bluuebunny/crossref_metadata_2025_split huggingface dataset:
Pig heart short chain L‐3‐hydroxyacyl‐CoA dehydrogenase revisited: Sequence analysis and crystal structure determination
Authors: Barycki Joseph J. , O'Brien Laurie K. , Birktoft Jens J. , Strauss Arnold W. , Banaszak Leonard J. 
Published: February 2009

AbstractShort chain L‐3‐hydroxyacyl CoA dehydrogenase (SCHAD) is a soluble dimeric enzyme critical for oxidative metabolism of fatty acids. Its primary sequence has been reported to be conserved across numerous tissues and species with the notable exception of the pig heart homologue. Preliminary efforts to solve the crystal structure of the dimeric pig heart SCHAD suggested the unprecedented occurrence of three enzyme subunits within the asymmetric unit, a phenomenon that was thought to have hampered refinement of the initial chain tracing. The recently solved crystal coordinates of human heart SCHAD facilitated a molecular replacement solution to the pig heart SCHAD data. Refinement of the model, in conjunction with the nucleotide sequence for pig heart SCHAD determined in this paper, has demonstrated that the previously published pig heart SCHAD sequence was incorrect. Presented here are the corrected amino acid sequence and the high resolution crystal structure determined for pig heart SCHAD complexed with its NAD+ cofactor (2.8 Å; Rcryst = 22.4%, Rfree = 28.8%). In addition, the peculiar phenomenon of a dimeric enzyme crystallizing with three subunits contained in the asymmetric unit is described.
X‐ray crystal structures of a severely desiccated protein
Authors: Bell Jeffrey A. 
Published: February 2009

AbstractUnlike most protein crystals, form IX of bovine pancreatic ribonuclease A diffracts well when severely dehydrated. Crystal structures have been solved after 2.5 and 4 days of desiccation with CaSO4, at 1.9 and 2.0 Å resolution, respectively. The two desiccated structures are very similar. An RMS displacement of 1.6 Å is observed for main‐chain atoms in each structure when compared to the hydrated crystal structure with some large rearrangements observed in loop regions. The structural changes are the result of intermolecular contacts formed by strong electrostatic interactions in the absence of a high dielectric medium. The electron density is very diffuse for some surface loops, consistent with a very disordered structure. This disorder is related to the conformational changes. These results help explain conformational changes during the lyophilization of protein and the associated phenomena of denaturation and molecular memory.
Outer membrane protein A of E. coli folds into detergent micelles, but not in the presence of monomeric detergent
Authors: Kleinschmidt Jörg H. , Wiener Michael C. , Tamm Lukas K. 
Published: February 2009

AbstractOuter membrane protein A (OmpA) of Escherichia coli is a β‐barrel membrane protein that unfolds in 8 M urea to a random coil. OmpA refolds upon urea dilution in the presence of certain detergents or lipids. To examine the minimal requirements for secondary and tertiary structure formation in β‐barrel membrane proteins, folding of OmpA was studied as a function of the hydrophobic chain length, the chemical structure of the polar headgroup, and the concentration of a large array of amphiphiles. OmpA folded in the presence of detergents only above a critical minimal chain length of the apolar chain as determined by circular dichroism spectroscopy and a SDS‐PAGE assay that measures tertiary structure formation. Details of the chemical structure of the polar headgroup were unimportant for folding. The minimal chain length required for folding correlated with the critical micelle concentration in each detergent series. Therefore, OmpA requires preformed detergent micelles for folding and does not adsorb monomeric detergent to its perimeter after folding. Formation of secondary and tertiary structure is thermodynamically coupled and strictly dependent on the interaction with aggregated amphiphiles.
Barrel structures in proteins: Automatic identification and classification including a sequence analysis of TIM barrels
Authors: Nagano Nozomi , Gail HUTCHINSON E. , Thornton Janet M. 
Published: February 2009

