Construction of a dictionary of sequence motifs that characterize groups of related proteins

An automatic procedure is proposed to identify, from the proteinsequence database, conserved amino acid patterns (or sequencemotifs) that are exclusive to a group of functionally relatedproteins. This procedure is applied to the PIR database anda dictionary of sequence motifs that relate to specific superfamiliesconstructed. The motifs have a practical relevance in identifyingthe membership of specific superfamilies without the need toperform sequence database searches in 20% of newly determinedsequences. The sequence motifs identified represent functionallyimportant sites on protein molecules. When multiple blocks existin a single motif they are often close together in the 3-D structure.Furthermore, occasionally these motif blocks were found to besplit by introns when the correlation with exon structures wasexamined.     

DOMPLOT: a program to generate schematic diagrams of the structural domain organization within proteins, annotated by ligand contacts

A program is described for automatically generating schematiclinear representations of protein chains in terms of their structuraldomains. The program requires the co-ordinates of the chain,the domain assignment, PROSITE information and a file listingall intermolecular interactions in the protein structure. Theoutput is a PostScript file in which each protein is representedby a set of linked boxes, each box corresponding to all or partof a structural domain. PROSITE motifs and residues involvedin ligand interactions are highlighted. The diagrams allow immediatevisualization of the domain arrangement within a protein chain,and by providing information on sequence motifs, and metal ion,ligand and DNA binding at the domain level, the program facilitatesdetection of remote evolutionary relationships between proteins.     

Searching for common sequence patterns among distantly related proteins

We have developed a program Gap Allowing Pattern Explorer (GAPE)to extract amino acid sequence motifs conserved among distantlyrelated proteins. The GAPE program is designed to allow gapsin the sequences. First, this program generates all possibleamino acid patterns comprising up to five amino acids. Sequencescontaining the amino acid residues in the same order as a generatedpattern are selected as subsequences, where the differencesin the distances between two consecutive amino acids are ignored.Next, the motifs are extracted from the subsequences under conditionsin which all four distances between the five amino acids arefixed. At this stage, motifs with gaps in their subsequenceare also found by relaxing one of the four fixed distances.The statistical significance for a motif obtained is calculatedbased on the amino acid composition of the sequences under consideration.When the GAPE program was applied to 59 pyridoxal-phosphaterelatedsequences and 64 ATP (AMP-forming)-related sequences, motifsextracted with a low expectation of occurrence contained someof the amino acid residues chemically proved to be involvedin the ligand recognition.     

The application of hydrogen bonding analysis in X-ray crystallography to help orientate asparagine, glutamine and histidine side chains

Protein X-ray crystallography produces an electron density mapthat rarely detects individual hydrogen atoms or distinguishesbetween carbon, nitrogen and oxygen atoms in the electron density.This makes it difficult to orientate the side chains of Asn,Gln and His, which appear symmetrical in the electron density;their orientation is usually judged on the basis of hydrogenbonding. Based on the observation that almost all buried donorsand acceptors are satisfied, we have developed a simple algorithmto compare the alternative conformations of these residues and,where possible, identify the most favourable. In a cross-sectionof protein structures we found a few side chains (15.0% of Asnand Gln and 9.9% of His) which would be more favourable in thealternative orientation. We have also found that this proportionrises slightly with worsening resolution.     

Local polarity analysis: a sensitive method that discriminates between native proteins and incorrectly folded models

The evaluation of calculated protein structures is an importantstep in the protein design cycle. Known criteria for this assessmentof proteins are the polar and apolar, accessible and buriedsurface area, electrostatic interactions and other interactionsbetween the protein atoms (e.g. HO, S-S),atomic packing, analysisof amino acid environment and surface charge distribution. Weshow that a powerful test of accuracy of protein structure canbe derived by analysing the water contact of atoms and additionallytaking into account their polarity. On the basis of estimatedreference values of the polar fraction of typical globular proteinswith known structure (mean, SD and distribution), the evaluationof misfolded structures can be improved significantly. The referencevalues are derived by moving windows of different length (3–99amino acid residues) over the amino acid sequence. Model proteins,which are included in the Brookhaven protein structure databank,deliberately misfolded proteins, hypothetical proteins and predictedprotein structures are diagnosed as at least partially incorrectlyfolded. The local fault, mostly observed, is that polar groupsare buried too frequently in the interior of the protein. Thedatabase-derived quantities are useful in screening the designedproteins prior to experimentation and may also be useful inthe assessment of errors in the experimentally determined proteinstructures.     

