Structural interpretation of site-directed mutagenesis and specificity of the catalytic subunit of protein kinase CK2 using comparative modelling

The catalytic subunit of protein kinase casein kinase 2 (CK2),which has specificity for both ATP and GTP, shows significantamino acid sequence similarity to the cyclin-dependent kinase2 (CDK2). We constructed site-directed mutants of CK2 and useda three-dimensional model to investigate the basis for the dualspecificity. Introduction of Phe and Gly at positions 50 and51, in order to restore the pattern of the glycine-rich motif,did not seriously affect the specificity for ATP or GTP. Weshow that the dual specificity probably originates from theloop situated around the position His115 to Asp120 (HVNNTD).The insertion of a residue in this loop in CK2 subunits, comparedwith CDK2 and other kinases, might orient the backbone to interactwith the base A and G; this insertion is conserved in all knownCK2. The mutant N118, the design of which was based on the modelling,showed reduced affinity for GTP as predicted from the model.Other mutants were intended to probe the integrity of the catalyticloop, alter the polarity of a buried residue and explore theimportance of the carboxy terminus. Introduction of Arg to replaceAsn189, which is mapped on the activation loop, results in amutant with decreased k_cat, possibly as a result of disruptionof the interaction between this residue and basic residues inthe vicinity. Truncation at position 331 eliminates the last60 residues of the subunit and this mutant has a reduced catalyticefficiency compared with the wild-type. Catalytic efficiencyis restored in the truncation mutant by the replacement of apotentially buried Glu at position 252 by Lys, probably owingto a higher stability resulting from the formation of a saltbridge between Lys252 and Asp208.     

An object-oriented database for protein structure analysis

An object-oriented database system has been developed whichis being used to store protein structure data. The databasecan be queried using the logic programming language Prolog orthe query language Daplex. Queries retrieve information by navigatingthrough a network of objects which represent the primary, secondaryand tertiary structures of proteins. Routines written in bothProlog and Daplex can integrate complex calculations with theretrieval of data from the database, and can also be storedin the database for sharing among users. Thus object-orienteddatabases are better suited to prototyping applications andanswering complex queries about protein structure than relationaldatabases. This system has been used to find loops of varyinglength and anchor positions when modelling homologous proteinstructures.     

Pattern-induced multi-sequence alignment (PUMA) algorithm employing secondary structure-dependent gap penalties for use in comparative protein modelling

A multiple sequence alignment algorithm is described that usesa dynamic programming-based pattern construction method to aligna set of homologous sequences based on their common patternof conserved sequence elements. This pattern-induced multi-sequencealignment (PUMA) algorithm can employ secondary-structure dependentgap penalties for use in comparative modelling of new sequenceswhen the three-dimensional structure of one or more membersof the same family is known. We show that the use of secondarystructure information can significantly improve the accuracyof aligning structure boundaries in a set of homologous sequenceseven when the structure of only one member of the family isknown     

Towards an automatic method of predicting protein stucture by homology: an evaluation of suboptimal sequence alignments

A major problem in predicting protein structure by homologymodelling is that the sequence alignment from which the modelis built may not be the best one in terms of the correct equivalencingof residues assessed by structural or functional criteria. Auseful strategy is to generate and examine a number of suboptimalalignments as better alignments can often be found away fromthe optimal. A procedure to filter rapidly suboptimal alignmentsbased on measurement of core volumes and packing pair potentialsis investigated. The approach is benchmarked on three pairsof sequences which are non-trivial to align correctly, namelytwo immunoglobulin domains, plastocyanin with azurin and twodistant globin sequences. It is shown to be useful to reducea large ensemble of possible alignments down to a few whichcorrespond more closely to the correct (structure based) alignment.     