AbstractAutomated methods for identifying and characterizing regular β‐barrels from coordinate data have been developed to analyze and classify various kinds of barrel structures based on geometric parameters such as the barrel strand number (n) and shear number (S). In total, we find 1,316 barrels in the January 1998 release of Protein Data Bank. Of 1,316 barrels, 1,277 barrels had an even shear number, corresponding to 50 nonhomologous families. The (βα)8 triose phosphate isomerase (TIM) barrel (n = 8, S = 8) fold has the largest number of apparently nonhomologous entries, 16, although the trypsin like antiparallel (n = 6, S = 8) barrels (representing only three families) are the most common with 527 barrels. Of all the protein families that exhibit barrel structures, 68% are found to be various kinds of enzymes, the remainder being binding proteins and transport membrane proteins. In addition, the layers of side chains, which form the cores of barrels with S = n and S = 2n, are also analyzed. More sophisticated methods were developed for detecting TIM barrels specifically, including consideration of the amino acid propensities for the side chains that form the layers. We found that the residues on the outside of the eight stranded parallel β‐barrel, buried by the α‐helices, are much more hydrophobic than the residues inside the barrel.
Mutants of ETS domain PU.1 and GGAA/T recognition: Free energies and kinetics
Authors: Pio F. , Assa‐Munt N. , Yguerabide J. , Maki R. A. 
Published: February 2009

AbstractThe ETS family members display specific DNA binding site preferences. As an example, PU.1 and ETS‐1 recognize different DNA sequences with a core element centered over 5′‐GGAA‐3′ and 5′‐GGAA/T‐3′, respectively. To understand the molecular basis of this recognition, we carried out site‐directed mutagenesis experiments followed by DNA binding studies that use electrophoretic mobility shift assay (EMSA) and surface plasmon resonance methods. EMSA experiments identified amino acid changes A231S and/or N236Y as being important for PU.1 recognition of both 5′‐GGAA‐3′ and 5′‐GGAT‐3′ containing oligonucleotides. To confirm these data and obtain accurate binding parameters, we performed kinetic studies using surface plasmon resonance on these mutants. The N236Y substitution revealed a weak protein‐DNA interaction with the 5′‐GGAA‐3′ containing oligonucleotide caused by a faster release of the protein from the DNA (koff tenfold higher than the wild‐type protein). With the double mutant A231S‐N236Y, we obtained an increase in binding affinity and stability toward both 5′‐GGAA‐3′ and 5′‐GGAT‐3′ containing oligonucleotides. We propose that substitution of alanine for serine introduces an oxygen atom that can accept hydrogen and interact with potential water molecules or other atoms to make an energetically favorable hydrogen bond with both 5′‐GGAA‐3′ and 5′‐GGAT‐3′ oligonucleotides. The free energy of dissociation for the double mutant A231S‐N236Y with 5′‐GGAA‐3′ (ΔΔG((A231S‐N236Y) – (N236Y)) = ‐ 1.2 kcal mol−1) confirm the stabilizing effect of this mutant in the protein‐DNA complex formation. We conclude that N236Y mutation relaxes the specificity toward 5′‐GGAA‐3′ and 5′‐GGAT‐3′ sequences, while A231S mutation modulates the degree of specificity toward 5′‐GGAA‐3′ and 5′GGAT‐3′ sequences. This study explains why wild‐type PU.1 does not recognize 5′‐GGAT‐3′ sequences and in addition broadens our understanding of 5′‐GGAA/T‐3′ recognition by ETS protein family members.
Photoaffinity labeling probe for the substrate binding site of human phenol sulfotransferase (SULT1A1): 7‐Azido‐4‐methylcoumarin
Authors: Chen Guangping , Battaglia Eric , Senay Claire , Falany Charles N. , Radominska‐Pandya Anna 
Published: February 2009

AbstractA novel fluorescent photoactive probe 7‐azido‐4‐methylcoumarin (AzMC) has been characterized for use in photoaffinity labeling of the substrate binding site of human phenol sulfotransferase (SULT1A1 or P‐PST‐1). For the photoaffinity labeling experiments, SULT1A1 cDNA was expressed inEscherichia colias a fusion protein to maltose binding protein (MBP) and purified to apparent homogeneity over an amylose column. The maltose moiety was removed by Factor Xa cleavage. Both MBSULT1A1 and SULT1A1 were efficiently photolabeled with AzMC. This labeling was concentration dependent. In the absence of light, AzMC competitively inhibited the sulfation of 4MU catalyzed by SULT1A1 (Ki= 0.47 ± 0.05 mM). Moreover, enzyme activity toward 2‐naphthol was inactivated in a timeand concentration‐dependent manner. SULT1A1 inactivation by AzMC was protected by substrate but was not protected by cosubstrate. These results indicate that photoaffinity labeling with AzMC is highly suitable for the identification of the substrate binding site of SULT1A1. Further studies are aimed at identifying which amino acids modified by AzMC are localized in the binding site.
Mutagenic analysis of conserved arginine residues in and around the novel sulfate binding pocket of the human Theta class glutathione transferase T2‐2
Authors: Flanagan J. U. , Rossjohn J. , Parker M. W. , Board P. G. , Chelvanayagam G. 
Published: February 2009

