bluuebunny/crossref_metadata_2025_split huggingface dataset:
OLDERADO: On‐line database of ensemble representatives and domains
Authors: Kelley Lawrence A. , Sutcliffe Michael J. 
Published: February 2009

AbstractIn cases where the structure of a single protein is represented by an ensemble of conformations, there is often a need to determine the common features and to choose a “representative” conformation. This occurs, for example, with structures determined by NMR spectroscopy, analysis of the trajectory from a molecular dynamics simulation, or an ensemble of structures produced by comparative modeling. We reported previously automatic methods for (1) defining the atoms with low spatial variance across an ensemble (i.e., the “core” atoms) and the domains in which these atoms lie, and (2) clustering an ensemble into conformationally related subfamilies. To extend the utility of these methods, we have developed a freely available server on the World Wide Web at http://neon.chem.le.ac.uk/olderado/. This (1) contains an automatically generated database of representative structures, core atoms, and domains determined for 449 ensembles of NMR‐derived protein structures in the Protein Data Bank (PDB) in May 1997, and (2) allows the user to upload a PDB‐formatted file containing the coordinates of an ensemble of structures. The server returns in real time: (1) information on the residues constituting domains; (2) the structures that constitute each conformational subfamily; and (3) an interactive java‐based three‐dimensional viewer to visualise the domains and clusters. Such information is useful, for example, when selecting conformations to be used in comparative modeling and when choosing parts of structures to be used in molecular replacement. Here we describe the OLDERADO server.
A diverse superfamily of enzymes with ATP‐dependent carboxylate—amine/thiol ligase activity
Authors: Galperin Michael Y. , Koonin Eugene V. 
Published: February 2009

AbstractThe recently developed PSI‐BLAST method for sequence database search and methods for motif analysis were used to define and expand a superfamily of enzymes with an unusual nucleotide‐binding fold, referred to as palmate, or ATP‐grasp fold. In addition to d‐alanine‐d‐alanine ligase, glutathione synthetase, biotin carboxylase, and carbamoyl phosphate synthetase, enzymes with known three‐dimensional structures, the ATP‐grasp domain is predicted in the ribosomal protein S6 modification enzyme (RimK), urea amidolyase, tubulin‐tyrosine ligase, and three enzymes of purine biosynthesis. All these enzymes possess ATP‐dependent carboxylate‐amine ligase activity, and their catalytic mechanisms are likely to include acylphosphate intermediates. The ATP‐grasp superfamily also includes succinate‐CoA ligase (both ADP‐forming and GDP‐forming variants), malate‐CoA ligase, and ATP‐citrate lyase, enzymes with a carboxylate‐thiol ligase activity, and several uncharacterized proteins. These findings significantly extend the variety of the substrates of ATP‐grasp enzymes and the range of biochemical pathways in which they are involved, and demonstrate the complementarity between structural comparison and powerful methods for sequence analysis.
Crystal structure of heat‐labile enterotoxin from Escherichia coli with increased thermostability introduced by an engineered disulfide bond in the A subunit
Authors: Van Den Akker F. , Feil I. K. , Roach C. , Platas A. A. , Merritt E. A. , Hol Wim G. J. 
Published: February 2009

AbstractCholera toxin (CT) produced by Vibrio cholerae and heat‐labile enterotoxin (LT‐I), produced by enterotoxigenic Escherichia coli, are AB5 heterohexamers with an ADP‐ribosylating A subunit and a GM1 receptor binding B pentamer. These toxins are among the most potent mucosal adjuvants known and, hence, are of interest both for the development of anti‐diarrheal vaccines against cholera or enterotoxigenic Escherichia coli diarrhea and also for vaccines in general. However, the A subunits of CT and LT‐I are known to be relatively temperature sensitive. To improve the thermostability of LT‐I an additional disulfide bond was introduced in the A1 subunit by means of the double mutation N40C and G166C. The crystal structure of this double mutant of LT‐I has been determined to 2.0 Å resolution. The protein structure of the N40C/G166C double mutant is very similar to the native structure except for a few local shifts near the new disulfide bond. The introduction of this additional disulfide bond increases the thermal stability of the A subunit of LT‐I by 6°C. The enhancement in thermostability could make this disulfide bond variant of LT‐I of considerable interest for the design of enterotoxin‐based vaccines.
Expression, characterization, and crystallization of a member of the novel phospholipase D family of phosphodiesterases
Authors: Zhao Yi , Stuckey Jeanne A. , Lohse Daniel L. , Dixon Jack E. 
Published: February 2009

