bluuebunny/crossref_metadata_2025_split huggingface dataset:
Predicted structure of the extracellular region of ligand‐gated ion‐channel receptors shows SH2‐like and SH3‐like domains forming the ligand‐binding site
Authors: Gready Jill E. , Ranganathan Shoba , Schofield Peter R. , Matsuo Yo , Nishikawa Ken 
Published: February 2009

AbstractFast synaptic neurotransmission is mediated by ligand‐gated ion‐channel (LGIC) receptors, which include receptors for acetylcholine, serotonin, GABA, glycine, and glutamate. LGICs are pentamers with extracellular ligand‐binding domains and form integral membrane ion channels that are selective for cations (acetylcholine and serotonin 5HT3receptors) or anions (GABAAand glycine receptors and the invertebrate glutamate‐binding chloride channel). They form a protein superfamily with no sequence similarity to any protein of known structure. Using a 1D‐3D structure mapping approach, we have modeled the extracellular ligand‐binding domain based on a significant match with the SH2 and SH3 domains of the biotin repressor structure. Refinement of the model based on knowledge of the large family of SH2 and SH3 structures, sequence alignments, and use of structure templates for loop building, allows the prediction of both monomer and pentamer models. These are consistent with medium‐resolution electron microscopy structures and with experimental structure/function data from ligand‐binding, antibody‐binding, mutagenesis, protein‐labeling and subunitlinking studies, and glycosylation sites. Also, the predicted polarity of the channel pore calculated from electrostatic potential maps of pentamer models of superfamily members is consistent with known ion selectivities. Using the glycine receptor α1 subunit, which forms homopentamers, the monomeric and pentameric models define the agonist and antagonist (strychnine) binding sites to a deep crevice formed by an extended loop, which includes the invariant disulfide bridge, between the SH2 and SH3 domains. A detailed binding site for strychnine is reported that is in strong agreement with known structure/function data. A site for interaction of the extracellular ligand‐binding domain with the activation of the M2 transmembrane helix is also suggested.
Template‐based docking of a prolactin receptor proline‐rich motif octapeptide to FKBP12: Implications for cytokine receptor signaling
Authors: Soman K. V. , Hanks B. A. , Tien H. , Chari M. V. , O'Neal K. D. , Morrisett J. D. 
Published: February 2009

AbstractA conserved proline‐rich motif (PRM) in the cytoplasmic domain of cytokine receptors has been suggested to be a signaling switch regulated by the action of the FK506 binding protein (FKBP) family of peptidylprolyl isomerases (O'Neal KD, Yu‐Lee LY, Shearer WT, 1995, Ann NY Acad Sci 766:282–284). We have docked the prolactin receptor PRM (Ile1‐Phe2‐Pro3‐Pro4‐Val5‐Pro6‐Gly7‐Pro8) to the ligand binding site of FKBP12. The procedure involved conformational search restricted by NMR restraints (O'Neal KD et al., 1996, Biochem J 315:833–844), energy minimization of the octapeptide conformers so obtained, template‐based docking of a selected conformer to FKBP12, and energy refinement of the resulting complex. The template used was the crystal structure of a cyclic FK506‐peptide hybrid bound to FKBP12. Val5‐Pro6 of the PRM was taken to be the biologically relevant Xaa‐Pro bond. The docked conformer is stabilized by two intramolecular hydrogen bonds, N7H7 → O4 and N2H2 → O8, and two intermolecular ones, Ile56: N‐H → O=C:Pro6 and Tyr82:O‐H → O=C:Gly7. This conformer features a Type I β‐turn and has extensive hydrophobic contacts with the FKBP12 binding surface. The observed interactions support the hypothesis that FKBP12 catalyzes cis‐trans isomerization in the PRM when it is part of the longer cytoplasmic domain of a cytokine receptor, and suggest a significant role for the PRM in signal transduction.
Identification of a novel bond between a histidine and the essential tyrosine in catalase HPII of Escherichia coli
Authors: Bravo Jeronimo , Fita Ignacio , Ferrer Juan C. , Ens Werner , Hillar Alex , Switala Jacek , Loewen Peter C. 
Published: February 2009

