bluuebunny/crossref_metadata_2025_split huggingface dataset:
The effect of multiple binding modes on empirical modeling of ligand docking to proteins
Authors: Brem Rachel , Dill Ken A. 
Published: February 2009

AbstractA popular approach to the computational modeling of ligand/receptor interactions is to use an empirical free energy like model with adjustable parameters. Parameters are learned from one set of complexes, then used to predict another set. To improve these empirical methods requires an independent way to study their inherent errors. We introduce a toy model of ligand/receptor binding as a workbench for testing such errors. We study the errors incurred from the two state binding assumption—the assumption that a ligand is either bound in one orientation, or unbound. We find that the two state assumption can cause large errors in free energy predictions, but it does not affect rank order predictions significantly. We show that fitting parameters using data from high affinity ligands can reduce two state errors; so can using more physical models that do not use the two state assumption. We also find that when using two state models to predict free energies, errors are more severe on high affinity ligands than low affinity ligands. And we show that two state errors can be diagnosed by systematically adding new binding modes when predicting free energies: if predictions worsen as the modes are added, then the two state assumption in the fitting step may be at fault.
Amino acid substitutions at the subunit interface of dimeric escherichia coli alkaline phosphatase cause reduced structural stability
Authors: Martin Donna Carolan , Pastra‐Landis S. C. , Kantrowitz Evan R. 
Published: February 2009

AbstractThe consequences of amino acid substitutions at the dimer interface for the strength of the interactions between the monomers and for the catalytic function of the dimeric enzyme alkaline phosphatase from Escherichia coli have been investigated. The altered enzymes R10A, R10K, R24A, R24K, T59A, and R10A/R24A, which have amino acid substitutions at the dimer interface, were characterized using kinetic assays, ultracentrifugation, and transverse urea gradient gel electrophoresis. The kinetic data for the wild‐type and altered alkaline phosphatases show comparable catalytic behavior with kcat values between 51.3 and 69.5 s–1 and Km values between 14.8 and 26.3 μM. The ultracentrifugation profiles indicate that the wild‐type enzyme is more stable than all the interface‐modified enzymes. The wild‐type enzyme is dimeric in the pH range of pH 4.0 and above, and disassembled at pH 3.5 and below. All the interface‐modified enzymes, however, are apparently monomeric at pH 4.0, begin assembly at pH 5.0, and are not fully assembled into the dimeric form until pH 6.0. The results from transverse urea gradient gel electrophoresis show clear and reproducible differences both in the position and the shape of the unfolding patterns; all these modified enzymes are more sensitive to the denaturant and begin to unfold at urea concentrations between 1.0 and 1.5 M; the wild‐type enzyme remains in the folded high mobility form beyond 2.5 M urea. Alkaline phosphatase H370A, modified at the active site and not at the dimer interface, resembles the wild‐type enzyme both in ultracentrifugation and electrophoresis studies. The results obtained suggest that substitution of a single amino acid at the interface sacrifices not only the integrity of the assembled dimer, but also the stability of the monomer fold, even though the activity of the enzyme at optimal pH remains unaffected and does not appear to depend on interface stability.
Diversity of functions of proteins with internal symmetry in spatial arrangement of secondary structural elements
Authors: Kinoshita Kengo , Kidera Akinori , Go Nobuhiro 
Published: February 2009

AbstractWe carry out a systematic analysis of the correlation between similarity of protein three‐dimensional structures and their evolutionary relationships. The structural similarity is quantitatively identified by an all‐against‐all comparison of the spatial arrangement of secondary structural elements in nonredundant 967 representative proteins, and the evolutionary relationship is judged according to the definition of superfamily in the SCOP database. We find the following symmetry rule: a protein pair that has similar folds but belong to different superfamilies has (with a very rare exception) certain internal symmetry in its common similar folds. Possible reasons behind the symmetry rule are discussed.
Oligomeric integrity—the structural key to thermal stability in bacterial alcohol dehydrogenases
Authors: Korkhin Yakov , Joseph KALB(GILBOA) A. , Peretz Moshe , Bogin Oren , Burstein Yigal , Frolow Felix 
Published: February 2009

