Incorporating intermolecular distance into protein-protein docking

In this work, intermolecular distance was integrated into the docking of protein-protein complexes. To develop an efficient docking procedure, 22 enzyme-inhibitor targets and 15 antibody-antigen targets were taken from a benchmark set. A three-step approach was adopted, which included global sampling by FTDOCK, filtering by intermolecular distance and ranking by a composite scoring function. For the enzyme-inhibitor targets, the composite scoring function consists of geometry and energy terms. In the set composed of the approximately 100 highest ranked candidates for each target, correct complexes were identified for all of the 22 enzyme-inhibitor targets. This docking strategy also succeeded on the four test targets, of which three are CAPRI targets with the same receptor but different binding modes. Interestingly, all three binding modes were correctly predicted. For the antibody-antigen targets, CDR and physical energy were also used in the filtering process and informatics terms were added to the scoring function. The composite score had successful prediction for 13 of the 15 antibody-antigen targets.     

An analysis of conformational changes on proteinprotein association: implications for predictive docking

Conformational changes on complex formation have been measuredfor 39 pairs of structures of complexed proteins and unboundequivalents, averaged over interface and non-interface regionsand for individual residues. We evaluate their significanceby comparison with the differences seen in 12 pairs of independentlysolved structures of identical proteins, and find that justover half have some substantial overall movement. Movementsinvolve main chains as well as side chains, and large changesin the interface are closely involved with complex formation,while those of exposed non-interface residues are caused byflexibility and disorder. Interface movements in enzymes aresimilar in extent to those of inhibitors. All eight of the complexes(six enzyme–inhibitor and two antibody–antigen) thathave structures of both components in an unbound form availableshow some significant interface movement. However, predictivedocking is successful even when some of the largest changesoccur. We note however that the situation may be different insystems other than the enzyme–inhibitors which dominatethis study. Thus the general model is induced fit but, becausethere is only limited conformational change in many systems,recognition can be treated as lock and key to a first approximation.     

Prediction of protein-protein interaction sites using support vector machines

The identification of protein-protein interaction sites is essential for the mutant design and prediction of protein-protein networks. The interaction sites of residue units were predicted using support vector machines (SVM) and the profiles of sequentially/spatially neighboring residues, plus additional information. When only sequence information was used, prediction performance was highest using the feature vectors, sequentially neighboring profiles and predicted interaction site ratios, which were calculated by SVM regression using amino acid compositions. When structural information was also used, prediction performance was highest using the feature vectors, spatially neighboring residue profiles, accessible surface areas, and the with/without protein interaction sites ratios predicted by SVM regression and amino acid compositions. In the latter case, the precision at recall = 50% was 54-56% for a homo-hetero mixed test set and >20% higher than for random prediction. Approximately 30% of the residues wrongly predicted as interaction sites were the closest sequentially/spatially neighboring on the interaction site residues. The predicted residues covered 86-87% of the actual interfaces (96-97% of interfaces with over 20 residues). This prediction performance appeared to be slightly higher than a previously reported study. Comparing the prediction accuracy of each molecule, it seems to be easier to predict interaction sites for stable complexes.     

Optimal protein library design using recombination or point mutations based on sequence-based scoring functions

In this paper, we introduce and test two new sequence-based protein scoring systems (i.e. S1, S2) for assessing the likelihood that a given protein hybrid will be functional. By binning together amino acids with similar properties (i.e. volume, hydrophobicity and charge) the scoring systems S1 and S2 allow for the quantification of the severity of mismatched interactions in the hybrids. The S2 scoring system is found to be able to significantly functionally enrich a cytochrome P450 library over other scoring methods. Given this scoring base, we subsequently constructed two separate optimization formulations (i.e. OPTCOMB and OPTOLIGO) for optimally designing protein combinatorial libraries involving recombination or mutations, respectively. Notably, two separate versions of OPTCOMB are generated (i.e. model M1, M2) with the latter allowing for position-dependent parental fragment skipping. Computational benchmarking results demonstrate the efficacy of models OPTCOMB and OPTOLIGO to generate high scoring libraries of a prespecified size.     

