internal motions of eotaxin were derived using the Lipari‐Szabo model free analysis. The most ordered regions of the protein correspond to the known secondary structure elements. However, surrounding the core, the regions known to be functionally important in chemokines show a range of motions on varying timescales. These include extensive subnanosecond to picosecond motions in the N‐terminus, C‐terminus, and the N‐loop succeeding the disulfides. Analysis of rotational diffusion anisotropy of eotaxin and chemical exchange terms at multiple fields also allowed the confident identification of slow conformational exchange through the “30s” loop, disulfides, and adjacent residues. In addition, we show that these motions may be attenuated in the dimeric form of a synthetic eotaxin. The structure and dynamical basis for eotaxin receptor binding is discussed in light of the dynamics data.