AbstractPrinciples of protein thermostability have been studied by comparing structures of thermostable proteins with mesophilic counterparts that have a high degree of sequence identity. Two tetrameric NADP(H)‐dependent alcohol dehydrogenases, one from Clostridium beijerinckii (CBADH) and the other from Thermoanaerobacter brockii (TBADH), having exceptionally high (75%) sequence identity, differ by 30° in their melting temperatures. The crystal structures of CBADH and TBADH in their holo‐enzyme form have been determined at a resolution of 2.05 and 2.5 Å, respectively. Comparison of these two very similar structures (RMS difference in Ca = 0.8 Å) revealed several features that can account for the higher thermal stability of TBADH. These include additional ion pairs, “charged‐neutral” hydrogen bonds, and prolines as well as improved stability of α‐helices and tighter molecular packing. However, a deeper structural insight, based on the location of stabilizing elements, suggests that enhanced thermal stability of TBADH is due mainly to the strategic placement of structural determinants at positions that strengthen the interface between its subunits. This is also supported by mutational analysis of structural elements at critical locations. Thus, it is the reinforcement of the quaternary structure that is most likely to be a primary factor in preserving enzymatic activity of this oligomeric bacterial ADH at elevated temperatures.
The schiff base complex of yeast 5‐aminolaevulinic acid dehydratase with laevulinic acid
Authors: Erskine Peter T. , Newbold Richard , Roper Jenny , Coker Alun , Warren Martin J. , Shoolingin‐Jordan Peter M. , Wood Steve P. , Cooper Jon B. 
Published: February 2009

AbstractThe X‐ray structure of the complex formed between yeast 5‐aminolaevulinic acid dehydratase (ALAD) and the inhibitor laevulinic acid has been determined at 2.15 Å resolution. The inhibitor binds by forming a Schiff base link with one of the two invariant lysines at the catalytic center: Lys263. It is known that this lysine forms a Schiff base link with substrate bound at the enzyme' so‐called P‐site. The carboxyl group of laevulinic acid makes hydrogen bonds with the side‐chain‐OH groups of Tyr329 and Ser290, as well as with the main‐chain >NH group of Ser290. The aliphatic moiety of the inhibitor makes hydrophobic interactions with surrounding aromatic residues in the protein including Phe219, which resides in the flap covering the active site. Our analysis strongly suggests that the same interactions will be made by P‐side substrate and also indicates that the substrate that binds at the enzyme' A‐site will interact with the enzyme' zinc ion bound by three cysteines (133, 135, and 143). Inhibitor binding caused a substantial ordering of the active site flap (residues 217–235), which was largely invisible in the native electron density map and indicates that this highly conserved yet flexible region has a specific role in substrate binding during catalysis.
The Ubp6 family of deubiquitinating enzymes contains a ubiquitin‐like domain: SUb
Authors: Wyndham Allison M. , Baker Rohan T. , Chelvanayagam Gareth 
Published: February 2009

AbstractA sequence motif that is Similar to Ubiquitin (SUb) has been identified in the Saccharomyces cerevisiae ubiquitin‐specific protease Ubp6. SUb is conserved in all known Ubp6 homologues from a spectrum of eukaryotic species and is also present in a group of hypothetical proteins of unknown function (Unk1–3) present in sequence databases. An N‐terminal deletion mutant of Ubp6 that lacks SUb is still capable of cleaving α‐linked ubiquitin fusions, suggesting that SUb forms a separate domain to the catalytic core of Ubp6 and demonstrating that it is not required for in vitro cleavage activity. A homology model of the 78 N‐terminal amino acids of human Ubp6, based on the known fold of ubiquitin, is presented. In human Ubp6, SUb shares only 20% sequence identity with ubiquitin. Even weaker similarity occurs between S. cerevisiae SUb and ubiquitin. The homology model supports a ubiquitin‐like fold for SUb and suggests that two conserved Lys residues, corresponding to Lys48 and Lys63 of ubiquitin, are functionally important.
Probing the role of water in the tryptophan repressor‐operator complex
Authors: Brown Martha P. , Grillo Adeola O. , Boyer Mireille , Royer Catherine A. 
Published: February 2009