AbstractA family of phospholipase D (PLD) proteins has recently been identified (Koonin, 1996; Ponting & Kerr, 1996) based upon amino acid sequence identity. This family includes human and plant PLDs, proteins encoded by open reading frames in pathogenic viruses and bacteria, as well as an endonuclease. The endo‐nuclease, known as Nuc, is encoded by the IncN plasmid, pKM101, present in Salmonella typhimurium. The recombinant Nuc protein has been expressed and purified from Escherichia coli. The amino‐terminal sequencing of the purified protein indicated that the mature protein started from the 23rd residue of the predicted sequence, suggesting that the protein is proteolytically processed during export to the periplasmic space. The recombinant enzyme was able to hydrolyze both double and single‐strand DNA and an artificial substrate, bis(4‐nitrophenyl) phosphate, which contains a phosphodiester bond. The enzyme activity was not inhibited in the presence of EDTA and was not regulated by divalent cations. The purified protein has been crystallized by hanging drop vapor diffusion methods, and those crystals diffract to 1.9 Å resolution.
Complete measurement of the pKavalues of the carboxyl and imidazole groups inBacillus circulansxylanase
Authors: Joshi Manish D. , Hedberg Anders , Mcintosh Lawrence P. 
Published: February 2009

AbstractElectrostatic interactions in proteins can be dissected experimentally by determining the pKavalues of their constituent ionizable amino acids. To complement previous studies of the glutamic acid and histidine residues inBacillus circulansxylanase (BCX), we have used NMR methods to measure the pKas of the seven aspartic acids and the C‐terminus of this protein. The pKas of these carboxyls are all less than the corresponding values observed with random coil polypeptides, indicating that their ionization contributes favorably to the stability of the folded enzyme. In general, the aspartic acids with the most reduced pKas are those with limited exposure to the solvent and a high degree of conservation among homologous xylanases. Most dramatically, Asp 83 and Asp 101 have pKas < 2 and thus remain deprotonated in native BCX under all conditions examined. Asp 83 is completely buried, forming a strong salt bridge with Arg 136. In contrast, Asp 101 is located on the surface of the protein, stabilized in the deprotonated form by an extensive network of hydrogen bonds involving an internal water molecule and the neutral side‐chain and main‐chain atoms of Ser 100 and Thr 145. These data provide a complete experimental database for theoretical studies of the ionization behavior of BCX under acidic conditions.
HIV‐1 Nef protein: Purification, crystallizations, and preliminary X‐ray diffraction studies
Authors: Franken P. , Arold S. , Padilla A. , Hoh E , Strub M. P. , Boyer M. , Jullien M. , Dumas C. , Bodeus M. , Benarous R. 
Published: February 2009

AbstractHuman immunodeficiency virus Nef protein accelerates virulent progression of AIDS by its interaction with specific cellular proteins involved in cellular activation and signal transduction. Here we report the purification and crystallization of the conserved core of HTV‐1LAI Nef protein in the unliganded form and in complex with the wild‐type SH3 domain of the p59fyn protein‐tyrosine kinase. One‐dimensional NMR experiments show that full‐length protein and truncated fragment corresponding to the product of HIV‐1 protease cleavage have a well‐folded compact tertiary structure. The ligand‐free HIV‐1 Nefcore protein forms cubic crystals belonging to space group P23 with unit cell dimensions of a = b = c = 86.4 Å. The Nef‐Fyn SH3 cocrystals belong to the space group P6122 or its enantiomorph, P6522, with unit cell dimensions of a = b = 108.2 Å and c = 223.7 Å. Both crystal forms diffract to a resolution limit of 3.0 Å resolution using synchrotron radiation, and are thus suitable for X‐ray structure determination.
Refolding rate of stability‐enhanced cytochrome c is independent of thermodynamic driving force
Authors: McGee William A. , Nall Barry T. 
Published: February 2009

