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.     

Permuteins of interleukin 1{beta}--a simplified approach for the construction of permutated proteins having new termini

A technique for the rapid and simple generation of permutatedversions of the interleukin-1ß (IL-1ß) geneis described. In this method, the human IL-1ß cDNAis twice amplified by the polymerase chain reaction (PCR) andthe resulting DNA fragments are ligated in tandem. Between thetwo genes, the DNA sequence encodes a short four amino acidloop to link the native N- and C-terminal ends of the IL-1ßprotein. By using PCR amplification from this starting template,a new version of the IL-1ß cDNA was obtained thatencodes a permutated form of the IL-1ß protein wherethe new N- and C-terminal amino acids correspond to residues65 and 64 of the native IL-1ß sequence, respectively.The name ‘permutein’ is proposed to describe proteinsgenerated by this technology. The molecular profile (IL-1 receptorbinding, biologic activity and solution properties) of the IL-1permutein produced by this technology, permutein 65/64, is shownto be identical to that of native IL-1ß The approachshould be useful to define further the structural features ofthis protein that are important for its function.     

Improved alignment of weakly homologous protein sequences using structural information

Protein sequence alignments can be unproved when at least oneof the proteins to be aligned has a known 3-D structure. Inthis work, geometrical constraints extracted from the targetfold are evaluated in independent units that deal with complementarystructural features. This information is used to set up mutationtables specific to the locally observed structural environments.The resulting partial evaluations are then combined linearlyinto a global function which is optimized by dynamic programming.Eventually, a score based on tertiary interactions can be usedas a selection criterion to discriminate among a set of suboptimalalignments. The relevance of the scores given by each unit istested on a representative set of protein families. Finally,a method for combining the different scores is described andits efficiency is evaluated on a few pairs of weakly homologousproteins.     

A model for vicilin solubility at mild acidic pH, based on homology modelling and electrostatics calculations

The crystallographic structures of jack bean canavalin and Frenchbean phaseolin have been used to construct a homology modelof the storage vicilin of cocoa. Reported molecular weightsfor cocoa storage protein subunits correlate with proteolysisat the site of a large hydrophilic insert in the mature protein.Burial of the hydrophobic amino acids on trimer formation isa strongly conserved feature in the vicilin family. Histidineresidues also sit at the monomer-monomer interfaces of the trimerand are likely to contribute to the decreased solubility ofcocoa vicilin at mild acidic pH, which is generally consideredto be caused solely by aggregation near to the isoelectric point.Electrostatic calculations suggest that such an arrangementof histidine residues in the absence of specific counterionbinding will not favour the particular geometry of trimer formationbelow neutral pH. Higher order aggregates that do not excludehistidine charge from the solvent may be favoured, aiding theprecipitation of cocoa vicilin at mild acidic pH. This suggestionis considered for the vicilin family. The hypothesis could contributeto an understanding of the pH and ionic strength dependenceof vicilin solubility in vitro, and possibly of the behaviourof vicilins in the seed storage environment     

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     

Homology modelling of rat kallikrein rK9, a member of the tissue kallikrein family: implications for substrate specificity and inhibitor binding

The rat kallikrein rK9 is one of the six members of the rattissue kallikrein family isolated to date. It is 84% identicalto rK2 (tonin), and both proteinases are thought to have vasoconstrictiveproperties. Recently we have shown that rK9 and rK2 have distinctsubstrate specificities and sensitivities to inhibitors, despitetheir similar sequences. Unlike all other mammalian kallikrein-relatedproteinases, rK9 is resistant to inhibition by aprotinin. Wehave developed a 3-D model of rK9, based on the known X-raystructures of rK2, porcine kallikrein and bovine trypsin, toidentify the structural features underlying this functionaldiversity. The final rK9 model is structurally similar to rK2,but variable regions surrounding the active site differ quitemarkedly from the reference proteins. The kallikrein loop, whichdiffers from that in porcine kallikrein by a seven-residue insertion,has been generated de novo and subjected to simulated annealingto assess its influence on the restricted substrate specificityof these proteinases. The proposed conformation of the specificitypocket in rK9 differs from that of other serine proteinases,but it can still accommodate both aromatic and basic amino acidside chains at the substrate P₁ position, thus explaining thedual chymotrypsin and trypsin-like activity of rK9. The electrostaticpotentials of rK9 and aprotinin were calculated using the finitedifference Poisson–Boltzmann method. They indicated alarge positive region near the active site of rK9 not foundin related proteinases because of positively charged residuesat positions 61 and 65 in rK9. They generate a positive region,which overlaps a positive region in aprotinin, and may preventaprotinin binding. A single mutation in aprotinin is suggestedthat might allow kallikrein rK9 inhibition by aprotinin. Thismodel contributes significantly to our understanding of thestructure-function relationships among proteinases of the tissuekallikrein family.     

