bluuebunny/crossref_metadata_2025_split huggingface dataset:
Solution structure of DinI provides insight into its mode of RecA inactivation
Authors: Ramirez Benjamin E. , Bax Ad , Voloshin Oleg N. , Camerini‐otero R. Daniel 
Published: February 2009

AbstractThe Escherichia coli RecA protein triggers both DNA repair and mutagenesis in a process known as the SOS response. The 81‐residue E. coli protein DinI inhibits activity of RecA in vivo. The solution structure of DinI has been determined by multidimensional triple resonance NMR spectroscopy, using restraints derived from two sets of residual dipolar couplings, obtained in bicelle and phage media, supplemented with J couplings and a moderate number of NOE restraints. DinI has an α/β fold comprised of a three‐stranded β‐sheet and two α‐helices. The β‐sheet topology is unusual: the central strand is flanked by a parallel and an antiparallel strand and the sheet is remarkably flat. The structure of DinI shows that six negatively charged Glu and Asp residues on DinI's kinked C‐terminal α‐helix form an extended, negatively charged ridge. We propose that this ridge mimics the electrostatic character of the DNA phospodiester backbone, thereby enabling DinI to compete with single‐stranded DNA for RecA binding. Biochemical data confirm that DinI is able to displace ssDNA from RecA.
The identification of conserved interactions within the SH3 domain by alignment of sequences and structures
Authors: Larson Stefan M. , Davidson Alan R. , Davidson Alan R. 
Published: February 2009

AbstractThe SH3 domain, comprised of approximately 60 residues, is found within a wide variety of proteins, and is a mediator of protein–protein interactions. Due to the large number of SH3 domain sequences and structures in the databases, this domain provides one of the best available systems for the examination of sequence and structural conservation within a protein family. In this study, a large and diverse alignment of SH3 domain sequences was constructed, and the pattern of conservation within this alignment was compared to conserved structural features, as deduced from analysis of eighteen different SH3 domain structures. Seventeen SH3 domain structures solved in the presence of bound peptide were also examined to identify positions that are consistently most important in mediating the peptide‐binding function of this domain. Although residues at the two most conserved positions in the alignment are directly involved in peptide binding, residues at most other conserved positions play structural roles, such as stabilizing turns or comprising the hydrophobic core. Surprisingly, several highly conserved side‐chain to main‐chain hydrogen bonds were observed in the functionally crucial RT‐Src loop between residues with little direct involvement in peptide binding. These hydrogen bonds may be important for maintaining this region in the precise conformation necessary for specific peptide recognition. In addition, a previously unrecognized yet highly conserved β‐bulge was identified in the second β‐strand of the domain, which appears to provide a necessary kink in this strand, allowing it to hydrogen bond to both sheets comprising the fold.
Free energy determinants of tertiary structure and the evaluation of protein models
Authors: Petrey Donald , Honig Barry 
Published: February 2009

AbstractWe develop a protocol for estimating the free energy difference between different conformations of the same polypeptide chain. The conformational free energy evaluation combines the CHARMM force field with a continuum treatment of the solvent. In almost all cases studied, experimentally determined structures are predicted to be more stable than misfolded “decoys.” This is due in part to the fact that the Coulomb energy of the native protein is consistently lower than that of the decoys. The solvation free energy generally favors the decoys, although the total electrostatic free energy (sum of Coulomb and solvation terms) favors the native structure. The behavior of the solvation free energy is somewhat counterintuitive and, surprisingly, is not correlated with differences in the burial of polar area between native structures and decoys. Rather, the effect is due to a more favorable charge distribution in the native protein, which, as is discussed, will tend to decrease its interaction with the solvent. Our results thus suggest, in keeping with a number of recent studies, that electrostatic interactions may play an important role in determining the native topology of a folded protein. On this basis, a simplified scoring function is derived that combines a Coulomb term with a hydrophobic contact term. This function performs as well as the more complete free energy evaluation in distinguishing the native structure from misfolded decoys. Its computational efficiency suggests that it can be used in protein structure prediction applications, and that it provides a physically well‐defined alternative to statistically derived scoring functions.
Three‐dimensional model of the extracellular domain of the type 4a metabotropic glutamate receptor: New insights into the activation process
Authors: Bessis Anne‐Sophie , Acher Francine , Bertrand Hugues‐Olivier , Galvez Thierry , De Colle Cyril , Pin Jean‐Philippe 
Published: February 2009