AbstractN52I iso‐2 cytochrome c is a variant of yeast iso‐2 cytochrome c in which asparagine substitutes for isoleucine 52 in an alpha helical segment composed of residues 49–56. The N52I substitution results in a significant increase in both stability and cooperativity of equilibrium unfolding, and acts as a “global suppressor” of destabilizing mutations. The equilibrium m‐value for denaturant‐induced unfolding of N52I iso‐2 increases by 30%, a surprisingly large amount for a single residue substitution. The folding/unfolding kinetics for N52I iso‐2 have been measured by stopped‐flow mixing and by manual mixing, and are compared to the kinetics of folding/unfolding of wild‐type protein, iso‐2 cytochrome c. The results show that the observable folding rate and the guanidine hydrochloride dependence of the folding rate are the same for iso‐2 and N52I iso‐2, despite the greater thermodynamic stability of N52I iso‐2. Thus, there is no linear free‐energy relationship between mutation‐induced changes in stability and observable refolding rates. However, for N52I iso‐2 the unfolding rate is slower and the guanidine hydrochloride dependence of the unfolding rate is smaller than for iso‐2. The differences in the denaturant dependence of the unfolding rates suggest that the N52I substitution decreases the change in the solvent accessible hydrophobic surface between the native state and the transition state. Two aspects of the results are inconsistent with a two‐state folding/unfolding mechanism and imply the presence of folding intermediates: (1) observable refolding rate constants calculated from the two‐state mechanism by combining equilibrium data and unfolding rate measurements deviate from the observed refolding rate constants; (2) kinetically unresolved signal changes (“burst phase”) are observed for both N52I iso‐2 and iso‐2 refolding. The “burst phase” amplitude is larger for N52I iso‐2 than for iso‐2, suggesting that the intermediates formed during the “burst phase” are stabilized by the 52I substitution.
Motifs and structural fold of the cofactor binding site of human glutamate decarboxylase
Authors: Qu Kunbin , Martin David L. , Lawrence Charles E. 
Published: February 2009

AbstractThe pyridoxal‐P binding sites of the two isoforms of human glutamate decarboxylase (GAD65 and GAD67) were modeled by using PROBE (a recently developed algorithm for multiple sequence alignment and database searching) to align the primary sequence of GAD with pyridoxal‐P binding proteins of known structure. GAD's cofactor binding site is particularly interesting because GAD activity in the brain is controlled in part by a regulated interconversion of the apo‐ and holoenzymes. PROBE identified six motifs shared by the two GADs and four proteins of known structure: bacterial ornithine decarboxylase, dialkylglycine decarboxylase, aspartate aminotransferase, and tyrosine phenol‐lyase. Five of the motifs corresponded to the α/β elements and loops that form most of the conserved fold of the pyridoxal‐P binding cleft of the four enzymes of known structure; the sixth motif corresponded to a helical element of the small domain that closes when the substrate binds. Eight residues that interact with pyridoxal‐P and a ninth residue that lies at the interface of the large and small domains were also identified. Eleven additional conserved residues were identified and their functions were evaluated by examining the proteins of known structure. The key residues that interact directly with pyridoxal‐P were identical in ornithine decarboxylase and the two GADs, thus allowing us to make a specific structural prediction of the cofactor binding site of GAD. The strong conservation of the cofactor binding site in GAD indicates that the highly regulated transition between apo‐ and holoGAD is accomplished by modifications in this basic fold rather than through a novel folding pattern.
Crystal structures of the Klenow fragment of Thermus aquaticus DNA polymerase I complexed with deoxyribonucleoside triphosphates
Authors: Li Ying , Kong Yong , Korolev Sergey , Waksman Gabriel 
Published: February 2009

AbstractThe crystal structures of the Klenow fragment of the Thermus aquaticus DNA polymerase I (Klentaq 1) complexed with four deoxyribonucleoside triphosphates (dNTP) have been determined to 2.5 Å resolution. The dNTPs bind adjacent to the O helix of Klentaq 1. The triphosphate moieties are at nearly identical positions in all four complexes and are anchored by three positively charged residues, Arg659, Lys663, and Arg587, and by two polar residues, His639 and Gln613. The configuration of the base moieties in the Klentaq 1/dNTP complexes demonstrates variability suggesting that dNTP binding is primarily determined by recognition and binding of the phosphate moiety. However, when superimposed on the Taq polymerase/blunt end DNA complex structure (Eom et al., 1996), two of the dNTP/Klentaql structures demonstrate appropriate stacking of the nucleotide base with the 3′ end of the DNA primer strand, suggesting that at least in these two binary complexes, the observed dNTP conformations are functionally relevant.
The role of polar interactions in the molecular recognition of CD40L with its receptor CD40
Authors: Singh Juswinder , Garber Ellen , Vlijmen Herman Van , Karpusas Michael , Hsu Yen‐Ming , Zheng Zhongli , Thomas David , Naismith James H. 
Published: February 2009