Citrate synthase from the hyperthermophilic Archaeon, Pyrococcus furiosus

The gene encoding the enzyme citrate synthase has been clonedand sequenced from the hyperthermophilic Archaeon Pyrococcusfuriosus, and the derived amino acid sequence has been phylogeneticallycompared with citrate synthases from archaeal, bacterial andeukaryal organisms.The gene has been over-expressed in Escherichiacoli to produce an active enzyme that has then been characterizedwith respect to its kinetic, oligomeric and hyperthermost-ableproperties. A structurally-based sequence alignment was madeto the citrate synthase from the thermophilic Archaeon Thermoplasmaacidophilum, the crystal structure of which we have determinedrecently. From this alignment,a homology-modelled structurefor the P.furiosus citratesynthase was generated and analysed.     

Analysis of the reaction mechanism and substrate specificity of haloalkane dehalogenases by sequential and structural comparisons

Haloalkane dehalogenases catalyse environmentally importantdehalogenation reactions. These microbial enzymes representobjects of interest for protein engineering studies, attemptingto improve their catalytic efficiency or broaden their substratespecificity towards environmental pollutants. This paper presentsthe results of a comparative study of haloalkane dehalogenasesoriginating from different organisms. Protein sequences andthe models of tertiary structures of haloalkane dehalogenaseswere compared to investigate the protein fold, reaction mechanismand substrate specificity of these enzymes. Haloalkane dehalogenasescontain the structural motifs of /ß-hydrolases and epoxidaseswithin their sequences. They contain a catalytic triad withtwo different topological arrangements. The presence of a structurallyconserved oxyanion hole suggests the two-step reaction mechanismpreviously described for haloalkane dehalogenase from Xanthobacterautotrophicus GJ10. The differences in substrate specificityof haloalkane dehalogenases originating from different speciesmight be related to the size and geometry of an active siteand its entrance and the efficiency of the transition stateand halide ion stabilization by active site residues. Structurallyconserved motifs identified within the sequences can be usedfor the design of specific primers for the experimental screeningof haloalkane dehalogenases. Those amino acids which were predictedto be functionally important represent possible targets forfuture site-directed mutagenesis experiments.     

Structural basis for the difference in thermodynamic properties between the two cysteine proteinase inhibitors human stefins A and B

Homology modelling has been used to model stefin A based onthe X-ray structure of stefin B. Several models have been producedby interactive modelling or positioning of the side chains byMonteCarlo procedure with simulated annealing.The quality of modelswas evaluated by calculation of the free energy of hydration,3D-1D potential or buried area of surface accessibility. StefinA is a thermostable protein, exhibiting a two-state denaturation,while stefin B denaturesat a 40°C lower temperature andforms a stable molten globule intermediate under mild denaturingconditions. From the tertiary structures, thermodynamic functionswere predicted, conforming closely to the experimental calorimetrkresults. Polar and apolar buried areas of surface accessibilitywere obtained by structural deconvolution of the thermograms.It is suggested that the bask difference between the stefinsis the domination of hydrophobic interaction in the stabilizationof stefin B, which is due to its non-specific nature leadingto the formation of a molten globule intermediate. Modellingof stefin A predicts increased numbers of hydrogen bonds whichstabilize it and the increase the cooperativity of its denaturation.     