A system based on specific protein-RNA interactions for analysis of target protein-protein interactions in vitro: successful selection of membrane-bound Bak-Bcl-xL proteins in vitro

Ribosome display systems are very effective and powerful tools for in vitro screening of transcribed mRNAs that encode proteins (or peptides) with specific (known or unknown) functions. We have modified such a system by exploiting the interaction between a tandemly fused MS2 coat-protein (MSp) dimer and the RNA sequence of the corresponding specific binding motif, C-variant (or Cv). We placed the MSp dimer at the N-terminus of a nascent protein and the Cv binding motif was attached to the 5' end of the protein's mRNA. This configuration enhanced the stability of the ribosome-mRNA complex. We demonstrate here that this improved ribosome display system provides an effective method for identifying the gene for a protein that binds to a protein of interest. We visualized the formation of polysome complexes in this advanced polysome display by atomic force microscopy (AFM) and found that the AFM images of polysomes in our system were different from those observed in the case of conventional ribosome display systems. Our results suggest that our technology might usefully complement yeast two-hybrid assays.     

A novel search method for protein sequence-structure relations using property profiles

In protein engineering and design it is very important thatresidues can be inspected in their specific environment. A standardrelational database system cannot serve this purpose adequatelybecause it cannot handle relations between individual residues.With SCAN3D we introduce a new database system for integratedsequence and structure analysis of proteins. It uses the relationalparadigm wherever possible. Its main power, however, stems fromthe ability to retrieve stretches of consecutive residues withcertain properties by comparing a property profile with allstretches of residues in the database, exploiting the orderedcharacter of proteins. In doing so, it bypasses the large numberof join operations that would be required by relational databasesystems. An additional advantage of using property profile matchingis that searches can be carried out allowing a pre-set numberof mismatches. Also, as the database is read-only, SCAN3D doesnot need interactive data update mechanisms. Queries typicalof a molecular engineering environment are demonstrated withspecific examples: analysis of peptides that induce local structure,analysis of site-dependent rotamers and residue-residue contactanalysis     

Frame shuffling: a novel method for in vitro protein evolution

We describe 'frame shuffling', a novel method for preparing artificial protein libraries. With this method, a Y-family DNA polymerase known to introduce frame shift mutations at high rates is utilized to scramble the reading frames of a parental gene. The resultant progeny produce mutant proteins having segmental sequence changes. Such frame-shuffled mutant proteins exhibit physicochemical properties that differ from those of proteins obtained using conventional mutagenesis.     

Refinement of protein cores and protein-peptide interfaces using a potential scaling approach

Refinement of side chain conformations in protein model structures and at the interface of predicted protein-protein or protein-peptide complexes is an important step during protein structural modelling and docking. A common approach for side chain prediction is to assume a rigid protein main chain for both docking partners and search for an optimal set of side chain rotamers to optimize the steric fit. However, depending on the target-template similarity in the case of comparative protein modelling and on the accuracy of an initially docked complex, the main chain template structure is only an approximation of a realistic target main chain. An inaccurate rigid main chain conformation can in turn interfere with the prediction of side chain conformations. In the present study, a potential scaling approach (PS-MD) during a molecular dynamics (MD) simulation that also allows the inclusion of explicit solvent has been used to predict side chain conformations on semi-flexible protein main chains. The PS-MD method converges much faster to realistic protein-peptide interface structures or protein core structures than standard MD simulations. Depending on the accuracy of the protein main chain, it also gives significantly better results compared with the standard rotamer search method.     

Functional classification of protein kinase binding sites using Cavbase

Increasingly, drug-discovery processes focus on complete gene families. Tools for analyzing similarities and differences across protein families are important for the understanding of key functional features of proteins. Herein we present a method for classifying protein families on the basis of the properties of their active sites. We have developed Cavbase, a method for describing and comparing protein binding pockets, and show its application to the functional classification of the binding pockets of the protein family of protein kinases. A diverse set of kinase cavities is mutually compared and analyzed in terms of recurring functional recognition patterns in the active sites. We are able to propose a relevant classification based on the binding motifs in the active sites. The obtained classification provides a novel perspective on functional properties across protein space. The classification of the MAP and the c-Abl kinases is analyzed in detail, showing a clear separation of the respective kinase subfamilies. Remarkable cross-relations among protein kinases are detected, in contrast to sequence-based classifications, which are not able to detect these relations. Furthermore, our classification is able to highlight features important in the optimization of protein kinase inhibitors. Using small-molecule inhibition data we could rationalize cross-reactivities between unrelated kinases which become apparent in the structural comparison of their binding sites. This procedure helps in the identification of other possible kinase targets that behave similarly in "binding pocket space" to the kinase under consideration.     