Easy adaptation of protein structure to sequence

An investigation into the conservation of coarse, medium andfine grain structural properties has been performed over a dataset of 175 protein tertiary structures in 34 different families,each characterized by a common core fold and a library of conservedsites formed for each family. It is shown that, while the conservationof coarse and medium grain properties correlates to the structuraldeviation between the proteins, fine grain properties are poorlyconserved except in functional sites. This flexibility in finegrain properties suggests that folding can be viewed as an optimizationprocess whereby side chains have freedom to position themselvesas best as possible given environmental conformationa] constraintsand that given a basic framework, the local structure is ableto adapt easily to sequence variation. The conserved cores ofthe 34 families are used to estimate a minimal core size of35% of the fold, consistent with buried residue considerations.Finally, conservation in side chain l torsion angles is combinedwith structural deviation, sequence deviation and resolutionto suggest a set of example structure pairs suitable for testingautomatic homology modelling programs     

Protein modelling using a chimera reference protein derived from exons

Bovine pancreatic /S-trypsin (PDB ID-code: 1TPO) which is registeredin the Brookhaven Protein Data Bank (PDB) consists of four exons.The results of homology searches for each exon in the PDB showedthat homologous proteins were tonin (PDB ID-code: 1TON), ratmast cell protease (PDB ID-code: 3RP2_A), kaffikrein A (PDBID-code: 2PKA_B) and kallikrein A (2PKA_B) respectively. Thus,for the three-dimensional structure prediction of 1TPO, a chimeraprotein was constructed from the three proteins mentioned aboveand the 3-D structure prediction was performed using this chimerareference protein. The modelled structure of 1TPO was energeticallyoptimized by molecular mechanics and molecular dynamics simulationand was compared with its X-ray crystal structure registeredin the PDB. The root mean square deviations (r.m.s.d.) of mainchain atoms and the neighbouring active site (5 sphere fromHis57, AsplO2 and Serl95) between the modelled structure andthe X-ray structure were 1.66 and 0.94 respectively. Porcinepancreatic elastase (PDB ID-code: 3EST) which is registeredin the PDB was used as the reference protein and the modelledstructure from 3EST was also compared with the X-ray data. Ther.m.s.d. of main chain atoms and that of the active site were2.14 and 1.18 respectively. These results dearly support thepropriety of this method using the chimera reference protein.     

Enhanced dead-end elimination in the search for the global minimum energy conformation of a collection of protein side chains

Although the conformational states of protein side chains canbe described using a library of rotamers, the determinationof the global minimum energy conformation (GMEC) of a largecollection of side chains, given fixed backbone coordinates,represents a challenging combinatorial problem with importantapplications in the field of homology modelling. Recently, wehave developed a theoretical framework, called the dead-endelimination method, which allows us to identify efficientlyrotamers that cannot be members of the GMEC. Such dead-endingrotamers can be iteratively removed from the system under studythereby tracking down the size of the combinatorial problem.Here we present new developments to the dead-end eliminationmethod that allow us to handle larger proteins and more extensiverotamer libraries. These developments encompass (i) a procedureto determine weight factors in the generalized dead-end eliminationtheorem thereby enhancing the elimination of dead-ending rotamersand (ii) a novel strategy, mainly based on logical argumentsderived from the logic pairs theorem, to use dead-ending rotamerpairs in the efficient elimination of single rotamers. Thesedevelopments are illustrated for proteins of various sizes andthe flow of the current method is discussed in detail. The effectivenessof dead-end elimination is increased by two orders of magnitudeas compared with previous work. In addition, it now becomesfeasible to use extremely detailed libraries. We also providean appendix in which the validity of the generalized dead-endcriterion is shown. Finally, perspectives for further applicationswhich may now become within reach are discussed.     

