Local electrostatic optimization in proteins

A simple electrostatic model has been used to investigate theextent to which the structure of protein molecules is organizedto optimize the internal electrostatic interactions. We findthat the model provides a favorable total intra-protein electrostaticenergy for almost all polar and charged groups of atoms, suggestinga high degree of structural optimization. By contrast, a significantfraction of individual group–group interactions are foundto be unfavorable. An analysis as a function of the range ofinteractions included shows the electrostatic organization isgenerally relatively short range (up to 6 or 7 Å betweengroup centers). Although the model is very simple, it is usefulfor assessing the overall quality of protein experimental structures,for pin-pointing some types of errors and as a guide to improvingprotein design.     

Influence of secondary structure on the hydration of serine, threonine and tyrosine residues in proteins

Previous analysis of experimental data on the solvation of highresolution protein structures has shown that preferred interactionsites for water molecules exist around most amino acid sidechains. We have extended this analysis to look in more detailat the distributions around serine, threonine and tyrosine.We find that for serine and threonine side chains the preferredinteraction sites of solvent molecules with the hydroxyl groupdepends on secondary structure and the _X1 torsion angle of theside chain. For tyrosine side chains the hydroxyl group is toofar from the main chain to reflect secondary structure influences.Specific patterns of hydration are observed in which water molecules‘bridge’ between the hydroxyl side-chain atom andanother main chain or side-chain atom.     

Protein design to understand peptide ligand recognition by tetratricopeptide repeat proteins

Protein design aims to understand the fundamentals of protein structure by creating novel proteins with pre-specified folds. An equally important goal is to understand protein function by creating novel proteins with pre-specified activities. Here we describe the design and characterization of a tetratricopeptide (TPR) protein, which binds to the C-terminal peptide of the eukaryotic chaperone Hsp90. The design emphasizes the importance of both direct, short-range protein-peptide interactions and of long-range electrostatic optimization. We demonstrate that the designed protein binds specifically to the desired peptide and discriminates between it and the similar C-terminal peptide of Hsp70.     

Conservation of residue interactions in a family of Ca-binding proteins

In the TNC family of Ca-binding proteins (calmodulin, parvalbumin,intestinal calcium binding protein and troponin C) {small tilde}70 well-conserved amino acid sequences and six crystal structuresare known. We find a clear correlation between residue contactsin the structures and residue conservation in the sequences:residues with strong sidechain–sidechain contacts in thethree-dimensional structure tend to be the more conserved inthe sequence. This is one way to quantify the intuitive notionof the importance of sidechain interactions for maintainingprotein three-dimensional structure in evolution and may usefullybe taken into account in planning point mutations in proteinengineering.     

An unequivocal example of cysteine proteinase activity affected by multiple electrostatic interactions

The role of electrostatic interactions between the ionizableAsp158 and the active site thiolate-imidazolium ion pair ofsome cysteine proteinases has been the subject of controversyfor some time. This study reports the expression of wild typeprocaricain and Asp158Glu, Asp158Asn and Asp158Ala mutants fromEscherichia coli. Purification of autocatalytically maturedenzymes yielded sufficient fully active material for pH (k_cat/K_m)profiles to be obtained. Use of both uncharged and charged substratesallowed the effects of different reactive enzyme species tobe separated from the complications of electrostatic effectsbetween enzyme and substrate. At least three ionizations aredetectable in the acid limb of wild type caricain and the Gluand Asn mutants. Only two pK_a, values, however, are detectablein the acid limb using the Ala mutant. Comparison of pH activityprofiles shows that whilst an ionizable residue at position158 is not essential for the formation of the thiolate-imidazoliumion pair, it does form a substantial part of the electrostaticfield responsible for increased catalytic competence. Changingthe position of this ionizable group in any way reduces activity.Complete removal of the charged group reduces catalytic competenceeven further. This work indicates that hydronations distantto the active site are contributing to the electrostatic effectsleading to multiple active ionization states of the enzyme.     

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.     

Correlation between occupancy and B factor of water molecules in protein crystal structures

An empirical relationship between occupancy and the atomic displacementparameter of water molecules in protein crystal structures hasbeen found by comparing a set of well refined sperm whale myoglobincrystal structures. The relationship agrees with a series ofindependent structural features whose impact on water occupancycan easily be predicted as well as with other known data andis independent of the protein fold. The estimation of the wateroccupancy in protein crystal structures may help in understandingthe physico-chemical properties of the protein–solventinterface and can allow the monitoring of the accuracy of theprotein crystal structure refinement.     