AbstractThe Escherichia coli tryptophan repressor protein (TR) represses the transcription of several genes in response to the concentration of tryptophan in the environment. In the co‐crystal structure of TR bound to a DNA fragment containing its target very few direct contacts between TR and the DNA were observed. In contrast, a number of solvent mediated contacts were apparent. NMR solution structures, however, did not resolve any solvent mediated bonds at the complex interface. To probe for the role of water in TR operator recognition, the effect of osmolytes on the interactions between TR and a target oligonucleotide bearing the operator site was examined. In the absence of specific solvent mediated hydrogen bonding interactions between the protein and the DNA, increasing osmolyte concentration is expected to strongly stabilize the TR operator interaction due to the large amount of macromolecular surface area buried upon complexation. The results of our studies indicate that xylose did not alter the binding affinity significantly, while glycerol and PEG had a small stabilizing effect. A study of binding as a function of betaine concentration revealed that this osmolyte at low concentration results in a stabilization of the 1:1 TR/operator complex, but at higher concentrations leads to a switching between binding modes to favor tandem binding. Analysis of the effects of betaine on the 1:1 complex suggest that this osmolyte has about 78% of the expected effect. If one accepts the analysis in terms of the number of water molecules excluded upon complexation, these results suggest that about 75 water molecules remain at the interface of the 1:1 dimer/DNA complex. This value is consistent with the number of water molecules found at the interface in the crystallographically determined structure and supports the notion that interfacial waters play an important thermodynamic role in the specific complexation of one TR dimer with its target DNA. However, the complexity of the effects of betaine and the small or negligible effects of the other osmolytes could also arise from osmolyte induced competition between antagonistic coupled reactions.
The 80s loop of the catalytic chain of Escherichia coli aspartate transcarbamoylase is critical for catalysis and homotropic cooperativity
Authors: Macol Christine , Dutta Malini , Stec Boguslaw , Tsuruta Hiro , Kantrowitz Evan R. 
Published: February 2009

AbstractThe X‐ray structure of the Escherichia coli aspartate transcarbamoylase with the bisubstrate analog phosphonacetyl‐L‐aspartate (PALA) bound shows that PALA interacts with Lys84 from an adjacent catalytic chain. To probe the function of Lys84, site‐specific mutagenesis was used to convert Lys84 to alanine, threonine, and asparagine. The K84N and K84T enzymes exhibited 0.08 and 0.29% of the activity of the wild‐type enzyme, respectively. However, the K84A enzyme retained 12% of the activity of the wild‐type enzyme. For each of these enzymes, the affinity for aspartate was reduced 5‐ to 10‐fold, and the affinity for carbamoyl phosphate was reduced 10‐ to 30‐fold. The enzymes K84N and K84T exhibited no appreciable cooperativity, whereas the K84A enzyme exhibited a Hill coefficient of 1.8. The residual cooperativity and enhanced activity of the K84A enzyme suggest that in this enzyme another mechanism functions to restore catalytic activity. Modeling studies as well as molecular dynamics simulations suggest that in the case of only the K84A enzyme, the lysine residue at position 83 can reorient into the active site and complement for the loss of Lys84. This hypothesis was tested by the creation and analysis of the K83A enzyme and a double mutant enzyme (DM) that has both Lys83 and Lys84 replaced by alanine. The DM enzyme has no cooperativity and exhibited 0.18% of wild‐type activity, while the K83A enzyme exhibited 61% of wild‐type activity. These data suggest that Lys84 is not only catalytically important, but is also essential for binding both substrates and creation of the high‐activity, high‐affinity active site. Since low‐angle X‐ray scattering demonstrated that the mutant enzymes can be converted to the R‐structural state, the loss of cooperativity must be related to the inability of these mutant enzymes to form the high‐activity, high‐affinity active site characteristic of the R‐functional state of the enzyme.
New insight into the pH‐dependent conformational changes in bovine β‐lactoglobulin from Raman optical activity
Authors: Blanch Ewan W. , Hecht Lutz , Barron Laurence D. 
Published: February 2009