AbstractMetabotropic glutamate receptors (mGluRs) belong to the family 3 of G‐protein‐coupled receptors. On these proteins, agonist binding on the extracellular domain leads to conformational changes in the 7‐transmembrane domains required for G‐protein activation. To elucidate the structural features that might be responsible for such an activation mechanism, we have generated models of the amino terminal domain (ATD) of type 4 mGluR (mGlu4R). The fold recognition search allowed the identification of three hits with a low sequence identity, but with high secondary structure conservation: leucine isoleucine valine‐binding protein (LIVBP) and leucine‐binding protein (LBP) as already known, and acetamide‐binding protein (AmiC). These proteins are characterized by a bilobate structure in an open state for LIVBP/LBP and a closed state for AmiC, with ligand binding in the cleft. Models for both open and closed forms of mGlu4R ATD have been generated. ACPT‐I (1‐aminocyclopentane 1,3,4‐tricarboxylic acid), a selective agonist, has been docked in the two models. In the open form, ACPT‐I is only bound to lobe I through interactions with Lys74, Arg78, Ser159, and Thr182. In the closed form, ACPT‐I is trapped between both lobes with additional binding to Tyr230, Asp312, Ser313, and Lys317 from lobe II. These results support the hypothesis that mGluR agonists bind a closed form of the ATDs, suggesting that such a conformation of the binding domain corresponds to the active conformation.
Enhanced internal dynamics of a membrane transport protein during substrate translocation
Authors: Döring KLAUS , Surrey Thomas , Grünewald Sylvia , John Edgar , Jähnig Fritz 
Published: February 2009

AbstractConformational changes are essential for the activity of many proteins. If, or how fast, internal fluctuations are related to slow conformational changes that mediate protein function is not understood. In this study, we measure internal fluctuations of the transport protein lactose permease in the presence and absence of substrate by tryptophan fluorescence spectroscopy. We demonstrate that nanosecond fluctuations of α‐helices are enhanced when the enzyme transports substrate. This correlates with previously published kinetic data from transport measurements showing that millisecond conformational transitions of the substrate‐loaded carrier are faster than those in the absence of substrate. These findings corroborate the hypothesis of the hierarchical model of protein dynamics that predicts that slow conformational transitions are based on fast, thermally activated internal motions.
Exploring steric constraints on protein mutations using MAGE/PROBE
Authors: Word J. Michael , Presley Brent K. , Lovell Simon C. , Richardson David C. , Batemanm Robert C. 
Published: February 2009

AbstractWhen planning a mutation to test some hypothesis, one crucial question is whether the new side chain is compatible with the existing structure; only if it is compatible can the interpretation of mutational results be straightforward. This paper presents a simple way of using the sensitive geometry of all‐atom contacts (including hydrogens) to answer that question. The interactive MAGE/PROBE system lets the biologist explore conformational space for the mutant side chain, with an interactively updated kinemage display of its all‐atom contacts to the original structure. The Autobondrot function in PROBE systematically explores that same conformational space, outputting contact scores at each point, which are then contoured and displayed. These procedures are applied here in two types of test cases, with known mutant structures. In ricin A chain, the ability of a neighboring glutamate to rescue activity of an active‐site mutant is modeled successfully. In T4 lysozyme, six mutations to Leu are analyzed within the wild‐type background structure, and their Autobondrot score maps correctly predict whether or not their surroundings must shift significantly in the actual mutant structures; interactive examination of contacts for the conformations involved explains which clashes are relieved by the motions. These programs are easy to use, are available free for UNIX or Microsoft Windows operating systems, and should be of significant help in choosing good mutation experiments or in understanding puzzling results.
Evaluation of PSI‐BLAST alignment accuracy in comparison to structural alignments
Authors: Friedberg Iddo , Kaplan Tommy , Margalit Hanah 
Published: February 2009