Novel protein structural motifs containing two-turn and longer 310-helices

The 3₁₀-helix constitutes a small but significant fraction ofsecondary structural elements in proteins. Protein data basesurveys have shown these helices to be present as -helical extensions,in loops and as connectors between ß-strands. The presentwork focuses on two-turn and longer 3₁₀-helices where we establishthat two-turn and longer 3₁₀ helices, unlike the more abundantsingle-turn 3₁₀-helices, frequently occur independent of anyother contiguous secondary structural elements. More importantly,a large fraction of these independent two-turn and longer 3₁₀-helices,along with -helices and ß-strands, are found to form novelsuper-secondary structural motifs in several proteins with possibleimplications for protein folding, local conformational relaxationand biological functions.     

Solvent interactions with n ring systems in proteins

The interaction of water molecules with apolar amino acids isan important aspect of the hydrophobic effect and hence of proteinfolding. Our distributed multipole electrostatic model for waterinteracting with phenylalanine dipeptides shows that minimumenergy sites exist above the aromatic ring such that a solventmolecule can interact with the electrons, but only when thissite is not blocked by mainchain atoms or disturbed by main-chainpolar atoms. This is consistent with the experimental evidenceof others that water can hydrogen bond to aromatic n electrons.In contrast, our analysis of solvent interactions with phenylalanineresidues based on 48 high-resolution, well-refined protein structuresshows that the dominant interaction of solvent molecules iswith the edge of the ring and not with the 7i electrons. Asthe faces of phenylalanine rings tend to be buried, and solventinteractions with neighbouring polar atoms are more favourable,the interaction of water molecules with the faces of aromatic rings appears not to occur frequently in proteins     

Grafting of discontinuous sites: a protein modeling strategy

A strategy for modeling continuous as well as discontinuoussites in protein structures has been developed. Central to thismodeling strategy is the search algorithm of FITSITE, a programto search a given target structure for suitable combinationsof backbone positions mirroring as closely as possible the geometricrelationships of a source structural motif of interest. Alltarget sites detected by FITSITE are further refined to mimicthe source geometry. The side-chain rotamer library conceptfails to precisely describe side chains involved in coordinativebonding (e.g. metal binding sites). Therefore an algorithm usingdetailed database bonding parameter information was appliedfor the side-chain construction. The FITSITE program and thesubsequent processing of the program output are presented ina test case. The Rop protein, a four-helix bundle structure,served as the target protein. It was searched for candidatesites to model a variety of metal binding sites, with structuresextracted from Brookhaven Protein Database entries. The preliminaryprotein models were investigated for structural overlaps withneighboring residues by interactive computer graphics; if required,additional changes were performed. A set of parameters for energyminimization with AMBER (including metal ions) was developed,and the completed Rop variants were energy minimized. Finally,12 potentially metal binding Rop variants were selected forproduction via genetic engineering.     

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.     

Cumulative stabilizing effects of hydrophobic interactions on the surface of the neutral protease from Bacillus subtilis

Using genetically engineered mutants of the neutral pro-teasefrom Bacillus stearothermophilus (BsteNP), it had been shownthat the surface-exposed structural motif constituted by Phe63embedded in a four amino acid hydrophobic pocket is criticalfor the thermal stability of the thermophilic neutral proteasesfrom Bacilli. To measure the stabilizing contribution of eachhydrophobic interaction taking place between Phe63 and the hydrophobicpocket, we grafted this structural motif in the neutral proteasefrom the mesophile Bacillus subtilis (BsubNP). This was accomplishedby first creating the Thr63Phe mutant of BsubNP and then generatinga series of mutants in which the four amino acids which in thermolysinsurround Phe63 and form the hydrophobic pocket were added oneafter the other. By analysing the thermal stability of eachmutant it was found that the 2°C destabilizing effect ofthe Thr63Phe substitution was completely suppressed by the additionof the four amino acid hydrophobic pocket, each replacementproviding a stabilizing contribution of approxi mately 0.8–1°C.These results are discussed in the light of the peculiar mechanismof thermal inactivation of proteolytic enzymes.     

Recombinant anti-polyamine antibodies: identification of a conserved binding site motif