Comparative modelling of major house dust mite allergen Der p I: structure validation using an extended environmental amino acid propensity table

A model of the 3-D structure of a major house dust mite allergenDer p I associated with hypersensitivity reactions in humanswas built from its amino acid sequence and its homology to threeknown structures, papain, actinidin and papaya proteinase flof the cysteine proteinase family. Comparative modelling usingCOMPOSER was used to arrive at an initial model. This was refinedusing interactive graphics and energy minimization with theAMBER force field incorporated in SYBYL (Tripos Associates).Compatibility of the Der p I amino add sequence with the cysteineproteinase fold was checked using an environment-dependent aminoadd propensity table incorporated into a new program HARMONYwith a variable length windowing facility. A fiveresidue windowwas used to probe local conformational integrity. Propensitieswere derived from a structural alignment database of homologousproteins using a robust entropy-driven smoothing procedure.Der p I shares essential structural and mechanistic featureswith other papain-like cysteine proteinases, including cathepsinB. The active-site t iolate-imidazolium ion pair comprises theside chains of Cys34 and Hisl70. A cystine disulfide not presentin other known structures bridges residue 4 of an N-terminalextension and the core residue 117. Two conserved disulfidebridges are formed by residues 31 and 71 and residues 65 and103. Model building of peptide substrate analogue complexessuggests a preference for phenylalanyl or bask residues at theP₂ position, whilst selectivity may be of minor importance atthe S₁ subsite. The electrostatic influences on the Der p Iactive-site ion pair and extended peptide binding region aremarkedly different from those in known structures. A highlyimmunogenic surface exposed region (residues 107–131),comprising several overlapping T cell epitope sites, has noshared sequence identity with human liver cathepsin B and containsthree insertion-deletion sites. The structure provides a basisfor testing the substrate specificity of Der p I and the potentialrole of proteinase activity in hypersensitivity reactions. Thesestudies may offer a new treatment strategy by hyposensitizationwith inactive mutants or mutants with significantly alteredproteinase activity, either alone or complexed with antibody.     

A study of simulated annealing protocols for use with molecular dynamics in protein structure prediction

The method of simulated annealing can be of use in protein structureprediction by homology modelling where side chain conformationsmust be predicted. In this study an attempt has been made tooptimize a molecular dynamics method for this purpose. Heatingand cooling protocols to maximize the accuracy of the predictionshave been developed. The optimized protocol involves coolingfrom 3000 to 0 K over 20 ps while simultaneously introducingthe non-bonded energy term. The use of a 'soft' non-bonded interactionenergy term in place of a standard 6–12 potential is foundto be important. The reliability of the predictions has beenanalysed in terms of the environment of the residues (solventaccessibility) and the degree of uncertainty in the structure(number of unknown torsion angles). Depending on these factorsthe percentage of unknown side chain torsion angles that arecorrectly predicted within 30° ranges from –50 to75%. Potential problems and limitations of the method are discussed.     

A relational database of protein structures designed for flexible enquiries about conformation

A relational database of protein structure has been developedto enable rapid and flexible enquiries about the occurrenceof many aspects of protein architecture. The coordinates of294 proteins from the Brookhaven Data Bank have been processedby standard computer programs to generate many additional termsthat quantify aspects of protein structure. These terms includesolvent accessibility, main-chain and side-chain dihedral angles,and secondary structure. In a relational database, the informationis stored in tables with columns holding the different termsand rows holding the different entries for the terms. The differentrelational base tables store the information about the proteincoordinate set, the different chains in the protein, the aminoacid residues and ligands, the atomic coordinates, the saltbridges, the hydrogen bonds, the disulphide bridges and theclose tertiary contacts. The database was established underORACLE management system. Enquiries are constructed in ORACLEusing SQL (structured query language) which is simple to useand alleviates the need for extensive computer programs. A singletable can be searched for entries that meet various criteria,e.g. all protein solved to better than a given resolution. Thepower of the database occurs when several tables, or the entriesin a single table, are cross-correlated. For example the dihedralangles of proline in the fourth position in an -helix in highresolution structures can be rapidly obtained. The structuraldatabase provides a powerful tool to obtain empirical rulesabout protein conformation. This database of protein structuresis part of a joint project between Birkbeck College and LeedsUniversity to establish an integrated data resource of proteinsequences and structures (ISIS) that encodes the complex patternsof residues and coordinates that define protein conformation.The entire data resource (ISIS) will provide a system to guideall areas of protein modelling including structure prediction,site-directed mutagenesis and de novo protein design. The availabilityof ISIS is described in the paper.     