AbstractA bond between the Nδ of the imidazole ring of His 392 and the Cβ of the essential Tyr 415 has been found in the refined crystal structure at 1.9 Å resolution of catalase HPII of Escherichia coli. This novel type of covalent linkage is clearly defined in the electron density map of HPII and is confirmed by matrix‐assisted laser desorption/ionization mass spectrometry analysis of tryptic digest mixtures. The geometry of the bond is compatible with both the sp3 hybridization of the Cβ atom and the planarity of the imidazole ring. Two mutated variants of HPII active site residues, H128N and N201H, do not contain the His 392‐Tyr 415 bond, and their crystal structures show that the imidazole ring of His 392 was rotated, in both cases, by 80° relative to its position in HPII. These mutant forms of HPII are catalytically inactive and do not convert heme b to heme d, suggesting a relationship between the self‐catalyzed heme conversion reaction and the formation of the His‐Tyr linkage. A model coupling the two processes and involving the reaction of one molecule of H2O2 on the proximal side of the heme with compound I is proposed.
Hsc66 and Hsc20, a new heat shock cognate molecular chaperone system from Escherichia coli
Authors: Vickery Larry E. , Silberg Jonathan J. , Ta Dennis T. 
Published: February 2009

AbstractThe hscA and hscB genes of Escherichia coli encode novel chaperone and co‐chaperone proteins, designated Hsc66 and Hsc20, respectively. We have overproduced and purified Hsc66 and Hsc20 in high yield in E. coli and describe their initial characterization including absorbance, fluorescence, and circular dichroism spectra. Immunoblot analyses of E. coli cultures using antisera to Hsc66 and Hsc20 raised in rabbits establish that Hsc66 and Hsc20 are constitutively expressed at levels corresponding to cell concentrations ˜20 μM and ˜10 μM, respectively. The levels do not change appreciably following heat shock (44°C), but a small increase in Hsc20 is observed following a shift to 10°C. Purified Hsc66 exhibits a low intrinsic ATPase activity (˜0.6 min−1 at 37° C), and Hsc20 was found to stimulate this activity up to 3.8‐fold with half‐maximal stimulation at a concentration ˜5 μM. These findings suggest that Hsc66 and Hsc20 comprise a molecular chaperone system similar to the prokaryotic DnaK/DnaJ and eukaryotic hsp70/hsp40 systems. Sequence differences between Hsc66 and Hsc20 compared to other members of this chaperone family, however, suggest that the Hsc66/Hsc20 system will display different peptide binding specificity and that it is likely to be subject to different regulatory mechanisms. The high level of constitutive expression and the lack of a major response to temperature changes suggest that Hsc66 and Hsc20 play an important cellular role(s) under non‐stress conditions.
Consistency in structural energetics of protein folding and peptide recognition
Authors: Zhang Chao , Cornette James L. , Delisi Charles 
Published: February 2009

AbstractWe report a new free energy decomposition that includes structure‐derived atomic contact energies for the desolvation component, and show that it applies equally well to the analysis of single‐domain protein folding and to the binding of flexible peptides to proteins. Specifically, we selected the 17 single‐domain proteins for which the three‐dimensional structures and thermodynamic unfolding free energies are available. By calculating all terms except the backbone conformational entropy change and comparing the result to the experimentally measured free energy, we estimated that the mean entropy gain by the backbone chain upon unfolding (ΔSbb) is 5.3 cal/K per mole of residue, and that the average backbone entropy for glycine is 6.7 cal/K. Both numbers are in close agreement with recent estimates made by entirely different methods, suggesting a promising degree of consistency between data obtained from disparate sources. In addition, a quantitative analysis of the folding free energy indicates that the unfavorable backbone entropy for each of the proteins is balanced predominantly by favorable backbone interactions. Finally, because the binding of flexible peptides to receptors is physically similar to folding, the free energy function should, in principle, be equally applicable to flexible docking. By combining atomic contact energies, electrostatics, and sequence‐dependent backbone entropy, we calculated a priori the free energy changes associated with the binding of four different peptides to HLA‐A2.1 MHC molecule and found agreement with experiment to within 10% without parameter adjustment.
Hexafluoroacetone hydrate as a structure modifier in proteins: Characterization of a molten globule state of hen egg‐white lysozyme
Authors: Bhattacharjya Surajit , Balaram Padmanabhan 
Published: February 2009