Comparison of solvent-inaccessible cores of homologous proteins: definitions useful for protein modelling

The three-dimensional structures of 41 homologous proteins (belongingto eight families) were compared by pairwise superposition.A subset of "core" residues was defined as those whose sidechains have <7% of their surface exposed to solvent. Thissubset has significantly higher sequence identity and lowerroot mean square (RMS) carbon separation than for all topologicallyequivalent residues in the structure, when members of a proteinfamily are superposed. For such superpositions the relationshipbetween RMS distance and percentage sequence identity of thissubset of residues is similar to that for all equivalent residues,although some variation is observed between families of proteinswhich are predominantly ß sheet and those which aremainly helix. The definition of a structurally more conservedcore may be ueful in model building proteins from an homologousfamily. The RMS differences of coordinates of structures ofproteins with identical sequences are found to be related tothe resolutions of the structures.     

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.     

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.     

Favorable scaffolds: proteins with different sequence, structure and function may associate in similar ways

Proteins with similar structures may have different functions. Here, using a non-redundant two-chain protein-protein interface dataset containing 103 clusters, we show that this paradigm extends to interfaces. Whereas usually similar interfaces are obtained from globally similar chains, this is not always the case. Remarkably, in some interface clusters, although the interfaces are similar, the overall structures and functions of the chains are different. Hence, our work suggests that different folds may combinatorially assemble to yield similar local interface motifs. The preference of different folds to associate in similar ways illustrates that the paradigm is universal, whether for single chains in folding or for protein-protein association in binding. We analyze and compare the two types of clusters. Type I, with similar interfaces, similar global structures and similar functions, is better packed, less planar, has larger total and non-polar buried surface areas, better complementarity and more backbone-backbone hydrogen bonds than Type II (similar interfaces, different global structures and different functions). The dataset clusters may provide rich data for protein-protein recognition, cellular networks and drug design. In particular, they should be useful in addressing the difficult question of what the favorable ways for proteins to interact are.     

Amino acid neighbours and detailed conformational analysis of cysteines in proteins

Here we present an investigation of the contacts that cysteinesmake with residues in their three-dimensional environment anda comprehensive analysis of the conformational features of 351disulphide bridges in 131 non-homologous single-chain proteinstructures. Upstream half-cystines preferentially have downstreamneighbours, whereas downstream half-cystines have mainly upstreamneighbours. Non-disulphide bridged cysteines (free cysteines)have no preference for upstream or downstream neighbours. Freecysteines have more contacts to non-polar residues and fewercontacts to polar/charged residues than half-cystines, whichcorrelates with our observation that free cysteines are moreburied than half-cystines. Free cysteines prefer to be locatedin -helices while no clear preference is observed for half-cystines.Histidine and methionine are preferentially seen nearby freecysteines. Tryptophan is found preferentially nearby half-cystines.We have merged sequential and spatial information, and highlyinteresting novel patterns have been discovered. The numberof cysteines per protein is typically an even number, peakingat four. The number of residues separating two half-cystinesis preferentially 11 and 16. Left-handed and right-handed disulphidebridges display different conformational parameters. Here wepresent side chain torsion angle information based on a 5–12times larger number of disulphide bridges than has previouslybeen published. Considering the importance of cysteines formaintaining the 3D-structural scaffold of proteins, it is essentialto have as accurate information as possible concerning the packingand conformational preferences. The present work may providekey information for engineering the protein environment aroundcysteines.     

Contribution of a salt bridge to binding affinity and dUMP orientation to catalytic rate: mutation of a substrate-binding arginine in thymidylate synthase

Invariant arginine 179, one of four arginines that are conservedin all thymidylate synthases (TS) and that bind the phosphatemoiety of the substrate 2'-deoxyuridine-5'-monophosphate (dUMP),can be altered even to a negatively charged glutainic acid withlittle effect on k_cat. In the mutant structures, ordered wateror the other phosphate binding arginines compensate for thehydrogen bonds made by Arg179 in the wild-type enzyme and thereis almost no change in the conformation or binding site of dUMP.Correlation of dUMP K_ds for TS R179A and TS R179K with the structuresof their binary complexes shows that the positive charge onArg179 contributes significantly to dUMP binding affinity. k_cat/Kmfor dUMP measures the rate of dUMP binding to TS during theordered bi-substrate reaction, and in the ternary complex dUMPprovides a binding surface for the cofactor. k_cat/Km reflectsthe ability of the enzyme to accept a properly oriented dUMPfor catalysis and is less sensitive than is K_d to the changesin electrostatics at the phosphate binding site.     

Determination of the pKa values of titratable groups of an antigen-antibody complex, HyHEL-5-hen egg lysozyme

The titration behavior of the ionizable residues of the HyHEL-5–henegg lysozyme complex and its individual components has beenstudied using continuum electrostatic calculations. Severalresidues of HyHEL-5 had pK_a values shifted away from model valuesfor isolated residues by more than three pH units. Shifts awayfrom the model values were smaller for the residues of hen egglysozyme. A moderate variation in the pK_a values of the titratablegroups was observed upon increase of the ionic strength from0 to 100 mM, amounting to 1–2 pH units in most cases.Under physiological conditions, the net charge of HyHEL-5 wasopposite that for hen egg lysozyme. Several residues, includingthose involved in the Arg–Glu salt bridges that have beenproposed to be important in antibody-antigen binding, had pK_avalues that were changed significantly upon binding. The maintitration event upon antibody-antigen binding appears to beloss of a proton from residue GluH50 of the Fv molecule. Thelimitations of our calculation methods and the role they mightplay in the design of antibodies for use in assays, sensorsand separations are discussed     