Comparative modelling and analysis of amino acid substitutions suggests that the family of pregnancy-associated glycoproteins includes both active and inactive aspartic proteinases

The pregnancy-associated glycoproteins (PAGs) are secretoryproducts synthesized by the outer epithelial cell layer (chorion)of the placentas of various ungulate species. The amino acidsequences of eight PAGs have been inferred from cloned cDNAof cattle and sheep, as well as of the non-ruminant pig andhorse. We compare the PAG sequences and present results of thethree-dimensional models of boPAG-1 and ovPAG-1 that were constructedon the basis of the crystal structures of homologous porcinepepsin and bovine chymosin using a rule-based comparative modellingapproach. Further, we compare peptide binding subsites definedby interactions with pepstatin and a decapeptide inhibitor (CH-66)modelled on the basis of crystal structures of other asparticproteinases. We have extended our analysis of the peptide bindingsubsites to the other PAG molecules of known sequence by aligningthe PAG sequences to the structural template derived from thepepsin family and by making use of the three-dimensional modelsof the boPAG-1 and ovPAG-1. The residues that are likely toaffect peptide binding in the boPAG-1, ovPAG-1 and other PAGmolecules have been identified. Sequence comparisons revealthat all PAG molecules may have evolved from a pepsin-like progenitormolecule with the equine PAG most closely related to the pepsins.The presence of substitutions at the S₁ and other subsites relativeto pepsin make it unlikely that either bovine, ovine or theporcine PAG-1 have catalytic activity. Only two of the eightPAGs examined (porcine PAG-2 and equine PAG-1) retain featuresof active aspartic proteinases with pepsin-like activity. Ourresults indicate that in the PAGs so far characterized the peptidebinding specificities differ significantly from each other andfrom pepsin, despite their high sequence identities. Analysisof the various peptide binding subsites demonstrates why bothbovine and ovine PAG-1 are capable of binding pepstatin. Thestrong negative charge in the binding cleft of boPAG-1 and ovPAG-1indicates a preference for lysine- or arginine-rich peptides.PAGs represent a family where the possible peptide binding functionmay be retained through their binding specificities, but wherethe catalytic activity may be lost in some cases, such as theboPAG-1, ovPAG-1 and the poPAG-1.     

Large-scale modelling as a route to multiple surface comparisons of the CCP module family

Numerous mammalian proteins are constructed from a limited repertoire of module-types. Proteins belonging to the regulators of complement activation family--crucial for ensuring a complement-mediated immune response is targeted against infectious agents--are composed solely of complement control protein (CCP) modules. In the current study, CCP module sequences were grouped to allow selection of the most appropriate experimentally determined structures to serve as templates in an automated large-scale structure modelling procedure. The resulting 135 individual CCP module models, valuable in their own right, are available at the online database http://www.bru.ed.ac.uk/~dinesh/ccp-db.html. Comparisons of surface properties within a particular family of modules should be more informative than sequence alignments alone. A comparison of surface electrostatic features was undertaken for the first 28 CCP modules of complement receptor type 1 (CR1). Assignments to clusters based on surface properties differ from assignments to clusters based on sequences. This observation might reflect adaptive evolution of surface-exposed residues involved in protein-protein interactions. This illustrative example of a multiple surface-comparison was indeed able to pinpoint functional sites in CR1.     

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.     