AbstractThe PSI‐BLAST algorithm has been acknowledged as one of the most powerful tools for detecting remote evolutionary relationships by sequence considerations only. This has been demonstrated by its ability to recognize remote structural homologues and by the greatest coverage it enables in annotation of a complete genome. Although recognizing the correct fold of a sequence is of major importance, the accuracy of the alignment is crucial for the success of modeling one sequence by the structure of its remote homologue. Here we assess the accuracy of PSI‐BLAST alignments on a stringent database of 123 structurally similar, sequence‐dissimilar pairs of proteins, by comparing them to the alignments defined on a structural basis. Each protein sequence is compared to a nonredundant database of the protein sequences by PSI‐BLAST. Whenever a pair member detects its pair‐mate, the positions that are aligned both in the sequential and structural alignments are determined, and the alignment sensitivity is expressed as the per‐centage of these positions out of the structural alignment. Fifty‐two sequences detected their pair‐mates (for 16 pairs the success was bi‐directional when either pair member was used as a query). The average percentage of correctly aligned residues per structural alignment was 43.5 ± 2.2%. Other properties of the alignments were also examined, such as the sensitivity vs. specificity and the change in these parameters over consecutive iterations. Notably, there is an improvement in alignment sensitivity over consecutive iterations, reaching an average of 50.9 + 2.5% within the five iterations tested in the current study.
Prediction of a common β‐propeller catalytic domain for fructosyltransferases of different origin and substrate specificity
Authors: Pons Tirso , Hernández Lázaro , Batista Frank R. , Chinea Glay 
Published: February 2009

AbstractThe three‐dimensional (3D) structure of fructan biosyn‐thetic enzymes is still unknown. Here, we have explored folding similarities between reported microbial and plant enzymes that catalyze transfructosylation reactions. A sequence‐structure compatibility search using TOPITS, SDP, 3D‐PSSM, and SAM‐T98 programs identified a β‐propeller fold with scores above the confidence thresh‐old that indicate a structurally conserved catalytic domain in fruc‐tosyltransferases (FTFs) of diverse origin and substrate specificity. The predicted fold appeared related to that of neuraminidase and sialidase, of glycoside hydrolase families 33 and 34, respectively. The most reliable structural model was obtained using the crystal structure of neuraminidase (Protein Data Bank file: 5nn9) as template, and it is consistent with the location of previously identified functional residues of bacterial levansucrases (Batista et al., 1999; Song & Jacques, 1999). The sequence–sequence analysis presented here reinforces the recent inclusion of fungal and plant FTFs into glycoside hydrolase family 32, and suggests a modified sequence pattern {H‐x(2)‐[PTV]‐x(4)‐[LIVMA]‐[NSCAYG]‐[DE]‐P‐[NDSC]‐[GA]} for this family.
Design of a minimal protein oligomerization domain by a structural approach
Authors: Burkhard Peter , Meier M. , Lustig Ariel 
Published: February 2009

AbstractBecause of the simplicity and regularity of the α‐helical coiled coil relative to other structural motifs, it can be conveniently used to clarify the molecular interactions responsible for protein folding and stability. Here we describe the de novo design and characterization of a two heptad‐repeat peptide stabilized by a complex network of inter‐ and intrahelical salt bridges. Circular dichroism spectroscopy and analytical ultracentrifugation show that this peptide is highly α‐helical and 100% dimeric under physiological buffer conditions. Interestingly, the peptide was shown to switch its oligomerization state from a dimer to a trimer upon increasing ionic strength. The correctness of the rational design principles used here is supported by details of the atomic structure of the peptide deduced from X‐ray crystallography. The structure of the peptide shows that it is not a molten globule but assumes a unique, native‐like conformation. This de novo peptide thus represents an attractive model system for the design of a molecular recognition system.
Cysteine‐independent polymerization of metallothioneins in solutions and in crystals
Authors: Hou Tingjun , Xu Xiaojie , An Yu , Ru Binggen , Bi Ruchang 
Published: February 2009