Modelling of solvent positions around polar groups in proteins

Previous analysis of the distribution of experimental solventmolecule positions around amino acid side chains showed thatdistinct clustering occurred close to polar or charged atomsin proteins. We have used those data to predict likely solventpositions around proteins not used in our initial analysis.We envisage that this algorithm, AQUARIUS, will be useful forfinding solvent positions in electron density maps generatedby protein crystallography and as useful starting positionsfor solvent molecules in computer simulation studies of macromolecules.     

Detection of internal cavities in globular proteins

We have undertaken a study of internal cavities in five proteinstructure groups, each containing different crystallographicstructure determinations of the same protein, to understandbetter the nature of packing defects in protein tertiary architectures.Our results show that cavity detection and consistency of detectionare highly dependent on probe and cavity size, cavity positionwithin the globular protein and the local ‘quality’(r.m.s. deviation) of structural consistency within the group.The consistency of solvent placement within cavities has alsobeen examined. We provide guidelines for estimating the likelihoodof a given cavity to be an actual packing defect or to be aresult of experimental error.     

Structure activity relationships of monocyte chemoattractant proteins in complex with a blocking antibody

Monocyte chemoattractant proteins (MCPs) are cytokines that direct immune cells bearing appropriate receptors to sites of inflammation or injury and are therefore attractive therapeutic targets for inhibitory molecules. 11K2 is a blocking mouse monoclonal antibody active against several human and murine MCPs. A 2.5 A structure of the Fab fragment of this antibody in complex with human MCP-1 has been solved. The Fab blocks CCR2 receptor binding to MCP-1 through an adjacent but distinct binding site. The orientation of the Fab indicates that a single MCP-1 dimer will bind two 11K2 antibodies. Several key residues on the antibody and on human MCPs were predicted to be involved in antibody selectivity. Mutational analysis of these residues confirms their involvement in the antibody-chemokine interaction. In addition to mutations that decreased or disrupted binding, one antibody mutation resulted in a 70-fold increase in affinity for human MCP-2. A key residue missing in human MCP-3, a chemokine not recognized by the antibody, was identified and engineering the preferred residue into the chemokine conferred binding to the antibody.     

Model structures and action of interleukin 1 and its antagonist

A comparison has been made between the homology and hydrophobkityprofiles of six interleukin amino add sequences and that ofthe human interleukin 1ß (IL-lß) for whicha crystal structure exists. The resulting sequence alignmentwas used to build model structures for the sequences for threeIL-l, two IL-1ß and an interleukin receptor antagonist.Analysis of these structures demonstrates that the interleukinmolecule has a strong electric dipole which is generated bythe topological position of the amino acids in the sequence.Electrostatic surface calculations implicate a particular residues(Lysl45) as being fundamental to interleukin activity and thissupports site-directed mutation evidence that this residue isrequired for activity.     

The role of heat-shock and chaperone proteins in protein folding: possible molecular mechanisms

Recently some heat-shock proteins have been linked to functionsof ‘chaperoning’ protein folding in vivo. Here currentexperimental evidence is reviewed and possible requirementsfor such an activity are discussed. It is proposed that onemode of chaperone action is to actively unfold misfolded orbadly aggregated proteins to a conformation from whkh they couldrefold spontaneously; that improperly folded proteins are recognizedby excessive stretches of solvent-exposed backbone, rather thanby exposed hydrophobic patches; and that the molecular mechanismfor unfolding is either repeated binding and dissociation (‘plucking’)or translocation of the protein backbone through a binding cleft(‘threading’), allowing the threaded chain to refoldspontaneously. The observed hydrolysis of ATP would providethe energy for active unfolding. These hypotheses can be appliedto both monomeric folding and oligomeric assembly and are sufficientlydetailed to be open to directed experimental verification.     

Distance distributions in proteins: a six-parameter representation

We present a statistical analysis of protein structures basedon interatomic C_a distances. The overall distance distributionsreflect in detail the contents of sequence-specific substructuresmaintained by local interactions (such as -helixes) and longerrange interactions (such as disulfide bridges and ß-sheets).We also show that a volume scaling of the distances makes distancedistributions for protein chains of different length superimposable.Distance distributions were also calculated specifically foramino acids separated by a given number of residues. Specificfeatures in these distributions are visible for sequence separationsof up to 20 amino acid residues. A simple representation, whichpreserves most of the information in the distance distributions,was obtained using six parameters only. The parameters giverise to canonical distance intervals and when predicting coarse-graineddistance constraints by methods such as data-driven artificialneural networks, these should preferably be selected from theseintervals. We discuss the use of the six parameters for determiningor reconstructing 3-D protein structures.