Local protein sequence similarity does not imply a structural relationship

A database search often will find a seemingly strong sequencesimilarity between two fragments of proteins that are not expectedto have an evolutionary or functional relationship. It is temptingto suggest that the two fragments will adopt a similar conformationdue to a common pattern of residues that dictate a particularsubstructure. To investigate the likelihood of such a structuralsimilarity, local sequence similarities between proteins ofknown conformation were identified by a standard database searchalgorithm. Significant sequence similarity was identified aswhen the chance probability of obtaining the relatedness scorefrom a scan of the entire database was <1%. In this regionboth true homologies and false homologies are detected. A totalof 69 false homologies was located of length between 20 and262 aligned positions. Many of these alignments had 25% sequenceidentity and a further 25% of conservative changes. However,the results show in general these aligned fragments did nothave a significant similarity in secondary or tertiary structure.Thus local sequence does not indicate a structural similaritywhen there is neither an evolutionary nor functional explanationto support this. Accordingly structure predictions based onfinding a local sequence similarity with an evolutionary unrelatedprotein of known conformation are unlikely to be valid.     

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.     

Secondary structure prediction of the H5 pore of potassium channels

The ‘H5’ segment located between the putative fifthand sixth transmembrane helices is the most highly conservedregion in voltage-gated potassium channels and it is believedto constitute a major part of the ion conduction path (pore).Here we present a two-step procedure, comprising secondary structureprediction and hydrophobic moment profiling, to predict thestructure of this important region. Combined results from theapplication of the procedure to the H5 region of four voltage-gatedand five other K⁺ channel sequences lead to the prediction ofa ß-strand-turn-(3-strand structure for H5. The reasonsfor the application of these soluble protein methods to partsof membrane proteins are: (i) that pore-lining residues areaccessible to water and (ii) that a large enough database ofhighresolution membrane protein structures does not yet existThe results are compared with experimental results, in particularspectroscopic studies of two peptides based on the H5 sequenceof SHAKER potassium channel. The procedure developed here maybe applicable to wateraccessible regions of other membrane proteins.     

An investigation of protein subunit and domain interfaces

Protein structures were collected from the Brookhaven Databaseof tertiary architectures that displayed oligomeric association(24 molecules) or whose polypeptide folding revealed domains(34 proteins). The subunit and domain interfaces for these proteinswere respectively examined from the following aspects: percentagewater-accessible surface area buried by the respective associations,surface compositions and physical characteristics of the residuesinvolved in the subunit and domain contacts, secondary structuralstate of the interface amino acids, preferred polar and non-polarinteractions, spatial distribution of polar and non-polar residueson the interface surface, same residue interactions in the oligomeric:contacts, and overall cross-section and shape of the contactsurfaces. A general, consistent picture emerged for both thedomain and subunit interfaces.     

Predicting local structural changes that result from point mutations

Point mutations are frequently used to explore the structureand/or function of proteins. The ability to predict the structuraleffects of point mutations would make the planning of such experimentsmore reliable. We have now derived a set of detailed predictiverules based on the comparison of crystal structures of pointmutants and wild types in 83 cases. Despite the surprising simplicityof these rules, they describe well the conformational changesin 85% of all point mutant structures available at present.     

The evolutionary landscape of functional model proteins

To study the distinct influences of structure and function onevolution, we propose a minimalist model for proteins with bindingpockets, called functional model proteins, based on a shifted-HPmodel on a two-dimensional square lattice. These model proteinsare not maximally compact and contain an empty lattice sitesurrounded by at least three nearest neighbors, thus providinga binding pocket. Functional model proteins possess a uniquenative state, cooperative folding and tolerance to mutation.Due to the explicit functionality in these models (by design),we have been able to explore their fitness or evolutionary landscapes,as characterized by the size and distribution of homologousfamilies and by the complexity of the inter-relatedness of thefunctional model proteins. Mindful that these minimalist modelsare highly idealized and two-dimensional, functional model proteinsshould nevertheless provide a useful means for exploring theconstraints of maintaining structure and function on the evolutionof proteins.     