AbstractA molten globule‐like state of hen egg‐white lysozyme has been characterized in 25% aqueous hexafluoroacetone hydrate (HFA) by CD, fluorescence, NMR, and H/D exchange experiments. The far UV CD spectra of lysozyme in 25% HFA supports retention of native‐like secondary structure while the loss of near UV CD bands are indicative of the overall collapse of the tertiary structure. The intermediate state in 25% HFA exhibits an enhanced affinity towards the hydrophobic dye, ANS, and a native‐like tryptophan fluorescence quenching. 1‐D NMR spectra indicates loss of native‐like tertiary fold as evident from the absence of ring current‐shifted 1H resonances. CD, fluorescence, and NMR suggest that the transition from the native state to a molten globule state in 25% HFA is a cooperative process. A second structural transition from this compact molten globule‐like state to an “open” helical state is observed at higher concentrations of HFA (≥50%). This transition is characterized by a dramatic loss of ANS binding with a concomitant increase in far UV CD bands. The thermal unfolding of the molten globule state in 25% HFA is sharply cooperative, indicating a predominant role of side‐chain‐side‐chain interactions in the stability of the partially folded state. H/D exchange experiments yield higher protection factors for many of the backbone amide protons from the four α‐helices along with the C‐terminal 310 helix, whereas little or no protection is observed for most of the amide protons from the triple‐stranded antiparallel β‐sheet domain. This equilibrium molten globule‐like state of lysozyme in 25% HFA is remarkably similar to the molten globule state observed for α‐lactalbumin and also with the molten globule state transiently observed in the kinetic refolding experiments of hen lysozyme. These results suggest that HFA may prove generally useful as a structure modifier in proteins.
Cysteine reactivity in Thermoanaerobacter brockii alcohol dehydrogenase
Authors: Peretz Moshe , Weiner Lev M. , Burstein Yigal 
Published: February 2009

AbstractThe free cysteine residues in the extremely thermophilic Thermoanaerobacter brockii alcohol dehydrogenase (TBADH) were characterized using selective chemical modification with the stable nitroxyl biradical bis(1‐oxy‐2,2,5,5‐tetramethyl‐3‐imidazoline‐4‐yl)disulfide, via a thiol‐disulfide exchange reaction and with 2[14C]iodoacetic acid, via S‐alkylation. The respective reactions were monitored by electron paramagnetic resonance (EPR) and by the incorporation of the radioactive label. In native TBADH, the rapid modification of one cysteine residue per subunit by the biradical and the concomitant loss of catalytic activity was reversed by DTT. NADP protected the enzyme from both modification and inactivation by the biradical. RPLC fingerprint analysis of reduced and S‐carboxymethylated lysyl peptides from the radioactive alkylated enzyme identified Cys 203 as the readily modified residue. A second cysteine residue was rapidly modified with both modification reagents when the catalytic zinc was removed from the enzyme by o‐phenanthroline. This cysteine residue, which could serve as a putative ligand to the active‐site zinc atom, was identified as Cys 37 in RPLC. The EPR data suggested a distance of ≤ 10 Å between Cys 37 and Cys 203. Although Cys 283 and Cys 295 were buried within the protein core and were not accessible for chemical modification, the two residues were oxidized to cystine when TBADH was heated at 75 °C, forming a disulfide bridge that was not present in the native enzyme, without affecting either enzymatic activity or thermal stability. The status of these cysteine residues was verified by site directed mutagenesis.
Hydrogen exchange: The modern legacy of Linderstrøm‐Lang
Authors: Englander S. W. , Mayne L. , Bai Y. , Sosnick T. R. 
Published: February 2009

AbstractThis discussion, prepared for the Protein Society's symposium honoring the 100th anniversary of Kaj Linderstrøm‐Lang, shows how hydrogen exchange approaches initially conceived and implemented by Lang and his colleagues some 50 years ago are contributing to current progress in structural biology. Examples are chosen from the active protein folding field. Hydrogen exchange methods now make it possible to define the structure of protein folding intermediates in various contexts: as tenuous molten globule forms at equilibrium under destabilizing conditions, in kinetic intermediates that exist for less than one second, and as infinitesimally populated excited state forms under native conditions. More generally, similar methods now find broad application in studies of protein structure, energetics, and interactions. This article considers the rise of these capabilities from their inception at the Carlsberg Labs to their contemporary role as a significant tool of modern structural biology.
The uncharged surface features surrounding the active site ofEscherichia coliDsbA are conserved and are implicated in peptide binding
Authors: Guddat Luke W. , Martin Jennifer L. , Bardwell James C.A. , Zander Thomas 
Published: February 2009