Polyamines are small linear polycations found ubiquitously ineukaryotic cells. They are involved in nucleic acid and proteinsynthesis and rises in cellular polyamine levels have been correlatedwith cell proliferation. Antibodies to these molecules havepotential as prognostic indicators of disease conditions andindicators of treatment efficacy. Antipolyamine monoclonal antibodiesof differing but defined specificities have been generated inour laboratory using polyamine ovalbumin conjugates as immunogens.These antibodies show small but significant cross reactivitieswith other polyamine species; IAG-1 cross reacts with spermidine(8%), JAC-1 with spermine (6%) and JSJ-1 with both putrescine(11%) and spermine (6%). We have rescued and sequenced the heavyand light chain variable regions of all three of these antibodies.While the light chains of two antibodies, IAG-1 and JSJ-1, were93% homologous at the amino acid level, none of the heavy chainsdisplayed any significant sequence homology. However, computer-generatedmodels of all three antibody binding sites revealed a three-dimensionallyconserved polyamine binding site motif. The polyamine appearsto bind into a negatively charged cleft lined with acidic andpolar residues. The cleft is partially or completely closedat one end and the specificity of the interaction is determinedby placement of acidic residues in the cleft. Aromatic residuescontribute to polyamine binding interacting with the carbonbackbone. The polyamine-binding motif we have identified isvery similar to that observed in the crystal structure of PotD,the primary receptor of the polyamine transport system in Escherichiacoli.     

Spatial sign-alternating charge clusters in globular proteins

Large sign-alternating charge clusters formed by the chargedside groups of amino acid residues and N- and C-terminal groupswere found in the majority of considered globular proteins,namely 235 in a total of 274 protein structures, i.e. 85.8%.The clusters were determined by the criteria proposed earlier:charged groups were included in the cluster if their chargedN and O atoms were located at distances between 2.4 and 7.0Å. The set of selected proteins consisted of known non-homologousprotein structures from the Protein Data Bank with a resolutionless than or equal to 2.5 Å and pair sequence similarityless than 25%. Molecular masses of the proteins were from 5.5to 91.5 kDa and protein chain length from 50 to 830 residues.The distribution of charged groups on the protein surface betweenisolated charged groups, small clusters with two and three groups,and large clusters with four or more groups were found to beapproximately similar making 33, 35 and 32% of the total amountof protein charged groups, respectively. The large sign-alternatingcharge clusters with four or more charged groups were studiedin greater detail. The amount of such clusters depends on theprotein chain length. The small proteins contain 1–3 clusterswhile the large proteins display 4–6 or more clusters.On average, 1.5 clusters per each 100 residues were observed.In contrast with this, the size of a cluster, i.e. the numberof charged groups inside a cluster, does not depend on the proteinmolecular mass, and large clusters are observed for proteinsfrom a range of molecular masses. Clusters consisting of fourto six charged groups occur most frequently, although extralarge clusters are also often revealed. We can conclude thatsign-alternating charge clusters are a common feature of theprotein surface of globular protein. They are suggested to playa general functional role as a local polar factor of proteinsurface.     

Probing the structural plasticity of an archaeal primordial cobaltochelatase CbiX(S)

Insertion of metal ions into tetrapyrrole macrocycles is catalyzed by a diverse group of enzymes called chelatases. Structures are known for several chelatases catalyzing metal insertion into protoporphyrin IX or sirohydrochlorin. Despite a lack of significant amino acid sequence similarity, these ferro- and cobaltochelatases share a high degree of structural similarity. Cobaltochelatase CbiK and ferrochelatase HemH are bilobial enzymes with two alpha/beta domains, which were suggested to origin from a common ancestral protein via gene duplication. Small, single-domain chelatases (CbiX(S)) were recently described in archaea and are believed to represent primordial chelatases. Here, we tested the structural plasticity of an archaeal cobaltochelatase CbiX(S) by rearranging its structure with a novel method producing random in-frame deletions, duplications and insertions. A number of functional chelatase variants with insertion of duplicated sequence stretches, encompassing from one to nine secondary structural elements, were obtained. CbiX(S) was found to tolerate large sequence rearrangements in four out of the nine loop regions of the protein, indicating a high degree of structural plasticity. The predicted topologies of two variants (M51 and M518) are strikingly similar to CbiK and HemH, suggesting that we recreated duplication events that are believed to have created the bilobial chelatases.     

An attempt to unify the structure of polymerases

With the great availability of sequences from RNA- and DNA-dependentRNA and DNA polymerases, it has become possible to delineatea few highly conserved regions for various polymerase types.In this work a DNA polymerase sequence from bacteriophage SPO2was found to be homologous to the polymerase domain of the Klenowfragment of polymerase I from Escherichia coli, which is knownto be closely related to those from Staphylococcus pneumoniae,Thermits aquaticus and bacteriophages T7 and T5. The alignmentof the SPO2 polymerase with the other five sequences considerablynarrowed the conserved motifs in these proteins. Three of themotifs matched reasonably all the conserved motifs of anotherDNA polymerase type, characterized by human polymerase a. Itis also possible to find these three motifs in monomeric DNA-dependentRNA polymerases and two of them in DNA polymerase ßand DNA terminal transferases. These latter two motifs alsomatched two of the four motifs recently identified in 84 RNA-dependentpolymerases. From the known tertiary architecture of the Klenowfragment of E.coli pol I, a spatial arrangement can be impliedfor these motifs. In addition, numerous biochemical experimentssuggesting a role for the motifs in a common function (dNTPbinding) also support these inferences. This speculative hypothesis,attempting to unify polymerase structure at least locally, ifnot globally, under the pol I fold, should provide a usefulmodel to direct mutagenesis experiments to probe template andsubstrate specificity in polymerases.     

