Comparison of solvent-inaccessible cores of homologous proteins: definitions useful for protein modelling

The three-dimensional structures of 41 homologous proteins (belongingto eight families) were compared by pairwise superposition.A subset of "core" residues was defined as those whose sidechains have <7% of their surface exposed to solvent. Thissubset has significantly higher sequence identity and lowerroot mean square (RMS) carbon separation than for all topologicallyequivalent residues in the structure, when members of a proteinfamily are superposed. For such superpositions the relationshipbetween RMS distance and percentage sequence identity of thissubset of residues is similar to that for all equivalent residues,although some variation is observed between families of proteinswhich are predominantly ß sheet and those which aremainly helix. The definition of a structurally more conservedcore may be ueful in model building proteins from an homologousfamily. The RMS differences of coordinates of structures ofproteins with identical sequences are found to be related tothe resolutions of the structures.     

Knowledge based modelling of homologous proteins, part II: rules for the conformations of substituted sidechains

This paper describes a rapid, automated procedure which canbe used for model building sidechains using (i) spatial informationfrom sidechains in topologically equivalent positions as faras such a correlation is observed, and then (ii) most probableconformations of the sidechains in the respective secondarystructure type. Analysis of topologically equivalent residuesin the structurally conserved regions of a family of proteinsimplies that the spatial positions of the atoms in the sidechainsrather than conformations should be considered when model building.Rules for the modelling of all 20 sidechains from each otherin -helical, ß-Sheet and loop regions—a totalof 1200—are established. Cluster analysis is used on positionaldata from the sidechain atoms of structurally equivalent residuesin an homologous family to guide modelling. The most probableconformation for the sidechain is used for modelling atoms whereno useful guidance is obtainable from equivalent sidechainsof the homologous proteins. In order to test the procedure wehave modelled the sidechains of the residues in the structurallyconserved regions of myo globin from four other globins. Theautomated procedure described here has been incorporated intothe program COMPOSER.     

Dimeric character of a basic phospholipase A2 from cobra venom: experimental and modelling study

When it is gel filtered on Sephadex in the absence of calciumions, basic phospholipase A₂ from Naja nigricollis venom elutesas a dimer. In order to study the possibility of this dimerizationfrom a structural point of view, three-dimensional models ofboth monomeric and dimeric N. nigricollis phospholipases A havebeen graphically built on the basis of homologies with the phospholipasesA₂ from pancreatic bovine and Crotalus atrox venom. The buildingof a duneric model is made possible by the deletion of a particularloop of the bovine structure. The predicted models of N. nigricollisphospholipase A₂ have been checked using molecular mechanicsand molecular dynamics techniques according to a suitable protocolwhich has been developed starting from refined X-ray structuresof phospholipases A₂ as the test case. The observed stabilityof the dimeric model, in the absence of calcium, agrees withthe hypothesis of the dimerization of the basic phospholipaseA Particularly, Arg31, which replaces the hydrophobic residuepresent in pancreatic bovine and C.atrox venom phospholipasesA₂, contributes to this stability.     

Determination of three-dimensional structures of proteins by simulated annealing with interproton distance restraints. Application to crambin, potato carboxypeptidase inhibitor and barley serine proteinase inhibitor 2

An automated method, based on the principle of simulated annealing,is presented for determining the three-dimensional structuresof proteins on the basis of short (<5 Å) interprotondistance data derived from nuclear Overhauser enhancement (NOE)measurements. The method makes use of Newton's equations ofmotion to increase temporarily the temperature of the systemin order to search for the global minimum region of a targetfunction comprising purely geometric restraints. These consistof interproton distances supplemented by bond lengths, bondangles, planes and soft van der Waals repulsion terms. The latterreplace the dihedral, van der Waals, electrostatic and hydrogen-bondingpotentials of the empirical energy function used in moleculardynamics simulations. The method presented involves the implementationof a number of innovations over our previous restrained moleculardynamics approach [Clore,G.M., Brünger,A.T., Karplus,M.and Gronenborn,A.M. (1986) J. Mol. Biol., 191, 523–551].These include the development of a new effective potential forthe interproton distance restraints whose functional form isdependent on the magnitude of the difference between calculatedand target values, and the design and implementation of robustand fully automatic protocol. The method is tested on threesystems: the model system crambin (46 residues) using X-raystructure derived interproton distance restraints, and potatocarboxypeptidase inhibitor (CPI; 39 residues) and barley serineproteinase inhibitor 2 (BSPI-2; 64 residues) using experimentallyderived interproton distance restraints. Calculations were carriedout starting from the extended strands which had atomic r.m.s.differences of 57, 38 and 33 Å with respect to the crystalstructures of BSPI-2, crambin and CPI respectively. Unbiasedsampling of the conformational space consistent with the restraintswas achieved by varying the random number seed used to assignthe initial velocities. This ensures that the different trajectoriesdiverge during the early stages of the simulations and onlyconverge later as more and more interproton distance restraintsare satisfied. The average backbone atomic r.m.s. differencebetween the converged structures is 2.2 ± 0.3 Åfor crambin (nine structures), 2.4 ± 0.3 Å forCPI (eight structures) and 2.5 ± 0.2 Å for BSPI-2(five structures). The backbone atomic r.m.s. difference betweenthe mean structures derived by averaging the coordinates ofthe converged structures and the corresponding X-ray structuresis 1.2 Å for crambin, 1.6 Å for CPI and 1.7 Åfor BSPI-2.     