AbstractWe have studied the conformation of β‐lactoglobulin in aqueous solution at room temperature over the pH range ∼2.0–9.0 using vibrational Raman optical activity (ROA). The ROA spectra clearly show that the basic up and down β‐barrel core is preserved over the entire pH range, in agreement with other studies. However, from the shift of a sharp positive ROA band at ∼1268 to ∼1294 cm−1 on going from pH values below that of the Tanford transition, which is centered at pH ∼7.5, to values above, the Tanford transition appears to be associated with changes in the local conformations of residues in loop sequences possibly corresponding to a migration into the α‐helical region of the Ramachandran surface from a nearby region. These changes may be related to those detected in X‐ray crystal structures which revealed that the Tanford transition is associated with conformational changes in loops which form a doorway to the interior of the protein. The results illustrate how the ability of ROA to detect loop and turn structure separately from secondary structure is useful for studying conformational plasticity in proteins.
Selective association of protein molecules followed by mass spectrometry
Authors: Vis Hans , Dobson Christopher M. , Robinson Carol V. 
Published: February 2009

AbstractNanoflow electrospray mass spectrometry was used to monitor the formation of protein heterodimers of HU proteins from Bacillus stearothermophilus and Bacillus subtilis. This has enabled us to analyze both thermodynamic and kinetic features associated with the dissociation of homodimeric HU proteins. The results obtained correlate well with the kinetics of the protein dissociation process and the free energy difference between homo‐ and heterodimeric species anticipated from other studies. We suggest that this approach will have general applicability in studying protein association and dissociation under near‐equilibrium conditions and will be relevant to a wide range of biological systems.
Electrostatic interactions in the GCN4 leucine zipper: Substantial contributions arise from intramolecular interactions enhanced on binding
Authors: Hendsch Zachary S. , Tidor Bruce 
Published: February 2009

AbstractThe GCN4 leucine zipper is a peptide homodimer that has been the subject of a number of experimental and theoretical investigations into the determinants of affinity and specificity. Here, we utilize this model system to investigate electrostatic effects in protein binding using continuum calculations. A particularly novel feature of the computations made here is that they provide an interaction‐by‐interaction breakdown of the electrostatic contributions to the free energy of docking that includes changes in the interaction of each functional group with solvent and changes in interactions between all pairs of functional groups on binding. The results show that (1) electrostatic effects disfavor binding by roughly 15 kcal/mol due to desolvation effects that are incompletely compensated in the bound state, (2) while no groups strongly stabilize binding, the groups that are most destabilizing are charged and polar side chains at the interface that have been implicated in determining binding specificity, and (3) attractive intramolecular interactions (e.g., backbone hydrogen bonds) that are enhanced on binding due to reduced solvent screening in the bound state contribute significantly to affinity and are likely to be a general effect in other complexes. A comparison is made between the results obtained in an electrostatic analysis carried out calculationally and simulated results corresponding to idealized data from a scanning mutagenesis experiment. It is shown that scanning experiments provide incomplete information on interactions and, if overinterpreted, tend to overestimate the energetic effect of individual side chains that make attractive interactions. Finally, a comparison is made between the results available from a continuum electrostatic model and from a simpler surface‐area dependent solvation model. In this case, although the simpler model neglects certain interactions, on average it performs rather well.
Long‐range effects in protein–ligand interactions mediate peptide specificity inl the human major histocompatibility antigen HLA‐B27 (B*2701)
Authors: Krebs Stefan , Rognan Didier , Castro JOSÉ A.LÓPez De 
Published: February 2009