AbstractThe human Theta class glutathione transferase GSTT2‐2 has a novel sulfatase activity that is not dependent on the presence of a conserved hydrogen bond donor in the active site. Initial homology modeling and the crystallographic studies have identified three conserved Arg residues that contribute to the formation of (Arg107 and Arg239), and entry to (Arg242), a sulfate binding pocket. These residues have been individually mutated to Ala to investigate their potential role in substrate binding and catalysis. The mutation of Arg107 had a significant detrimental effect on the sulfatase reaction, while the Arg242 mutation caused only a small reduction in sulfatase activity. Surprisingly, the Arg239 had an increased activity resulting from a reduction in stability. Thus, Arg239 appears to play a role in maintaining the architecture of the active site. Electrostatic calculations performed on the wild‐type and mutant forms of the enzyme are in good agreement with the experimental results. These findings, along with docking studies, suggest that prior to conjugation, the location of 1‐menaphthyl sulfate, a model substrate for the sulfatase reaction, is approximately midway between the position ultimately occupied by the naphthalene ring of 1‐menaphthylglutathione and the free sulfate. It is further proposed that the Arg residues in and around the sulfate binding pocket have a role in electrostatic substrate recognition.
Incorporation of noncoded amino acids into the N‐terminal domain 1‐47 of hirudin yields a highly potent and selective thrombin inhibitor
Authors: Filippis Vincenzo De , Russo Ilaria , Vindigni Alessandro , Cera Enrico Di , Salmaso Stefano , Fontana Angelo 
Published: February 2009

AbstractHirudin is an anticoagulant polypeptide isolated from a medicinal leech that inhibits thrombin with extraordinary potency (Kd = 0.2‐1.0 pM) and selectivity. Hirudin is composed of a compact N‐terminal region (residues 1‐47, cross‐linked by three disulfide bridges) that binds to the active site of thrombin, and a flexible C‐terminal tail (residues 48‐64) that interacts with the exosite I of the enzyme. To minimize the sequence of hirudin able to bind thrombin and also to improve its therapeutic profile, several N‐terminal fragments have been prepared as potential anti‐coagulants. However, the practical use of these fragments has been impaired by their relatively poor affinity for the enzyme, as given by the increased value of the dissociation constant (Kd) of the corresponding thrombin complexes (Kd = 30‐400 nM). The aim of the present study is to obtain a derivative of the N‐terminal domain 1‐47 of hirudin displaying enhanced inhibitory potency for thrombin compared to the natural product. In this view, we have synthesized an analogue of fragment 1‐47 of hirudin HM2 in which Val1 has been replaced by tert‐butylglycine, Ser2 by Arg, and Tyr3 by β‐naphthylalanine, to give the BugArgNal analogue. The results of chemical and conformational characterization indicate that the synthetic peptide is able to fold efficiently with the correct disulfide topology (Cys6–Cys14, Cys16–Cys28, Cys22–Cys37), while retaining the conformational properties of the natural fragment. Thrombin inhibition data indicate that the effects of amino acid replacements are perfectly additive if compared to the singly substituted analogues (De Filippis V, Quarzago D, Vindigni A, Di Cera E, Fontana A, 1998, Biochemistry 37:13507‐13515), yielding a molecule that inhibits the fast or slow form of thrombin by 2,670‐ and 6,818‐fold more effectively than the natural fragment, and that binds exclusively at the active site of the enzyme with an affinity (Kd,fast = 15.4 pM, Kd,slow = 220 pM) comparable to that of full‐length hirudin (Kd,fast = 0.2 pM, Kd,slow = 5.5 pM). Moreover, BugArgNal displays absolute selectivity for thrombin over the other physiologically important serine proteases trypsin, plasmin, factor Xa, and tissue plasminogen activator, up to the highest concentration of inhibitor tested (10 μM).
Molecular dynamics investigation of the effect of an antiviral compound on human rhinovirus
Authors: Phelps Donald K. , Post Carol Beth 
Published: February 2009