Contribution of the C-terminal amino acid to the stability of Bacillus subtilis neutral protease

The role of the C-terminal Leu300 in maintaining thermal stabilityof the neutral protease of Bacillus subtilis was investigated.From model building studies based on the three dimensional structureof thermolysin, the neutral protease of B.thermoproteolyticus,it was conduded that this residue is located in a hydrophobicpocket composed of residues located in the C-terminal and themiddle domain. To test the hypothesis that Leu300, by contributingto a stabilizing interaction between these domains, is importantfor enzyme stability, several neutral protease mutants wereconstructed and characterized. The thermostability of the enzymewas lowered by deleting Leu300 or by replacing this residueby a smaller (Ala), a polar (Asn) or a sterically unfavourable(He) amino acid. Thermostabiity was increased upon replacingLeu300 by Phe. These results are in agreement with model-buildingstudies. The effects on thermostability observed after mutatingthe corresponding Val318 in the thermostable neutral proteaseof B.stearothermophilus were less pronounced.     

Computer modelling of the interaction between human choriogonadotropin and its receptor

Primary structural homology between the hormone binding siteof the LH/CG receptor and the enzyme binding site of chymotrypsininhibitor has been identified. This has led to the applicationof a knowledge-based approach of molecular modelling to describethe interaction of choriogonadotropin (CG) with the LH/CG receptor.A tertiary structural model for the mode of recognition betweenthe hormone and the receptor has been proposed. As in othersuch processes at the molecular level, the recognition betweenCG and its receptor is mediated through non-covalent interactions.The specificity of recognition is achieved by complementarityin van der Waals surfaces, hydrogen bonding and non-polar associations.The model shows nine hydrogen bonds between the hormone andthe receptor involving polar side chains as well as backboneamine and carbonyl groups. A hydropbobic cluster involving sidechain groups at the interface is also important in stabilizationof the intermolecular interactions     

Diphtheria toxin receptor-binding domain substitution with interleukin 6: genetic construction and interleukin 6 receptor-specific action of a diphtheria toxin-related interleukin 6 fusion protein

We have genetically replaced that portion of the diphtheriatoxin structural gene which encodes the native receptor-bindingdomain with a synthetic gene encoding the cytokine interleukin6 (IL-6/IFN-ß2/BSF-2). The resulting gene fusion encodesthe chimeric toxin DAB₃₈₉-IL-6. Following expression and purification,we demonstrate that DAB₃₈₉-IL-6 is selectively cytotoxic foreukaryotic cells bearing the interleukin 6 receptor. In addition,the cytotoxic action of DAB₃₈₉-IL-6 is shown to require bindingto the IL-6 receptor, internalization by receptor-mediated endocytosisand passage through an acidic compartment. Following the deliveryof the catalytically active fragment A to the cytosol of targetcells, cellular protein synthesis is inhibited by the ADP-ribosylationof elongation factor 2. While eukaryotic cells which are devoidof the IL-6 receptor are uniformly resistant to the action ofthis fusion toxin, the data presented suggest that a minimalnumber of IL-6 receptors may be necessary to mediate the internalizationof sufficient levels of DAB₃₈₉-IL-6 to result in the intoxicationof target cells.     

Model building of disulfide bonds in proteins with known three-dimensional structure

As an aid in the selection of sites in a protein where a disulfidebond might be engineered, a computer program has been developed.The algorithm starts with the generation of Cß positionsfrom the N, C and C atom coordinates available from a three-dimensionalmodel. A first set of residue pairs that might form a disulfidebond is selected on the basis of Cß–Cßdistances between residues. Then, for each residue in this set,S positions are generated, which satisfy the requirement that,with ideal values for the C–Cß and Cß–Sbond lengths and for the bond angle at Cß, the distancebetween S of residue 1 and Cß of residue 2 in a pair(determined by the bond angle at S2) is at, or very close toits ideal value. Usually two acceptable S positions are foundfor each half cystine, resulting in up to four different conformationsfor the disulfide bond. Finally, these conformations are subjectedto an energy minimization procedure to remove large deviationsfrom ideal geometry and their final energies are calculated.User input determines which final conformations are energeticallyacceptable. These conformations are written to a file to allowfurther analysis and e.g. inspection on a computer graphicsdevice.     