Modelling the structure of the calcitonin gene-related peptide

The calcitonin gene-related peptide (CGRP) is a 37 residue neuropeptidewhich causes vasodilatation, increases heart rate and inhibitsbone resorption. These effects make it an interesting lead fordrug discovery. We have combined current structural and biologicalinformation to model the structure of hCGRP-ß to beused as a basis for the rational design of novel analogues.Distinct regions of CGRP have been shown to be responsible forthe activity of the whole molecule. Thus, the structure of thepeptide was modelled in four parts which were finally combined.A random search of conformational space was performed for thefragments CGRP1–8 and CGRP30–37 which have beenshown to be central for receptor activation and binding, respectively.Five low-energy hCGRP-ß structures were obtained frommodelled fragments by molecular dynamics. The relevance of theapproach was verified by comparing the models with NMR structuresof CGRP and calcitonin. The models obtained for the N- and C-terminalfragments should enable the design of novel agonists and antagonistsof the CGRP receptor, respectively. Models of the whole moleculemay be used in the design of peptides with shortened spacersbetween the receptor-bound regions. The approach described isapplicable to several related peptide hormones, like growthhormone-releasing hormone and secretin.     

Predictive modelling of the 3-D structure of interleukin-13

Several atomic structures are now available for the family ofhelical cytokines, which includes growth hormone as well asmany of the interleukins. Using structural information fromfive members of this family, two alternative models of interleukin(IL)-13 are proposed. IL-13 has biological properties similarto those of IL-4 and, like the other interleukins, is a potentiallyimportant pharmaceutical target. The model of IL-13 is discussedand compared with the known interleukin structures.     

Neural network system for the evaluation of side-chain packing in protein structures

An artificial neural network system is used for pattern recognitionin protein side-chain-side-chain contact maps. A back-propagationnetwork was trained on a set of patterns which are popular inside-chain contact maps of protein structures. Several neuralnetwork architectures and different training parameters weretested to decide on the best combination for the neural network.The resulting network can distinguish between original (fromprotein structures) and randomized patterns with an accuracyof 84.5% and a Matthews' coefficient of 0.72 for the testingset. Applications of this system for protein structure evaluationand refinement are also proposed. Examples include structuresobtained after the application of molecular dynamics to crystalstructures, structures obtained from X-ray crystallography atvarious stages of refinement, structures obtained from a denovo folding algorithm and deliberately misfolded structures.     

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.     

A novel method for the modelling of peptide ligands to their receptors

A knowledge-based approach to the modelling of enzyme- peptideinhibitor comlexes is described. Given the structure of an enzyme,and knowledge of its bindings site, the method seeks to predictthe binding geometry of a peptide ligand. This novel methodinvolves using examples of sidechain packing derived from proteinsof known three dimensional structure to define possible packingarrangements of a peptide inhibitor group to its bindings site.Asuite of progams, GEMINI, was written and used to predict thepacking of pairs of amino acid groups from three inhibitorscomplexed to their enzymes for which the X-ray strutures wereavailable. These included the Phe group of the inhibitor H142bound to endothiapepsin, the Leu group of CLT complexed to thermolysinand the C-terminus of Gly-L-Tyr bound to carboxypeptidase A.A detailed comparison of the modelled and observed inhibitorcoordinates was made. This approach may be extended to modellingother types of protein interactions.     

Modelling the three-dimensional structure and the electrostatic potential field of two Cu,Zn superoxide dismutase variants from tomato leaves

The three-dimensional structure of tomato P31 and T10 Cu,Znsuperoxide dismutases (SODs) were computer modelled using thestructure of the bovine enzyme as a template. The structure-essentialresidues retain in the models the position occupied in the otherCu,Zn SODs of known 3D structure and the overall packing ofthe ß-barrel is maintained. Formation of ‘aromaticpairs’occurs between newly inserted aromatic residues.The number of total charges changes in the two variants andsome charged residues located in the proximity of the activesite in most Cu,Zn SODs disappear in tomato enzymes. Calculationof the electrostatic potential field, carried out by numericallysolving the Poisson-Boltzmann equation, indicates that in bothvariants a negative potential field surrounds all the proteinsurface except the active site areas, characterized by positivepotential values, as already observed in the bovine enzyme.This result confirms that coordinated mutations of charged residueshave occurred in the evolution of this enzyme giving rise toa peculiar electrostatic potential distribution common to allmembers of this protein family.