AbstractThe factors that influence the enhanced stability observed experimentally of human rhinovirus 14 (HRV14) upon binding a hydrophobic antiviral drug have been investigated by molecular dynamics. Simulations centered about the HRV14 drug‐binding pocket allow the reliable assessment of differences in capsid protein motions of HRV14 and drug‐bound HRV14. We propose that the experimentally observed stabilization of the ligated virus arises from higher entropy, rather than enthalpy. Time‐averaged interaction energies between the viral protein and molecules occupying the pocket are less favorable in the presence of the drug, consistent with the proposal that the observed stability arises from entropie effects. Interaction energies characterizing subunit‐subunit contacts within one viral protomer are found to be substantially stronger than those between two protomers. Such distinction in subunit interaction would have clear implications on assembly and disassembly. Drug binding is found to affect large‐scale, collective properties, while leaving local atomic properties unperturbed. Specifically, the simulations reveal a weakening of long‐range correlations in atomic motions upon drug binding. On the other hand, neither the fast time scale RMS fluctuations of individual atomic positions nor the fluctuation build‐up curves from the capsid β‐sandwich forming the drug‐binding pocket show a consistent distinction between the drug‐bound and drug‐free viral simulations. Collectively, the detailed description available from the simulations provides an understanding of the experimental observations on the drug‐induced changes in thermal stability and protease sensitivity reported for picornaviruses. The predicted significance of binding entropy can be explored experimentally and should be considered in the design of new antiviral compounds.
The role of position a in determining the stability and oligomerization state of α‐helical coiled coils: 20 amino acid stability coefficients in the hydrophobic core of proteins
Authors: Wagschal Kurt , Tripet Brian , Mant Colin , Hodges Robert S. , Lavigne Pierre 
Published: February 2009

AbstractWe describe here a systematic investigation into the role of position a in the hydrophobic core of a model coiled‐coil protein in determining coiled‐coil stability and oligomerization state. We employed a model coiled coil that allowed the formation of an extended three‐stranded trimeric oligomerization state for some of the analogs; however, due to the presence of a Cys‐Gly‐Gly linker, unfolding occurred from the same two‐stranded monomeric oligomerization state for all of the analogs. Denaturation from a two‐stranded state allowed us to measure the relative contribution of 20 different amino acid side chains to coiled‐coil stability from chemical denaturation profiles. In addition, the relative hydrophobicity of the substituted amino acid side chains was assessed by reversed‐phase high‐performance liquid chromatography and found to correlate very highly (R = 0.95) with coiled‐coil stability. We also determined the effect of position a in specifying the oligomerization state using ultracentrifugation as well as high‐performance size‐exclusion chromatography. We found that nine of the analogs populated one oligomerization state exclusively at peptide concentrations of 50 μM under benign buffer conditions. The Leu‐, Tyr‐, Gln‐, and His‐substituted analogs were found to be exclusively three‐stranded trimers, while the Asn‐, Lys‐, Orn‐, Arg‐, and Trp‐substituted analogs formed exclusively two‐stranded monomers. Modeling results for the Leu‐substituted analog showed that a three‐stranded oligomerization state is preferred due to increased side‐chain burial, while a two‐stranded oligomerization state was observed for the Trp analog due to unfavorable cavity formation in the three‐stranded state.
The complexed structure and antimicrobial activity of a non‐β‐lactam inhibitor of AmpC β‐lactamase
Authors: Powers Rachel A. , Blázquez Jesús , Weston G. Scott , Shoichet Brian K. , Morosini María‐Isabel , Baquero Fernando 
Published: February 2009

Abstractβ‐Lactamases are the major resistance mechanism to β‐lactam antibiotics and pose a growing threat to public health. Recently, bacteria have become resistant to β‐lactamase inhibitors, making this problem pressing. In an effort to overcome this resistance, non‐β‐lactam inhibitors of β‐lactamases were investigated for complementarity to the structure of AmpC β‐lactamase from Escherichia coli. This led to the discovery of an inhibitor, benzo (b)thiophene‐2‐boronic acid (BZBTH2B), which inhibited AmpC with a Kit of 27 nM. This inhibitor is chemically dissimilar to β‐lactams, raising the question of what specific interactions are responsible for its activity. To answer this question, the X‐ray crystallographic structure of BZBTH2B in complex with AmpC was determined to 2.25 Å resolution. The structure reveals several unexpected interactions. The inhibitor appears to complement the conserved, R1‐amide binding region of AmpC, despite lacking an amide group. Interactions between one of the boronic acid oxygen atoms, Tyr150, and an ordered water molecule suggest a mechanism for acid/base catalysis and a direction for hydrolytic attack in the enzyme catalyzed reaction. To investigate how a non‐β‐lactam inhibitor would perform against resistant bacteria, BZBTH2B was tested in antimicrobial assays. BZBTH2B significantly potentiated the activity of a third‐generation cephalosporin against AmpC‐producing resistant bacteria. This inhibitor was unaffected by two common resistance mechanisms that often arise against β‐lactams in conjunction with β‐lactamases. Porin channel mutations did not decrease the efficacy of BZBTH2B against cells expressing AmpC. Also, this inhibitor did not induce expression of AmpC, a problem with many β‐lactams. The structure of the BZBTH2B/AmpC complex provides a starting point for the structure‐based elaboration of this class of non‐β‐lactam inhibitors.
Mutations of endo‐β‐N‐acetylglucosaminidase H active site residues Asp 130 and Glu 132: Activities and conformations
Authors: Rao Vibha , Roey Patrick Van , Cui Tao , Guan Chudi 
Published: February 2009