Engineering of the substrate-binding region of the sublilisin-like, cell-envelop proteinase of Lactococcus lactis

The substrate-binding region of the cell-envelope proteinaseof Lactococcus lactis strain SK11 was modelled, based on sequencebomology of the catalytic domain with the serine proteinasessubtilisin and thermitase. Substitutions, deletions and insertionswere introduced, by site-directed and cassette mutagenesfe ofthe prtP gene encoding this enzyme, based on sequence comparisonboth with subtilisin and with the homologous L.lactis strainWg2 proteinase, which has different proteolytic properties.The engineered enzymes were investigated for thermal stability,proteolytic activity and cleavage specificity towards smallchromogenk peptide substrates and the peptide _g1-casein(l–23).Mutations in the subtilisin-like substrate-binding region showedthat Ser433 is the active site residue, and that residues 138and 166 at either side of the binding cleft play an importantrole in substrate specificity, particularly when these residuesand the substrate are oppositely charged. The K748T mutationin a different domain also affected specificity and stability,suggesting that this residue is in close proximity to the subtilisin-likedomain and may form part of the substratebinding site. Severalmutant SK11 proteinases have novel properties not previouslyencountered in natural variants. Replacements of residues 137–139AKTalong one side of the binding cleft produced the 137–139GPPmutant proteinase with reduced activity and narrowed specificity,and the 137–139GLA mutant with increased activity andbroader specificity. Furthermore, the 137–139GDT mutanthad a specificity towards _g1,-casein(l–23) closely resemblingthat of L.lactis Wg2 proteinase. Mutants with an additionalnegative charge in the binding region were more stable towardsautoproteolysis.     

Molecular modeling and mechanism of action of human decay-accelerating factor

A model of the regulatory region of human decay acceleratingfactor (DAF) was built based on the known coordinates of a fragmentof the structurally and functionally homologous serum protein,factor H. According to this model, the four short consensusrepeats (SCRs) in DAF are arranged in a helical fashion. A positivelycharged surface area on SCRs 2 and 3, two of the three repeatingunits essential for function, is postulated to be the primaryrecognition site for the C3 convertases C4b2a and C3bBb. Thisarea encompasses a cavity on SCR 2, as well as part of the grooveon the SCR 2–SCR 3 interface. Two additional surface depressionsare centered around the C-terminal disulfide bridges of SCRs3 and 4. These are likely to provide additional ligand bindingsites. Based on this model in conjunction with sequence homologyto the Ba fragment of factor B, a mechanism of DAF's acceleratedconvertase decay action is postulated.     

An evaluation of the performance of an automated procedure for comparative modelling of protein tertiary structure

A 3-D model of a protein can be constructed from its amino acidsequence and the 3-D structures of one or more homologues byannealing three sets of fragments: the structurally conservedregions, structurally variable regions and the side chains.The method encoded in the computer program COMPOSER was assessedby generating 3-D models of eight proteins whose crystal structuresare already known and for which 3-D structures of homologuesare available. In the structurally conserved regions, differencesbetween modelled and X-ray structures are smaller than the differencesbetween the X-ray structures of the modelled protein and thehomologues used to build the model. When several homologuesare used, the contributions of the known structures are weighted,preferably by the square of sequence similarity; this is especiallyimportant when the similarities of the homologues to the modelledstructure differ greatly. The ‘collar’ extensionapproach, in which a similar region of different length in ahomologue is used to extend the framework, can result in a moreaccurate model. If known homologues comprise more than one relatedgroup of proteins and they are both distantly related to theunknown, then alignment of the sequence to be modelled witheach group of homologues facilitates identification of structurallyconserved regions of the unknown and leads to an improved model.Models have root mean square differences (r.m.s.d.s) with thestructures defined by X-ray analysis of between 0.73 and 1.56Å for all C atoms, for seven of the eight models. Forthe model of mucor pepsin, where the closest homologue has 33%sequence identity and 20% of the residues are in structurallyvariable regions, the r.m.s.d. for the framework region is 1.71Å and the r.m.s.d. for all C atoms is 3.47 Â.     

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.     