Mutational and structural analysis of the lectin activity in binding domain 2 of ricin B chain

The study of the lectin binding sites of ricin B chain and ofother homologous members of the small gene family that makeup ricin-like molecules has revealed a number of key contactresidues involved in sugar binding. In particular, on the basisof data generated by the X-ray crystallographic structure ofricin, comparisons of sequence homologies to other ricin-likemolecules and substrate binding studies with these molecules,it has been proposed that His248 of Ricinus communis agglutinin(RCA) B chain may interfere with galactose binding in the secondbinding domain of that lectin. To test that hypothesis, singlebinding domain 2 (SBD2) of ricin B chain was expressed as agene 3 fusion protein on the surface of fd phage to measuredirectly the effect of mutational changes on this binding site.Replacement of tyrosine with histidine at amino acid position248 of SBD2 of ricin B chain was shown to reduce lectin activity.The sequences of RCA and ricin B chains were aligned and comparedwith the tertiary structure of ricin B chain to select variousmutations that were introduced as controls in the study. Oneof these controls, Leu247 to Val247, displayed increased affinityfor galactosides. The role of sequence changes is discussedin relation to the structural and functional divergence in thesemolecules.     

Search for the most stable folds of protein chains: I. Application of a self-consistent molecular field theory to a problem of protein three-dimensional structure prediction

We present a general approach to the prediction of 3-D foldsof protein chains from their amino acid sequences. The approachis based on the use of the self-consistent molecular field theoryfor long-range interactions, the use of 1-D statistical mechanicsfor short-range interactions and on the discovery that thereis and should only be a relatively small discrete set of foldingpatterns. This makes it possible to examine the full varietyof ‘potentially stable’ folds and to determine thethermodynamically stable structure. In this paper, we give thegeneral theoretical background of the approach. The encouragingresults of the application of this approach to ß-domainsare described in another paper.     

An analysis of structural instances of low complexity sequence segments

Amino acid sequence databases contain many low complexity, compositionallybiased sequence segments. However, only a limited number ofrelatively short instances of these segments occur in proteinsof known structure. An analysis is presented of structural instancesof these low complexity sequence segments in the BrookhavenProtein Data Bank with regard to preferences for sequence composition,secondary structural conformation and the local atomic environment.The complexity varies almost linearly with segment length, reflectingthe absence of very long, low complexity segments in the structuraldatabase. The low complexity segments identified are not disorderedand have temperature factors which are generally the same asthe rest of the protein. It is observed that these segmentsare predominantly exposed and either helical or coiled, in excessof what would be expected by chance. Secondary structure predictionmethods perform well in correctly predicting those low complexitysegments which are helical but poorly in correctly predictingsegments that are strands.     

An active single-chain antibody containing a cellulase linker domain is secreted by Escherichia coli

Single-chain antibodies consist of the variable, antigen-bindingdomains of antibodies joined to a continuous polypeptide bygenetically engineered peptide linkers. We have used the flexibleinterdomain linker region of a fungal cellulase to link togetherthe variable domains of an anti-2-phenyloxazolone IgGl and showhere that the resulting single-chain antibody is efficientlysecreted and released to the culture medium of Escherichia coli.The yield of affinity-purified single-chain antibody is 1 -2mg/1 of culture medium and its affinity and stability are comparableto those of the corresponding native IgG.     

Search for the most stable folds of protein chains: II. Computation of stable architectures of {beta}-proteins using a self-consistent molecular field theory

In a preceding paper we presented a novel approach to computationof 3-D folds of protein chains from their amino acid sequences.This approach is a physically correct generalization of the‘threading’ methods. It is based on a self-consistentmolecular field theory and on a physical theory of protein foldingpatterns, which make it possible to examine all the varietyof ‘potentially stable’ folding patterns and allthe variety of the chain conformations within each of them andto determine the thermodynamically stable structure. In thispaper, we apply this approach to single out stable folding patternsand conformations for the chains of ß-sandwich proteinsand show that the similarity of the calculated and observedstructures is usually rather close.     

Protein design for non-aqueous solvents

Improving protein stability in unnatural and suboptimal environmentsis a promising application of protein engineering technology.Carefully designed amino acid alterations may lead to dramaticpositive effects on the stability of proteins under highly perturbingconditions, such as in non-aqueous solvents. Applications ofbiocatalysts and proteins with specific binding capabilitiesin the chemical industry have been severely limited by constraintsplaced on the solvent environment. With the advent of convenientmethods for altering the amino acid composition and even synthesizingentirely new protein molecules, it is worthwhile to considerengineering proteins for stability in non-aqueous solvents.In order to identify the features that a protein would needfor stability in organic media, we have been studying the structureand properties of the hydrophobic protein crambin. Crambin isunique in that it is soluble and stable in very high concentrationsof polar organic solvents. Crambin and its water-soluble homologsoffer a powerful demonstration of protein engineering for non-aqueoussolvents. This paper describes the structural features thatcontribute to crambin's special properties. Based on these observationsand consideration of how non-aqueous solvents affect the interactionsimportant in protein folding, a set of rules for designing non-aqueoussolvent-stable proteins is proposed.     