An approach to protein homology modelling based on an ensemble of NMR structures: application to the Sox-5 HMG-box protein

A new approach has been developed to reduce multiple proteinstructures obtained from NMR structure analysis to a smallernumber of representative structures which still reflect thestructural diversity of the data sets. The method, based onthe clustering of similar structures, has been tested in thehomology model building of the structure of Sox-5, a sequence-specificDNA-binding protein belonging to the high mobility group (HMG)nuclear proteins family. Sox (SRY box) genes are the autosomalgenes related to the sex-determining SRY, Y chromosomal gene.The Sox-5 protein, encoded by one of the SRY-related genes,displays a 29% sequence identity with the HMG1 B-box domainwhose structure, determined previously by NMR, has been usedin our study to predict the structure of Sox-5. Two independentensembles of HMG1 structures, each represented by closely relatedcoordinate sets, were used. Nine representative structures forHMG1 were subsequently selected as starting points for the modellingof Sox-5. The model of the protein shows close similarity tothe HMG1 fold, with differences at the secondary structure levellocated mainly in a-helices 1 and 3. A left-handed, three residueper turn polyproline II helix, forming a conserved polyprolineII/-helix supersecondary motif, was identified in the N-terminalregion of Sox-5 and other HMG boxes.     

Structural basis for the extreme thermostability of D-glyceraldehyde-3-phosphate dehydrogenase from Thermotoga maritima: analysis based on homology modelling

D-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from a hyperthermophiliceubacterium, Thermotoga maritima, is remarkably heat stable(T_m = 109°C). In this work, we have applied homology modellingto predict the 3-D structure of Th.maritima GAPDH to revealthe structural basis of thermostability. Three known GAPDH structureswere used as reference proteins. First, the rough model of onesubunit was constructed using the identified structurally conservedand variable regions of the reference proteins. The holoenzymewas assembled from four subunits and the NAD molecules. Thestructure was refined by energy minimization and molecular dynamicssimulated annealing. No errors were detected in the refinedmodel using the 3-D profile method. The model was compared withthe structure of Bacillus stearothermophilus GAPDH to identifystructural details underlying the increased thermostability.In all, 12 extra ion pairs per subunit were found at the proteinsurface. This seems to be the most important factor responsiblefor thermostability. Differences in the non-specific interactions,including hydration effects, were also found. Minor changeswere detected in the secondary structure. The model predictsthat a slight increase in a-helical propensities and helix-dipoleinteractions also contribute to increased stability, but toa lesser degree.     

Mix'n'Match: an improved multiple sequence alignment procedure for distantly related proteins using secondary structure predictions, designed to be independent of the choice of gap penalty and scoring matrix

A new multiple sequence alignment procedure is presented. Severaldifferent multiple alignments are made using differing criteria.Having divided the sequences into strongly conserved regions(SCRs) and loosely conserved regions (LCRs), the ‘best’alignment for each LCR is chosen, independently of the otherLCRs, from a selection of possibilities in the multiple alignments.To help make this choice for each LCR, the secondary structureis predicted and shown alongside each different possible alignment.One advantage of this method over automatic, non-interactivemethods, is that the final alignment is not dependent on thechoice of a single set of scoring parameters. Another is that,by allowing interactive choice and by taking account of secondarystructural information, the final alignment is based more onbiological rather than mathematical factors. This method canproduce better alignments than any of the initial automaticmultiple alignment methods used.