AbstractDsbA is a protein‐folding catalyst from the periplasm ofEscherichia colithat interacts with newly translocated polypeptide substrate and catalyzes the formation of disulfide bonds in these secreted proteins. The precise nature of the interaction between DsbA and unfolded substrate is not known. Here, we give a detailed analysis of the DsbA crystal structure, now refined to 1.7 Å, and present a proposal for its interaction with peptide.The crystal structure of DsbA implies flexibility between the thioredoxin and helical domains that may be an important feature for the disulfide transfer reaction. A hinge point for domain motion is identified—the type IV β‐turn Phe 63‐Met 64‐Gly 65‐Gly 66, which connects the two domains.Three unique features on the active site surface of the DsbA molecule—a groove, hydrophobic pocket, and hydrophobic patch—form an extensive uncharged surface surrounding the active‐site disulfide. Residues that contribute to these surface features are shown to be generally conserved in eight DsbA homologues. Furthermore, the residues immediately surrounding the active‐site disulfide are uncharged in all nine DsbA proteins.A model for DsbA‐peptide interaction has been derived from the structure of a human thioredoxin:peptide complex. This shows that peptide could interact with DsbA in a manner similar to that with thioredoxin. The active‐site disulfide and all three surrounding uncharged surface features of DsbA could, in principle, participate in the binding or stabilization of peptide.
MultiCoil: A program for predicting two‐and three‐stranded coiled coils
Authors: Wolf Ethan , Kim Peter S. , Berger Bonnie 
Published: February 2009

AbstractA new multidimensional scoring approach for identifying and distinguishing trimeric and dimeric coiled coils is implemented in the MultiCoil program. The program extends the two‐stranded coiled‐coil prediction program PairCoil to the identification of three‐stranded coiled coils. The computations are based upon data gathered from a three‐stranded coiled‐coil database comprising 6,319 amino acid residues, as well as from the previously constructed two‐stranded coiled‐coil database. In addition to identifying coiled coils not predicted by the two‐stranded database programs, MultiCoil accurately classifies the oligomerization states of known dimeric and trimeric coiled coils. Analysis of the MultiCoil scores provides insight into structural features of coiled coils, and yields estimates that 0.9% of all protein residues form three‐stranded coiled coils and that 1.5% form two‐stranded coiled coils. The MultiCoil program is available at http://theory.Ics.mit.edu/multicoil.
Optimization of the electrostatic interactions between ionized groups and peptide dipoles in proteins
Authors: Ladenstein Rudolf , Karshikoff Andrej D. , Spassov Velin Z. 
Published: February 2009

AbstractThe three‐dimensional optimization of the electrostatic interactions between the charged amino acid residues and the peptide partial charges was studied by Monte‐Carlo simulations on a set of 127 nonhomologous protein structures with known atomic coordinates. It was shown that this type of interaction is very well optimized for all proteins in the data set, which suggests that they are a valuable driving force, at least for the native side‐chain conformations. Similar to the optimization of the charge‐charge interactions (Spassov VZ, Karshikoff AD, Ladenstein R, 1995, Protein Sci 41516‐1527), the optimization effect was found more pronounced for enzymes than for proteins without enzymatic function. The asymmetry in the interactions of acidic and basic groups with the peptide dipoles was analyzed and a hypothesis was proposed that the properties of peptide dipoles are a factor contributing to the natural selection of the basic amino acids in the chemical composition of proteins.
Refined structure of villin 14T and a detailed comparison with other actin‐severing domains
Authors: Wagner Gerhard , Markus Michelle A. , Matsudaira Paul 
Published: February 2009