Protein fold recognition by threading: comparison of algorithms and analysis of results

Optimal sequence threading can be used to recognize membersof a library of protein folds which are closely related in 3-Dstructure to the native fold of an input test sequence, evenwhen the test sequence is not significantly homologous to thesequence of any member of the fold library. The methods providean alignment between the residues of the test sequence and theresidue positions in a template fold. This alignment optimizesa score function, and the predicted fold is the highest scoringmember of the library of folds. Most score functions containa pairwise interaction energy term. This, coupled with the needto introduce gaps into the alignment, means that the optimizationproblem is NP hard. We report a comparison between two heuristicoptimization algorithms used in the literature, double dynamicprogramming and an iterative algorithm based on the so-calledfrozen approximation. These are compared in terms of both theranking of likely folds and the quality of the alignment produced.     

Use of a quantitative structure-property relationship to design larger model proteins that fold rapidly

A quantitative structure–property relationship (QSPR)was used to design model protein sequences that fold repeatedlyand relatively rapidly to stable target structures. The specificmodel was a 125-residue heteropolymer chain subject to MonteCarlo dynamics on a simple cubic lattice. The QSPR was derivedfrom an analysis of a database of 200 sequences by a statisticalmethod that uses a genetic algorithm to select the sequenceattributes that are most important for folding and a neuralnetwork to determine the corresponding functional dependenceof folding ability on the chosen attributes. The QSPR dependson the number of anti-parallel sheet contacts, the energy gapbetween the native state and quasi-continuous part of the spectrumand the total energy of the contacts between surface residues.Two Monte Carlo procedures were used in series to optimize boththe target structures and the sequences. We generated 20 fullyoptimized sequences and 60 partially optimized control sequencesand tested each for its ability to fold in dynamic MC simulations.Although sequences in which either the number of anti-parallelsheet contacts or the energy of the surface residues is non-optimalare capable of folding almost as well as fully optimized ones,sequences in which only the energy gap is optimized fold markedlymore slowly. Implications of the results for the design of proteinsare discussed.     

Functional diversity of the phosphoglucomutase superfamily: structural implications

Three-dimensional structural models of three members of thephosphoglucomutase (PGM) superfamily, parafusin, phosphoglucomutase-relatedprotein and sarcoplasmic reticulum phosphoglucomutase, wereconstructed by homology modeling based on the known crystalstructure of rabbit muscle phosphoglucomutase. Parafusin, phosphoglucomutase-relatedprotein and sarcoplasmic reticulum phosphoglucomutase each have50% or more identity with rabbit muscle phosphoglucomutase atthe amino acid level and all are reported to exhibit no or minorphosphoglucomutase activity. There are four major insertionsand two deletions in the parafusin sequence relative to PGM,all of which are located in surface-exposed loops connectingsecondary structural elements. The remaining amino acid substitutionsare distributed throughout the sequence and are not predictedto alter the polypeptide fold. Parafusin contains a putativeprotein kinase C site located on a surface loop in domain IIthat is not present in the homologs. Although the general domainstructure and the active site of rabbit muscle phosphoglucomutaseare preserved in the model of phosphoglucomutase-related protein,a major structural difference is likely to occur in domain 1due to the absence of 55 amino acid residues in PGM-RP. Thisdeletion predicts the loss of three -helices and one ß-strandfrom an anti-parallel ß-sheet in this domain as comparedwith the rabbit muscle phosphoglucomutase.     

Protein engineering of the high-alkaline serine protease PB92 from Bacillus alcalophilus: functional and structural consequences of mutation at the S4 substrate binding pocket