Development of an optimized refolding process for recombinant Ala-Glu-IGF-1

Denatured and reduced N-terminal extended insulin-like growthfactor-1 (AE-IGF-1) was purified from Escherichia coli extractsand subjected to in vitro folding. The renaturation processwas shown to be a function of the redox potential of the solution.Folding by different methods had no significant effect on therenaturation. A maximal yield of 60% (w/w)was obtained. Thefolded AE-IGF-1 was enzymatically converted to IGF-1. The majorby-product (20% w/w) was identified as scrambled IGF-1. Enzymaticdigestion at alkaline and acidic pH suggested two possible disulphidebond arrangements: (i) Cys⁶–Cys⁴⁷, Cys¹⁸–Cys⁶¹,Cys⁴⁸–Cys⁵²; or (if) Cys⁶–Cys⁵², Cys^l8–Cys⁶¹,Cys⁴⁷ and Cys⁴⁸ being in their reduced forms. Energy minimizationand molecular modelling suggested that the scrambled IGF-1,having reduced cysteines at positions 47 and 48, was the energeticallymost stable conformation of the two.     

Homology modelling of the Lactococcus lactis leader peptidase NisP and its interaction with the precursor of the lantibiotic nisin

A model is presented for the 3-D structure of the catalyticdomain of the putative leader peptidase NisP of Lactococcuslactis, and the interaction with its specific substrate, theprecursor of the lantibiotic nisin. This homology model is basedon the crystal structures of subtilisin BPN' and thermitasein complex with the inhibitor eglin. Predictions are made ofthe general protein fold, inserted loops, Ca²⁺ binding sites,aromatic interactions and electrostatic interactions of NisP.Cleavage of the leader peptide from precursor nisin by NisPis the last step in maturation of nisin. A detailed predictionof the substrate binding site attempts to explain the basisof specificity of NisP for precursor nisin. Specific acidicresidues in the SI subsite of the substrate binding region ofNisP appear to be of particular importance for electrostaticinteraction with the PI Arg residue of precursor nisin afterwhich cleavage occurs. The hydrophobic S4 subsite of NisP mayalso contribute to substrate binding as it does in subtilisins.Predictions of enzyme-substrate interaction were tested by proteinengineering of precursor nisin and determining susceptibilityof mutant precursors to cleavage by NisP. An unusual propertyof NisP predicted from this catalytic domain model is a surfacepatch near the substrate binding region which is extremely richin aromatic residues. It may be involved in binding to the cellmembrane or to hydrophobic membrane proteins, or it may serveas the recognition and binding region for the modified, hydrophobicC-terminal segment of precursor nisin. Similar predictions forthe tertiary structure and substrate binding are made for thehighly homologous protein EpiP, the putative leader peptidasefor the lantibiotic epidermin from Staphylococcus epidermidis,but EpiP lacks the aromatic patch. Based on these models, proteinengineering can be employed not only to test the predicted enzyme-substrateinteractions, but also to design lantibiotic leader peptidaseswith a desired specificity.     

Role of primary and secondary protein structure in neurotransmitter receptor activation mechanisms

A proton transfer triggered by a ligand interacting with thereceptor had been suggested as the initial step in the activationof a receptor for the neurotransmitter serotonin (5-hydroxy-tryptamine;5-HT). To evaluate the role of the receptor macromolecule inmodulating the primary molecular event in ligand-mediated activation,the process of proton transfer was analysed in the environmentof a protein model for the 5-HT receptor. In the absence ofa detailed receptor structure, the enzyme actinidin was chosenas the model for the receptor based on criteria obtained fromstructure-activity considerations on the ligands. The firstsimulation of a mechanism for receptor activation was performedon this model using methods of theoretical chemistry to studythe effect of specific structural elements. The premise is thatthe role of the elements of secondary structure of soluble proteins(e.g. actinidin) in determining structure-function relationsin these macromolecules is maintained when these elements arepart of membrane-bound receptor proteins. Results from the calculationsof the effects of the six alpha helices of actinidin on theproton transfer process from the imidazolium side chain of His162 to the thiol side chain of Cys 25 in the protein show thatthe helices contribute in different ways to modulate the energyof proton transfer. The largest helix, Al, opposes the protontransfer through the effect of the helix dipole. The chargedresidues (primary structure) in helix A3 favor the proton transfer,and mask the effect of its helix dipole (secondary structure)which opposes the transfer. The direction of the proton transfersimulated for the activation mechanism is opposite to that assumedin the catalytic process of the thiol protease, and the entireprotein environment opposes the transfer. This supports thespecific role of the ligand in triggering the proton transferas a response to its binding.