AbstractVillin 14T is the amino terminal actin monomer binding domain from the actin‐severing and bundling protein villin. Its structure has been determined in solution using heteronuclear multidimensional nuclear magnetic resonance (NMR) spectroscopy (Markus MA, Nakayama T, Matsudaira P, Wagner G. 1994. Solution structure of villin 14T, a domain conserved among actin‐severing proteins. Protein Science 3:70‐81). An additional nuclear Overhauser effect (NOE) spectroscopy data set, acquired using improved gradient techniques, and further detailed analysis of existing data sets, produced an additional 601 NOE restraints for structure calculations. The overall fold does not change significantly with the additional NOE restraints but the definition of the structure is improved, as judged by smaller deviations among an ensemble of calculated structures that adequately satisfy the NMR restraints. Some of the side chains, especially those in the hydrophobic core of the domain, are much more defined. This improvement in the detail of the solution structure of villin 14T makes it interesting to compare the structure with the crystal structure of gelsolin segment 1, which shares 58% sequence identity with villin 14T, in an effort to gain insight into villin 14T's weaker affinity for actin monomers. Villin 14T has smaller side chains at several positions that make hydrophobic contacts with actin in the context of gelsolin segment 1. The structure is also compared with the structure of the related actin‐severing domain, severin domain 2.
Characterization of the receptor binding determinants of granulocyte colony stimulating factor
Authors: Young Dennis C. , Cheng Qi‐Lin , Hou Jinzhao , Matthews David J. , Zhan Hangjun 
Published: February 2009

AbstractWe performed a series of experiments using alanine‐scanning mutagenesis to locate side chains within human granulocyte colony‐stimulating factor (G‐CSF) that are involved in human G‐CSF receptor binding. We constructed a panel of 28 alanine mutants that examined all surface exposed residues on helices A and D, as well as all charged residues on the surface of G‐CSF. The G‐CSF mutants were expressed in a transiently transfected mammalian cell line and quantitated by a sensitive biosensor method. We measured the activity of mutant proteins using an in vitro proliferation assay and an ELISA binding competition assay. These studies show that there is a region of five charged residues on helices A and C employed by G‐CSF in binding its receptor, with the most important residue in this binding patch being Glu 19. Both wild‐type G‐CSF and the E19A mutant were expressed in E. coli. The re‐folded proteins were found to have proliferative activities similar to the analagous proteins from mammalian cells: furthermore, biophysical analysis indicated that the E19A mutation does not cause gross structural perturbations in G‐CSF. Although G‐CSF is likely to signal through receptor homo‐dimerization, we found no compelling evidence for a second receptor binding region. We also found no evidence of self‐antagonism at high G‐CSF concentrations, suggesting that, in contrast to human growth hormone (hGH) and erythropoietin (EPO), G‐CSF probably does not signal via a pure 2.1 receptor:ligand complex. Thus, G‐CSF, while having a similar tertiary structure to hGH and EPO, uses different areas of the four helix bundle for high‐affinity interaction with its receptor.
Secondary and tertiary structural changes in γδ resolvase: Comparison of the wild‐type enzyme, the I11OR mutant, and the C‐terminal DNA binding domain in solution
Authors: Pan Borlan , Ghosh Susmita , Mullen Gregory P. , Deng Zhengwu , Liu Dingjiang 
Published: February 2009

Abstractγδ Resolvase is a site‐specific DNA recombinase (Mr 20.5 kDa) in Escherichia coli that shares homology with a family of bacterial resolvases and invertases. We have characterized the secondary and tertiary structural behavior of the cloned DNA binding domain (DBD) and a dimerization defective mutant in solution. Low‐salt conditions were found to destabilize the tertiary structure of the DBD dramatically, with concomitant changes in the secondary structure that were localized near the hinge regions between the helices. The molten ertiary fold appears to contribute significantly to productive DNA interactions and supports a mechanism of DNA‐induced folding of the tertiary structure, a process that enables the DBD to adapt in conformation for each of the three imperfect palindromic sites. At high salt concentrations, the monomeric I11OR resolvase shows a minimal perturbation to the three helices of the DBD structure and changes in the linker segment in comparison to the cloned DBD containing the linker. Comparative analysis of the NMR spectra suggest that the I11OR mutant contains a folded catalytic core of ∼60 residues and that the segment from residues 100 to 149 are devoid of regular structure in the I11OR resolvase. No increase in the helicity of the linker region of I11OR resolvase occurs on binding DNA. These results support a subunit rotation model of strand exchange that involves the partial unfolding of the catalytic domains.