A knowledge-based scoring function based on residue triplets for protein structure prediction

One of the general paradigms for ab initio protein structure prediction involves sampling the conformational space such that a large set of decoy (candidate) structures are generated and then selecting native-like conformations from those decoys using various scoring functions. In this study, based on a physical/geometric approach first suggested by Banavar and colleagues, we formulate a knowledge-based scoring function, which uses the radii of curvature formed among triplets of residues in a protein conformation. By analyzing its performance on various decoy sets, we determine a good set of parameters--the distance cutoff and the number of distance bins--to use for configuring such a function. Furthermore, we investigate the effect of using various approaches for compiling the prior distribution on the performance of the knowledge-based function. Possible extensions to the current form of the residue triplet scoring function are discussed.     

Spectroscopic investigation of structure in octarellin (a de novo protein designed to adopt the {alpha}/{beta}-barred packing)

We present here a spectroscopic structural characterizationof octarellin, a recently reported de novo protein modelledon /ß-barrel proteins [K. Go raj, A.Renard and J.A.Martial(1990) Protein Engng, 3, 259–266]. Infrared and Ramanspectra analyses of octarellin‘s secondary structure revealthe expected percentage of -helices (30%) and a higher ß-sheetcontent (40%) than predicted from the design. When the Ramanspectra obtained with octarellin and native triosephosphateisomerase (a natural /ß-barrel) are compared, similarpercentages of secondary structures are found. Thermal denaturationof octarellin monitored by CD confirms that its secondary structuresare quite stable, whereas its native-like tertiary fold is not.Tyrosine residues, predicted to be partially hidden from solvent,are actually exposed as revealed by Raman and UV absorptionspectra. We conclude that the attempted /ß-barrelconformation in octarellin may be loosely packed. The criteriaused to design octarellin are discussed and improvements suggested.     

A single Fc binding domain-alkaline phosphatase gene fusion expresses a protein with both IgG binding ability and alkaline phosphatase enzymatic activity

A recombinant gene fusion was created and cloned using a previouslyconstructed gene encoding a monodomain IgG Fc binding proteinand the gene coding for bacterial alkaline phosphatase. Theconstruct was able to express and secrete a fusion protein thatexhibited both IgG binding and alkaline phosphatase enzymaticactivities. Greater than 60% of the protein demonstrating bothbiological activities was detected from periplasmic space preparations.Nanogram concentrations of the Fc binding-alkaline phosphatasefusion protein allowed primary IgG antibody detection withoutthe use of conjugated secondary antibodies. Removal of the domaincoding for alkaline phosphatase resulted in decreased resistanceof the protein to proteolytic degradation and the loss of IgGFc binding ability. Using affinity-purified fusion protein,the specificity of binding to IgG, IgM and IgA was examined;binding was strong to IgG and barely detectable against IgMor IgA. Affinity for binding of the fusion protein to IgG (k_d= 6.7 x10^-8 M) was determined to be equal to or greater thanpreviously reported for protein A.     

Comparative modeling of the three CP modules of the {beta}-chain of C4BP and evaluation of potential sites of interaction with protein S

A computer model of the ß-chain of C4b-binding protein(C4BP) was constructed, using the backbone fold of the NMR structuresof the sixteenth CP module of factor H (H16) and of a pair ofmodules consisting of the fifteenth and sixteenth CPs of factorH (H15-16). The characteristic hydrophobic core responsiblefor dictating the three-dimensional structure of the CP familyis conserved in the amino acid sequence of C4BP ßl, ß2and ß3. The distribution of the electrostatic potentialshows that the model is mainly covered by a negative contour.Interestingly, a positive area is observed in the C-terminalregion of the first CP module, enclosing peptide 31-45, knownto be a binding site for protein S. This observation suggeststhat electrostatic interactions can be of importance for theinteraction of C4BP to protein S. A solvent-accessible hydrophobicpatch, located nearby and involving the peptide 31-45, was alsofound in the model, further confirming that this area is involvedin the interaction with protein S. The contribution of ß-chainresidues 31-45 to the affinity for protein S was studied furtherby means of synthetic mutant peptides. The results suggest thatboth electrostatic and hydrophobic interactions are importantfor the binding to protein S.     