Serine endoproteases such as trypsins and subtilisins are knownto have an extended substrate binding region that interactswith residues P6 to P3' of a substrate. In order to investigatethe structural and functional effects of replacing residuesat the S4 substrate binding pocket, the serine protease fromthe alkalophilic Bacillus strain PB92, which shows homologywith the subtilisins, was mutated at positions 102 and 126–128.Substitution of Val102 by Trp results in a 12–fold increasein activity towards succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide(sAAPFpNA). An X-ray structure analysis of the V102W mutantshows that the Trp side chain occupies a hydrophobic pocketat the surface of the molecule leaving a narrow crevice forthe P4 residue of a substrate. Better binding of sAAPFpNA bythe mutant compared with the wild type protein as indicatedby the kinetic data might be due to the hydrophobic interactionof Ala P4 of the substrate with the introduced Trp102 side chain.The observed difference in binding of sAAPFpNA by protease PB92and thermitase, both of which possess a Trp at position 102,is probably related to the amino acid substitutions at positions105 and 126 (in the protease PB92 numbering).Kinetic data forthe variants obtained by random mutation of residues Serl26,Prol27 and Serl28 reveal that the activity towards sAAPFpNAincreases when a hydrophobic residue is introduced at position126. An X-ray diffraction analysis was carried out for the threeprotease PB92 mutants which have residues Serl26-Prol27-Serl28replaced by Met-Ala-Gly(‘MAG’ mutant), Phe-Gln-Ser(‘FQS’ mutant) and Asn-Ser-Ala (‘NSA’mutant). Met 126 and Phel26 in the crystal structures of thecorresponding mutants are fixed in the same hydrophobic environmentas Trp102 in the V102W mutant.In contrast, Asnl26 in the ‘NSA’mutant is completely disordered in both crystal forms for whichthe structure has been determined. According to our kineticmeasurements none of the mutants with Met, Phe, Leu or Val atposition 126 binds sAAPFpNA better than the wild type enzyme.Resultsof the site-directed mutagenesis at position 127 imply thatpossible interaction of this residue with a substrate has almostno effect on activity towards sAAPFpNA and casein.     

Conservation and specialization in PAS domain dynamics

The PAS (Per-ARNT-Sim) superfamily is presented as a well-suited study case to demonstrate how comparison of functional motions among distant homologous proteins with conserved fold characteristics may give insight into their functional specialization. Based on the importance of structural flexibility of the receptive structures in anticipating the signal-induced conformational changes of these sensory systems, the dynamics of these structures were analysed. Molecular dynamics was proved to be an effective method to obtain a reliable picture of the dynamics of the crystal structures of HERG, phy3, PYP and FixL, provided that an extensive conformational space sampling is performed. Other reliable sources of dynamic information were the ensembles of NMR structures of hPASK, HIF-2alpha and PYP. Essential dynamics analysis was successfully employed to extract the relevant information from the sampled conformational spaces. Comparison of motion patterns in the essential subspaces, based on the structural alignment, allowed identification of the specialized region in each domain. This appears to be evolved in the superfamily by following a specific trend, that also suggests the presence of a limited number of general solutions adopted by the PAS domains to sense external signals. These findings may give insight into unknown mechanisms of PAS domains and guide further experimental studies.     

Recombinant C-terminal domain of pancreatic lipase retains full ability to bind colipase

The organization of the pancreatic lipase in two well defineddomains has been correlated to a specific function for eachdomain, catalytic activity for the N-terminal domain and colipasebinding for the C-terminal domain. In order to see if such anorganization implies that the two domains can behave as separateentities, we expressed the N- and C-terminal domains in insectcells. The recombinant proteins secreted in the cell supernatantspresent the expected molecular properties. However, whereasthe C-terminal domain retains its function of colipase binding,the N-terminal domain appears to be unable to ensure catalysis.The lack of activity of the recombinant N-terminal domain couldresult either from a (partially) incorrect folding or from anincapacity to function by itself. These results suggest that,although both are structurally well defined, the two domainsof the pancreatic lipase behave differently when they are expressedas separate entities.     

Protein engineering of the hydrophobic domain of human factor IX

Vitamin K-dependent plasma proteins contain a highly conservedhydrophobic domain located between the -carboxyglutamic acid(Gla) domain and the first epidermal growth factor (EGF)-likedomain. Here we have used protein engineering of the hydrophobicdomain in human factor IX to investigate its function in intactfactor IX. Mutant proteins were generated by site-directed mutagenesisand in vitro expression in Madin-Darby canine kidney (MDCK)cells. All of our mutants, including one with a deletion ofthe entire hydrophobic domain, were activated by factor XIa,showing that this domain is not required for factor IX activation.The results with the mutant Phe41 Val suggest that the hydrophobicdomain interacts with the adjacent EGF-like domain. Our datafor the Phe41 Asp mutant is consistent with, but cannot prove,a role for this residue in the maintenance of a phospholipid-bindingstructure required for factor IX function.