AbstractCD40 Ligand (CD40L) is transiently expressed on the surface of T‐cells and binds to CD40, which is expressed on the surface of B‐cells. This binding event leads to the differentiation, proliferation, and isotype switching of the B‐cells. The physiological importance of CD40L has been demonstrated by the fact that expression of defective CD40L protein causes an immunodeficiency state characterized by high IgM and low IgG serum levels, indicating faulty T‐cell dependent B‐cell activation. To understand the structural basis for CD40L/CD40 association, we have used a combination of molecular modeling, mutagenesis, and X‐ray crystallography. The structure of the extracellular region of CD40L was determined by protein crystallography, while the CD40 receptor was built using homology modeling based upon a novel alignment of the TNF receptor superfamily, and using the X‐ray structure of the TNF receptor as a template. The model shows that the interface of the complex is composed of charged residues, with CD40L presenting basic side chains (K143, R203, R207), and CD40 presenting acidic side chains (D84, El14, El17). These residues were studied experimentally through site‐directed mutagenesis, and also theoretically using electrostatic calculations with the program Delphi. The mutagenesis data explored the role of the charged residues in both CD40L and CD40 by switching to Ala (K143A, R203A, R207A of CD40L, and E74A, D84A, E114A, E117A of CD40), charge reversal (K143E, R203E, R207E of CD40L, and D84R, El 14R, El 17R of CD40), mutation to a polar residue (K143N, R207N, R207Q of CD40L, and D84N, El 17N of CD40), and for the basic side chains in CD40L, isosteric substitution to a hydrophobic side chain (R203M, R207M). All the charge‐reversal mutants and the majority of the Met and Ala substitutions led to loss of binding, suggesting that charged interactions stabilize the complex. This was supported by the Delphi calculations which confirmed that the CD40/CD40L residue pairs E74‐R203, D84‐R207, and El 17‐R207 had a net stabilizing effect on the complex. However, the substitution of hydrophilic side chains at several of the positions was tolerated, which suggests that although charged interactions stabilize the complex, charge per se is not crucial at all positions. Finally, we compared the electrostatic surface of TNF/TNFR with CD40L/CD40 and have identified a set of polar interactions surrounded by a wall of hydrophobic residues that appear to be similar but inverted between the two complexes.
Two “unrelated” families of ATP‐dependent enzymes share extensive structural similarities about their cofactor binding sites
Authors: Denessiouk Konstantin A. , Korpela Timo , Lehtonen Jukka V. , Johnson Mark S. 
Published: February 2009

AbstractTwo proteins, D‐alanine:D‐alanine ligase and cAMP‐dependent protein kinase, share a remarkable degree of structural convergence despite having different three‐dimensional folds and different enzymatic functions. Here we report that as many as 103 residues from 10 segments form two identical super‐secondary structures between which the cofactor ATP is bound. The cofactor, two bound metal cations, and several water molecules form a large network of electrostatic and hydrophobic interactions common to both enzymes, and these are mediated by the similar placement of equivalent amino acids within the common supersecondary structures.
Enhanced thermal stability of Clostridium beijerinckii alcohol dehydrogenase after strategic substitution of amino acid residues with prolines from the homologous thermophilic Thermoanaerobacter brockii alcohol dehydrogenase
Authors: Bogin Oren , Peretz Moshe , Hacham Yael , Burstein Yigal , Korkhin Yakov , Kalb(gilboa) A. Joseph , Frolow Felix 
Published: February 2009