The role of extra-membranous inter-helical loops in helix-helix interactions

The effect of a short loop connecting two transmembrane alpha-helices was studied using molecular dynamics simulations. Helices F and G from bacteriorhodopsin and two corresponding polyalanine helices were embedded in octane and POPC membranes in a transmembrane configuration both with and without the inter-helical loop. The results indicate that the membrane environment and the sequence of the loop are more influential on the dynamics and structure of the motif than the presence of a loop as such, at least for the time-scales investigated. The four residues in the FG loop are stabilized by four hydrogen bonds. These hydrogen bonds are not present in the polyalanine loop, causing it to be more flexible than the FG loop. This effect was observed independently of the protein environment, stressing the importance of the sequence. The structural analysis indicates that the loop has weak stabilizing properties in all environments. The stabilization due to the presence of the loop was strongest in a simulation of the FG fragment in a membrane-mimetic octane slab. In the simulations of the helix-loop-helix motif embedded in an explicit lipid bilayer model, the lipid bilayer interface compensates to a large extent for the absence of the loop.     

High-throughput construction method for expression vector of peptides for NMR study suited for isotopic labeling

Fusion protein constructs for labeled peptides were generated with the 114 amino acid thioredoxin (TRX), coupled with the incorporation of a histidine tag for affinity purification. Two tandem AhdI sites were designed in the multiple cloning site of the fusion vector according to our novel unidirectional TA cloning methodology named PRESAT-vector, allowing one-step background-free cloning of DNA fragments. Constructs were designed to incorporate the four residue sequence Ile-Asp-Gly-Arg to generate pure peptides following Factor Xa cleavage of the fusion protein. The system is efficient and cost-effective for isotopic labeling of peptides for heteronuclear NMR studies. Seven peptides of varying length, including pituitary adenylate cyclase activating polypeptide (PACAP), vasoactive intestinal peptide (VIP) and ubiquitin interacting motif (UIM), were expressed using this TRX fusion system to give soluble fusion protein constructs in all cases. Three alternative methods for the preparation of DNA fragments were applied depending on the length of the peptides, such as polymerase chain reaction, chemical synthesis or a 'semi-synthetic method', which is a combination of chemical synthesis and enzymatic extension. The ability easily to construct, express and purify recombinant peptides in a high-throughput manner will be of enormous benefit in areas of biomedical research and drug discovery.     

Conserved sequence and structure association motifs in antibody-protein and antibody-hapten complexes

In this paper, we present the association requirements across a wide variety of antibody-antigen complexes. Phylogenetic analysis clearly indicates the representative nature of our structural dataset. Antigen molecules range from small-molecule haptens to complete protein structures. Common association motifs identified include five conserved tyrosine residues and a single conserved arginine residue from CDR-H3. Further, specificity is refined by a diverse array of antibody-antigen electrostatic interactions that maximize complex specificity. Through analysis of calculated pKa shifts on antigen binding, we find that these interactions are conserved at 23 alignment 'hot-spot' positions. Despite consistent roles in defining substrate specificity, 16 hot-spot positions are conserved less than 50% of the time. On the other hand, because of the conserved functional role of these positions, mutant screening at hot-spots is more likely to result in increased antigen specificity than elsewhere. Therefore, we believe these results should facilitate subsequent antibody design experimentation.     

PRINTS-a protein motif fingerprint database

The PRINTS database of protein ‘fingerprints’ isdescribed. Fingerprints comprise sets of motifs excised fromconserved regions of sequence alignments, their diagnostic poweror potency being refined by iterative database scanning (inthis case the OWL composite sequence database). Generally, themotifs do not overlap, but are separated along a sequence, thoughthey may be contiguous in 3-D space. The use of groups of independent,linearly or spatially separate motifs allows particular proteinfolds and functionalities to be characterized more flexiblyand powerfully than conventional single-component patterns orregular expressions. The current version of the database (4.0)contains 150 entries (encoding >700 motifs), covering a widerange of globular and membrane proteins, modular polypeptidesand so on. The growth of the database is influenced by a numberof factors, e.g. the use of multiple motifs, the maximizationof sequence information through iterative database scanningand the fact that the database searched is a large composite.The information contained within PRINTS is distinct from butcomplementary to the single consensus expressions stored inthe widely used PROSITE dictionary of patterns.