AbstractB*2701 differs from all other HLA‐B27 subtypes of known peptide specificity in that, among its natural peptide ligands, arginine is not the only allowed residue at peptide position 2. Indeed, B*2701 is unique in binding many peptides with Gln2 in vivo. However, the mutation (Asp74Tyr) responsible for altered selectivity is far away from the B pocket of the peptide binding site to which Gln/Arg2 binds. Here, we present a model that explains this effect. It is proposed that a new rotameric state of the conserved Lys70 is responsible for the unique B*2701 binding motif. This side chain should be either kept away from pocket B through its interaction with Asp74 in most HLA‐B27 subtypes, or switched to this pocket if residue 74 is Tyr as in B*2701. Involvement of Lys70 in pocket B would thus allow binding of peptides with Gln2. Binding of Arg2‐containing peptides to B*2701 is also possible because Lys70 could adopt another conformation, H‐bonded to Asn97, which preserves the same binding mode of Arg2 as in B*2705. This model was experimentally validated by mutating Lys70 into Ala in B*2701. Edman sequencing of the B*2701(K70A) peptide pool showed only Arg2, characteristic of HLA‐B27‐bound peptides, and no evidence for Gln2. This supports the computational model and demonstrates that allowance of B*2701 for peptides with Gln2 is due to the long‐range effect of the polymorphic residue 74 of HLA‐B27, by inducing a conformational switch of the conserved Lys70.
Antibody variable region binding by Staphylococcal protein A: Thermodynamic analysis and location of the Fv binding site on E‐domain
Authors: Starovasnik Melissa A. , O'Connell Mark P. , Fairbrother Wayne J. , Kelley Robert F. 
Published: February 2009

AbstractImmunoglobulins of human heavy chain subgroup III have a binding site for Staphylococcal protein A on the heavy chain variable domain (VH), in addition to the well‐known binding site on the Fc portion of the antibody. Thermodynamic characterization of this binding event and localization of the Fv‐binding site on a domain of protein A is described. Isothermal titration calorimetry (ITC) was used to characterize the interaction between protein A or fragments of protein A and variants of the hu4D5 antibody Fab fragment. Analysis of binding isotherms obtained for titration of hu4D5 Fab with intact protein A suggests that 3–4 of the five immunoglobulin binding domains of full length protein A can bind simultaneously to Fab with a Ka of 5.5 ± 0.5 × 105 M–1. A synthetic single immunoglobulin binding domain, Z‐domain, does not bind appreciably to hu4D5 Fab, but both the E and D domains are functional for hu4D5 Fab binding. Thermodynamic parameters for titration of the E‐domain with hu4D5 Fab are n = 1.0 ± 0.1, Ka = 2.0 ± 0.3 × 105 M–1, and ΔH = –7.1 ± 0.4 kcal mol–1. Similar binding thermodynamics are obtained for titration of the isolated VH domain with E‐domain indicating that the E‐domain binding site on Fab resides within VH. E‐domain binding to an IgG1 Fc yields a higher affinity interaction with thermodynamic parameters n = 2.2 ± 0.1, Ka > 1.0 × 107 M–1, and ΔH = –24.6 ± 0.6 kcal mol–1. Fc does not compete with Fab for binding to E‐domain indicating that the two antibody fragments bind to different sites. Amide 1H and 15N resonances that undergo large changes in NMR chemical shift upon Fv binding map to a surface defined by helix‐2 and helix‐3 of E‐domain, distinct from the Fc‐binding site observed in the crystal structure of the B‐domain/Fc complex. The Fv‐binding region contains negatively charged residues and a small hydrophobic patch which complements the basic surface of the region of the VH domain implicated previously in protein A binding.
Relating structure to thermodynamics: The crystal structures and binding affinity of eight OppA‐peptide complexes
Authors: Davies Thomas G. , Hubbard Roderick E. , Tame JEREMY R.H. 
Published: February 2009

AbstractThe oligopeptide‐binding protein OppA provides a useful model system for studying the physical chemistry underlying noncovalent interactions since it binds a variety of readily synthesized ligands. We have studied the binding of eight closely related tripeptides of the type Lysine‐X‐Lysine, where X is an abnormal amino acid, by isothermal titration calorimetry (ITC) and X‐ray crystallography. The tripeptides fall into three series of ligands, which have been designed to examine the effects of small changes to the central side chain. Three ligands have a primary amine as the second side chain, two have a straight alkane chain, and three have ring systems. The results have revealed a definite preference for the binding of hydrophobic residues over the positively charged side chains, the latter binding only weakly due to unfavorable enthalpic effects. Within the series of positively charged groups, a point of lowest affinity has been identified and this is proposed to arise from unfavorable electrostatic interactions in the pocket, including the disruption of a key salt bridge. Marked entropy‐enthalpy compensation is found across the series, and some of the difficulties in designing tightly binding ligands have been highlighted.