AbstractPolymerization of metallothioneins is one of the usually encountered puzzles during the research process of metallothioneins' structure and function. Our work focuses on the cysteine independently occurred polymerization from metallothioneins monomers in different milieus, while it leaves out the aggregation caused by the oxidation of cysteine, because the latter circumstance is the result of purification lapsus. After the purification of metallothioneins monomers, a dynamic light‐scattering technique is used to detect the polymerized states of rabbit liver metallothionein I and II in different buffers, which is the first systematical detection of polymerized states of metallothioneins in solutions. The effects of different compositions of each buffer are discussed in details. Steric complementarity, hydrophobic, and electrostatic interaction characteristics are studied, following the modeling of monomers and relevant polymers of rat metallothionein II, rabbit liver metallothionein I and II. These theoretical calculations are the first complete computer simulations on different factors affecting metallothioneins' polymerization. A molecular recognition mechanism of metallothioneins' polymerization in solutions is proposed on the bases of experimental results and theoretical calculations. Preliminary X‐ray studies of two crystal forms of rabbit liver metallothionein II are compared with the crystal structure of rat metallothionein II, and the polymerized states in crystal packing are discussed with the knowledge of polymerization of metallothioneins in solutions. The hypothesis, which is consistent with theoretical calculations and experimental results, is expected to construct a connection between the biochemical characteristics and physiological functions of metallothioneins, and this research may give some enlightenment to the topics of protein polymerizations.
β‐Helix core packing within the triple‐stranded oligomerization domain of the P22 tailspike
Authors: Kreisberg Jason F. , Betts Scott D. , King Jonathan 
Published: February 2009

AbstractA right‐handed parallel β‐helix of 400 residues in 13 tightly packed coils is a major motif of the chains forming the trimeric P22 tailspike adhesin. The β‐helix domains of three identical subunits are side‐by‐side in the trimer and make predominantly hydrophilic inter‐subunit contacts (Steinbacher S et al., 1994, Science 265:383‐386). After the 13th coil the three individual β‐helices terminate and the chains wrap around each other to form three interdigitated β‐sheets organized into the walls of a triangular prism. The β‐strands then separate and form antiparallel β‐sheets, but still defining a triangular prism in which each side is a β‐sheet from a different subunit (Seckler R, 1998, J Struct Biol 122:216–222). The subunit interfaces are buried in the triangular core of the prism, which is densely packed with hydrophobic side chains from the three β‐sheets. Examination of this structure reveals that its packed core maintains the same pattern of interior packing found in the left‐handed β‐helix, a single‐chain structure. This packing is maintained in both the interdigitated parallel region of the prism and the following antiparallel sheet section. This oligomerization motif for the tailspike β‐helices presumably contributes to the very high thermal and detergent stability that is a property of the native tailspike adhesin.
Stability and peptide binding specificity of Btk SH2 domain: Molecular basis for X‐linked agammaglobulinemia
Authors: Tzeng Shiou‐Ru , Pai Ming‐Tao , Wu Chih‐Wei , Cheng Jya‐Wei , Lung Feng‐Di T. , Roller Peter P. , Lei Benfang , Wei Chih‐Jen , Tu Shiao‐Chun , Chen Shi‐Han , Soong Wen‐Jue 
Published: February 2009

AbstractX‐linked agammaglobulinemia (XLA) is caused by mutations in the Bruton's tyrosine kinase (Btk). The absence of functional Btk leads to failure of B‐cell development that incapacitates antibody production in XLA patients leading to recurrent bacterial infections. Btk SH2 domain is essential for phospholipase C‐γ phosphorylation, and mutations in this domain were shown to cause XLA. Recently, the B‐cell linker protein (BLNK) was found to interact with the SH2 domain of Btk, and this association is required for the activation of phospholipase C‐γ. However, the molecular basis for the interaction between the Btk SH2 domain and BLNK and the cause of XLA remain unclear. To understand the role of Btk in B‐cell development, we have determined the stability and peptide binding affinity of the Btk SH2 domain. Our results indicate that both the structure and stability of Btk SH2 domain closely resemble with other SH2 domains, and it binds with phosphopeptides in the order pYEEI > pYDEP > pYMEM > pYLDL > pYIIP. We expressed the R288Q, R288W, L295P, R307G, R307T, Y334S, Y361C, L369F, and I370M mutants of the Btk SH2 domain identified from XLA patients and measured their binding affinity with the phosphopeptides. Our studies revealed that mutation of R288 and R307 located in the phosphotyrosine binding site resulted in a more than 200‐fold decrease in the peptide binding compared to L295, Y334, Y361, L369, and I370 mutations in the pY + 3 hydrophobic binding pocket (∼3‐ to 17‐folds). Furthermore, mutation of the Tyr residue at the βD5 position reverses the binding order of Btk SH2 domain to pYIIP > pYLDL > pYDEP > pYMEM > pYEEI. This altered binding behavior of mutant Btk SH2 domain likely leads to XLA.
Resonant mirror biosensor analysis of type Iα cAMP‐dependent protein kinase B domain—Cyclic nucleotide interactions
Authors: Muhonen Wallace W. , Shabb John B. 
Published: February 2009