AbstractEndo‐β‐N‐acetylglucosaminidase H hydrolyzes the β‐(1‐4)‐glycosidic link of the N,N′‐diacetylchitobiose core of high‐mannose and hybrid asparagine‐linked oligosaccharides. Seven mutants of the active site residues, Asp130 and Glu132, have been prepared, assayed, and crystallized. They include single site mutants of each residue to the corresponding amide, to Ala and to the alternate acidic residue, and to the double amide mutant. The mutants of Asp130 are more active than the corresponding Glu132 mutants, consistent with the assignment of the latter residue as the primary catalytic residue. The amide mutants are more active than the alternate acidic residue mutants, which in turn are more active than the Ala mutants. The structures of the Asn mutant of Asp130 and the double mutant are very similar to that of the wild‐type enzyme. Several residues surrounding the mutated residues, including some that form part of the core of the β‐barrel and especially Tyr168 and Tyr244, adopt a very different conformation in the structures of the other two mutants of Asp 130 and in the Asp mutant of Glu132. The results show that the residues in the upper layers of the β‐barrel can organize into two very distinct packing arrangements that depend on subtle electrostatic and steric differences and that greatly affect the geometry of the substrate‐binding cleft. Consequently, the relative activities of several of the mutants are defined by structural changes, leading to impaired substrate binding, in addition to changes in functionality.
Immunochemical evidence that cholesteryl ester transfer protein and bactericidal/permeability‐increasing protein share a similar tertiary structure
Authors: Guyard‐Dangremont Valérie , Tenekjian Viken , Chauhan Vinita , Walter Stephanie , Milne Ross , Roy Patrice , Rassart Eric 
Published: February 2009

AbstractCholesteryl ester transfer protein (CETP) plays an important role in plasma lipoprotein metabolism through its ability to transfer cholesteryl ester, triglyceride, and phospholipid between lipoproteins. CETP is a member of a gene family that also includes bactericidal/permeability‐increasing protein (BPI). The crystal structure of BPI shows it to be composed of two domains that share a similar structural fold that includes an apolar ligand‐binding pocket. As structurally important residues are conserved between BPI and CETP, it is thought that CETP and BPI may have a similar overall conformation. We have previously proposed a model of CETP structure based on the binding characteristics of anti‐CETP monoclonal antibodies (mAbs). We now present a refined epitope map of CETP that has been adapted to a structural model of CETP that uses the atomic coordinates of BPI. Four epitopes composed of CETP residues 215‐219, 219‐223, 223‐227, and 444‐450, respectively, are predicted to be situated on the external surface of the central β‐sheet and a fifth epitope (residues 225‐258) on an extended linker that connects the two domains of the molecule. Three other epitopes, residues 317‐331, 360‐366, and 393‐410, would form part of the putative carboxyterminal β‐barrel. The ability of the corresponding mAbs to compete for binding to CETP is consistent with the proximity of the respective epitopes in the model. These results thus provide experimental evidence that is consistent with CETP and BPI having similar surface topologies.
X‐ray crystallographic analysis of the structural basis for the interaction of pokeweed antiviral protein with guanine residues of ribosomal RNA
Authors: Kurinov I. V. , Rajamohan F. , Venkatachalam T. K. , Uckun F. M. 
Published: February 2009

AbstractPokeweed antiviral protein (PAP) is a ribosome‐inactivating protein (RIP), which enzymatically removes a single adenine base from a conserved, surface exposed loop sequence of ribosomal rRNA. We now present unprecedented experimental evidence that PAP can release not only adenine but guanine as well from Escherichia coli rRNA, albeit at a rate 20 times slower than for adenine. We also report X‐ray structure analysis and supporting modeling studies for the interactions of PAP with guanine. Our modeling studies indicated that PAP can accommodate a guanine base in the active site pocket without large conformational changes. This prediction was experimentally confirmed, since a guanine base was visible in the active site pocket of the crystal structure of the PAP‐guanine complex.