AbstractA comparison of the three‐dimensional structures of the closely related mesophilic Clostridium beijerinckii alcohol dehydrogenase (CBADH) and the hyperthermophilic Thermoanaerobacter brockii alcohol dehydrogenase (TBADH) suggested that extra proline residues in TBADH located in strategically important positions might contribute to the extreme thermal stability of TBADH. We used site‐directed mutagenesis to replace eight complementary residue positions in CBADH, one residue at a time, with proline. All eight single‐proline mutants and a double‐proline mutant of CBADH were enzymatically active. The critical sites for increasing thermostability parameters in CBADH were Leu‐316 and Ser‐24, and to a lesser degree, Ala‐347. Substituting proline for His‐222, Leu‐275, and Thr‐149, however, reduced thermal stability parameters. Our results show that the thermal stability of the mesophilic CBADH can be moderately enhanced by substituting proline at strategic positions analogous to nonconserved prolines in the homologous thermophilic TBADH. The proline residues that appear to be crucial for the increased thermal stability of CBADH are located at a β‐turn and a terminating external loop in the polypeptide chain. Positioning proline at the N‐caps of α‐helices in CBADH led to adverse effects on thermostability, whereas single‐proline mutations in other positions in the polypeptide had varying effects on thermal parameters. The finding presented here support the idea that at least two of the eight extra prolines in TBADH contribute to its thermal stability.
Reactivation of thermally inactivated pre‐β‐lactamase by DnaK, DnaJ, and GrpE
Authors: Mccarthy Diane , Kramer Gisela , Hardesty Boyd 
Published: February 2009

AbstractTo understand the role of the 23‐amino acid signal sequence in the folding and stability of β‐lactamase, the precursor and a mutantβ‐lactamase with a 19‐amino acid signal sequence deletion were synthesized in vitro using anEscherichia colicell‐free coupled transcription/translation system. Approximately 30% of the newly synthesized full‐length precursor and 60% of the deletion mutant polypeptides were terminated and released from the ribosomes as active enzyme. Activity of the pre‐β‐lactamase, but not the mutant, was unstable at 37 °C, suggesting that the signal sequence causes the enzyme to unfold. This inactivation was independent of ATP. Pre‐β‐lactamase activity was stabilized by lowering the temperature to 30 °C. Furthermore, addition of the molecular chaperones DnaK/J and GrpE, in the presence of ATP and Mg2+, restored the activity of the temperature‐inactivated precursor. The precursor formed a stable complex with DnaK and GrpE. Both ATP and DnaJ were required for recovery of enzymatic activity, indicating that DnaJ may bind transiently to the complex. These results suggest that the signal sequence of the pre‐β‐lactamase causes a temperature‐dependent unfolding of the synthesized enzyme and that DnaK/J and GrpE interact with unfolded pre‐β‐lactamase to promote refolding of the protein into its native, enzymatically active conformation.
Topology of sarcoplasmic reticulum Ca2+‐ATPase: An infrared study of thermal denaturation and limited proteolysis
Authors: Echabe Izaskun , Prado Adelina , Goñi Fé Lix M. , Arondo José Luis R. , Dornberger Utz 
Published: February 2009

AbstractSarcoplasmic reticulum Ca2+‐ATPase structure and organization in the membrane has been studied by infrared spectroscopy by decomposition of the amide I band. Besides the component bands assignable to secondary structure elements such as α‐helix, β‐sheet, etc…, two unusual bands, one at 1,645 cm −1 in H2O buffer and the other at 1,625 cm−1 in D2O buffer are present. By perturbing the protein using temperature and limited proteolysis, the band at 1,645 cm−1 is tentatively assigned to α‐helical segments located in the cytoplasmic domain and coupled to β‐sheet structure, whereas the band at 1,625 cm−1 arises probably from monomer‐monomer contacts in the native oligomeric protein. The secondary structure obtained is 33% α‐helical segments in the transmembrane plus stalk domain; 20% α‐helix and 22% β‐sheet in the cytoplasmic domain plus 19% turns and 6% unordered structure. Thermal unfolding of Ca2+‐ATPase is a complex process that cannot be described as a two‐state denaturation. The results obtained are compatible with the idea that the protein is an oligomer at room temperature. The loss of the 1,625 cm −1 band upon heating would be consistent with a disruption of the oligomers in a process that later gives rise to aggregates (appearance of the 1,618 cm −1 band). This picture would also be compatible with early results suggesting that processes governing Ca2+ accumulation and ATPase activity are uncoupled at temperatures above 37 °C, so that while ATPase activity proceeds at high rates, Ca2+ accumulation is inhibited.