AbstractA resonant mirror biosensor was used to study cyclic nucleotide‐receptor interactions. In particular, a novel method was developed to determine inhibition constants (Ki) from initial rates of ligate association to immobilized ligand. This approach was applied to the comparison of cyclic nucleotide‐binding properties of the wild‐type isolated B domain of the cAMP‐dependent protein kinase type Iα regulatory subunit and its Ala‐334‐Thr (A334T) variant that has altered cyclic nucleotide specificity. A cUMP‐saturated form of the B domain was used for all measurements. Under the conditions used, cUMP did not affect the kinetics of B domain association to immobilized cAMP. Triton X‐100 was required to stabilize the protein at nanomolar concentrations. The association and dissociation rate constants for wild‐type and A334T B domains yielded equilibrium dissociation constants of 11 and 16 nM. Heterogeneity of ligate and immobilized ligand, mass transport effects, and other factors were evaluated for their influence on biosensor‐determined kinetic constants. Biosensor‐determined relative inhibition constants (K'i = KicAMP/K'ianalog) for 16 cyclic nucleotide analogs correlated well with those determined by a [3H]cAMP binding assay. Previously published K'i values for the B domain in the intact regulatory subunit were similar to those of the isolated B domain. The K'i values for the wild‐type and A334T B domains were essentially unchanged except for dramatic enhancements in affinity of cGMP analogs for the A334T B domain. These observations validate the isolated B domain as a simple model system for studying cyclic nucleotide‐receptor interactions.
The design of a hyperstable mutant of the Abp1p SH3 domain by sequence alignment analysis
Authors: Rath Arianna , Davidson Alan R. 
Published: February 2009

AbstractWe have characterized the thermodynamic stability of the SH3 domain from the Saccharomyces cerevisiae Abp1p protein and found it to be relatively low compared to most other SH3 domains, with a Tm of 60 °C and a ΔGu of 3.08 kcal/mol. Analysis of a large alignment of SH3 domains led to the identification of atypical residues at eight positions in the wild‐type Abp1p SH3 domain sequence that were subsequently replaced by the residue seen most frequently at that position in the alignment. Three of the eight mutants constructed in this way displayed increases in Tm ranging from 8 to 15 °C with concomitant increases in ΔGu of up to 1.4 kcal/mol. The effects of these substitutions on folding thermodynamics and kinetics were entirely additive, and a mutant containing all three was dramatically stabilized with a Tm greater than 90 °C and a ΔGu more than double that of the wild‐type domain. The folding rate of this hyperstable mutant was 10‐fold faster than wild‐type, while its unfolding rate was fivefold slower. All of the stabilized mutants were still able to bind a target peptide with wild‐type affinity. We have analyzed the stabilizing amino acid substitutions isolated in this study and several other similar sequence alignment based studies. In approximately 25% of cases, increased stability can be explained by enhanced propensity of the substituted residue for the local backbone conformation at the mutagenized site.
Thirty‐plus functional families from a single motif
Authors: Yu Lihua , Smith Temple F. , Gaitatzes Chrysanthe 
Published: February 2009

AbstractIt is now possible to identify over 30 functional subfamilies among the WD‐repeat‐containing proteins found in the completed genomes. The majority of these subfamilies have at least one member for which experimental data allow assignment to a cellular pathway or process. Half of the 63 WD‐repeat‐containing proteins in Saccharomyces cerevisiae, half of the 70 in Caenorhabditis elegans, and a third of the 100 plus predicted in Drosophila can be assigned to 23 of these functional subfamilies. Perhaps indicative of the future, 33 WD‐repeat‐containing proteins from the partial genome of Arabidopsis thaliana can now be assigned to 18 of these subfamilies. These assignments have been made possible by combining traditional sequence similarity with an implied common beta propeller structural context to obtain measures of protein‐protein surface similarity. The beta propeller structural context is represented in the form of a Hidden Markov Model. The procedure is completely automated.