A predicted three-dimensional structure for the human immunodeficiency virus binding domains of CD4 antigen

A predicted three-dimensional structure of the two N-terminalextracellular domains of human CD4 antigen, a cell surface glycoprotein,is reported. This region of CD4, particularly the first domain,has been identified as containing the binding region for theenvelope gp120 protein of the human immuno-deficiency virus.The model was predicted based on the sequence homology of eachdomain with the variable light chain of immunoglobulins. Theframework ß-sheet regions were taken from the crystalcoordinates of REI. For one region in the first domain of CD4there was an ambiguity in the alignment with REI and two alternatemodels are presented. Loops connecting the framework were modeledfrom fragments selected from a database of main chain coordinatesfrom all known protein structures. Residues identified as involvedin binding gp120 have been located in several other studieswithin the first domain of CD4. Epitopes from eight monoclonalantibodies have been mapped onto residues in both domains. Competitionof these antibodies with each other and with gp120 can be interpretedfrom the structural model.     

A new expression system for protein crystallization using trimeric coiled-coil adaptors

We repeatedly experienced difficulties in obtaining pure protein of a defined oligomeric state when expressing domains that consist partially or entirely of coiled coils. We therefore modified an established expression vector, pASK-IBA, to generate N- and C-terminal fusions of the cloned domain in heptad register with the GCN4 leucine zipper. GCN4 is a well-characterized coiled coil, for which stable dimeric, trimeric and tetrameric forms exist. To test this expression system, we produced a series of constructs derived from the trimeric autotransporter adhesin STM3691 of Salmonella (SadA), which has a highly repetitive structure punctuated by coiled-coil regions. The constructs begin and end with predicted coiled-coil segments of SadA, each fused in the correct heptad register to the trimeric form of GCN4, GCN4pII. All constructs were expressed at high levels, trimerized either natively or after refolding from inclusion bodies, and yielded crystals that diffracted to high resolution. Thus, fusion to GCN4pII allows for the efficient expression and crystallization of proteins containing trimeric coiled coils. The structure of short constructs can be solved conveniently by molecular replacement using the known GCN4 structure as a search model. The system can be adapted for constructs with dimeric or tetrameric coiled coils, using the corresponding GCN4 variants.     

A predicted three-dimensional structure of human cytochrome P450: implications for substrate specificity

A three-dimensional structure for human cytochrome P450IA1 waspredicted based on the crystal coordinates of cytochrome P450camfrom Pseudomonas putida. As there was only 15% residue identitybetween the two enzymes, additional information was used toestablish an accurate sequence alignment that is a prerequisitefor model building. Twelve representative eukaryotic sequenceswere aligned and a net prediction of secondary structure wasmatched against the known -helices and ß-sheets ofP450cam. The cam secondary structure provided a fixed main-chainframework onto which loops of appropriate length from the humanP450IA1 structure were added. The model-built structure of thehuman cytochrome conformed to the requirements for the segregationof polar and nonpolar residues between the core and the surface.The first 44 residues of human cytochrome P450 could not bebuilt into the model and sequence analysis suggested that residues1–26 formed a single membrane-spanning segment. Examinationof the sequences of cytochrome P450s from distinct gene familiessuggested specific residues that could account for the differencesin substrate specificity. A major substrate for P450IA1, 3-methyl-cholanthrene,was fitted into the proposed active site and this planar aromaticmolecule could be accommodated into the available cavity. Residuesthat are likely to interact with the haem were identified. Thesequence similarity between 59 eukaryotic enzymes was representedas a dendrogram that in general clustered according to genefamily. Until a crystallographic structure is available, thismodel-building study identifies potential residues in cytochromeP450s important in the function of these enzymes and these residuesare candidates for site-directed mutagenesis.     

Gene synthesis and functional expression of a protein exhibiting monodomain IgG Fc binding

A gene encoding a bacterial IgG Fc binding domain was designedand synthesized. The synthetic DNA fragment was cloned 3' toan inducible trpE promoter such that expression of the genein Escherichia coli produced abundant Fc binding protein fusedto the first seven amino acids of the trpE protein. The recombinantprotein contained a single Fc binding domain and demonstratedefficient binding to'human IgG in Western blot analysis. Thisprotein degraded rapidly following cell lysis in the absenceof protease inhibitors, but could be effectively protected bythe addition of protease inhibitor. After purification of theprotein by IgG affinity chromatography, IgG Fc binding abilitywas retained for at least 24 h at either 23 or 37°C andon heating for 15 min at temperatures up to 65°C. No immunoprecipitationwas observed in interactions between the monodomain Fc bindingprotein and IgG molecules. Unlike staphylococcal protein A,no detectable binding of the monodomain IgG Fc binding proteinwas observed to either IgM or IgA. Truncated proteins, expressedfrom a series of 3' deletions of the synthetic gene, were usedto estimate the minimum portion of a monodomain Fc binding proteinthat retained Fc binding ability.