Jiri Novotny

Empirical potentials and functions for protein folding and binding.

Simplified models and empirical potentials are being increasingly used for the analysis of proteins, frequently augmenting or replacing molecular mechanics approaches. Recent folding simulations have employed potentials that, in addition to terms assuring proper polypeptide geometry, include only two noncovalent effects-hydrogen bonding and hydrophobicity, with extremely simple approximations to the latter. The potentials that have been used in the free-energy ranking of protein-ligand complexes have generally been more involved. These potentials have more detailed solvation models and account for both local (hydrophobic and polar) solute-solvent phenomena and long range electrostatic solvation effects. The models of solvation that have been used most frequently are surface area related atomic parameters, knowledge-based models extracted from protein-structure data, and continum electrostatics with an additional area-related parameter. The knowledge-based approaches to solvation, although convenient and accurate enough, are suspect of double counting certain free-energy terms.

Finite difference Poisson-Boltzmann electrostatic calculations: Increased accuracy achieved by harmonic dielectric smoothing and charge antialiasing

A common problem in the calculation of electrostatic potentials with the Poisson‐Boltzmann equation using finite difference methods is the effect of molecular position relative to the grid. Previously a uniform charging method was shown to reduce the grid dependence substantially over the point charge model used in commercially available codes. In this article we demonstrate that smoothing the charge and dielectric values on the grid can improve the grid independence, as measured by the spread of calculated values, by another order of magnitude. Calculations of Born ion solvation energies, small molecule solvation energies, the electrostatic field of superoxide dismutase, and protein‐protein binding energies are used to demonstrate that this method yields the same results as the point charge model while reducing the positional errors by several orders of magnitude.

The antigenic structure of a scorpion toxin.

Scorpion toxins constitute a family of homologous proteins that exert potent pharmacological effects on ion channels. These proteins are immunogenic and constitute a good model for investigation of the molecular basis of antigenicity. In the first part of this article we summarize the results we have obtained in recent years concerning the location of the main antigenic regions of a model toxin, toxin II of the North African scorpion Androctonus australis Hector. Then, thanks to the recently available atomic coordinates of this toxin, we analyzed the relationships between the structural features of the protein and the location of the antigenic regions: we found that antigenic regions are located at exposed parts of the molecular surface, i.e. in reverse turns and the alpha-helix. These surface parts also correspond to segments of the polypeptide chain which are most accessible to a large spherical probe modelizing an antibody molecule. Finally, we obtained a general idea of what could be the main discontinuous antigenic determinants by looking for the neighboring relationships between the most exposed residues of the protein.

In Silico Pattern-Based Analysis of the Human Cytomegalovirus Genome

ABSTRACT More than 200 open reading frames (ORFs) from the human cytomegalovirus genome have been reported as potentially coding for proteins. We have used two pattern-based in silico approaches to analyze this set of putative viral genes. With the help of an objective annotation method that is based on the Bio-Dictionary, a comprehensive collection of amino acid patterns that describes the currently known natural sequence space of proteins, we have reannotated all of the previously reported putative genes of the human cytomegalovirus. Also, with the help of MUSCA, a pattern-based multiple sequence alignment algorithm, we have reexamined the original human cytomegalovirus gene family definitions. Our analysis of the genome shows that many of the coded proteins comprise amino acid combinations that are unique to either the human cytomegalovirus or the larger group of herpesviruses. We have confirmed that a surprisingly large portion of the analyzed ORFs encode membrane proteins, and we have discovered a significant number of previously uncharacterized proteins that are predicted to be G-protein-coupled receptor homologues. The analysis also indicates that many of the encoded proteins undergo posttranslational modifications such as hydroxylation, phosphorylation, and glycosylation. ORFs encoding proteins with similar functional behavior appear in neighboring regions of the human cytomegalovirus genome. All of the results of the present study can be found and interactively explored online (http://cbcsrv.watson.ibm.com/virus/ ).

Characterization of an anti-digoxin antibody binding site by site-directed in vitro mutagenesis☆

In vitro mutagenesis and immunoglobulin gene transfection were used to investigate the binding site of a monoclonal antibody, 2610, that binds to digoxin, a cardiac glycoside. A computer model was generated in order to select sites in the complementarity determining regions (CDR) that would participate in binding. Residues in the CDR segments were chosen that possess high solvent exposure and were located in a putative cleft. The cloned heavy and light chain variable regions were subjected to in vitro mutagenesis at these sites. The mutated variable regions in M13 were then subcloned into expression vectors and transfected. The affinities and specificity binding properties of the resultant expressed antibodies were measured. Many of the mutants of the putative contact residues showed significant but not major alterations of binding properties. Since most of the residues in the binding site are non-polar and aromatic and since many of the mutations resulted in only modest binding changes, we theorize that much of the high affinity binding (> 10(9)/M) is the cumulation of many weak interactions, arising from dispersion forces and hydrophobic effects in the pocket. Preliminary mutagenesis of two L chain positions proposed to bind to the lactone end of digoxin have larger binding effects. Specificity studies show that the mutants more frequently possess altered binding to the lactone ring of digoxin that altered binding to other digoxin moieties. The data are most suggestive of a model in which lactone is at the bottom of a binding pocket, followed by the steroid nucleus and then by the sugar moiety extruding out of the pocket. The binding information may be useful in understanding the immune response to large, hydrophobic haptens.

Conformational Similarity and Systematic Displacement of Complementarity Determining Region Loops in High Resolution Antibody X-ray Structures

Comparison of seven high resolution x-ray structures shows that the conformations of canonical complementarity determining region (CDR) loops, which are shared by these antibodies, are very similar. However, large spatial displacements (up to 2.7 Å) of the essentially identical CDR loops become evident when the antibody β-sheet frameworks, to which the loops are attached, are least-squares superposed. The loop displacements follow, and amplify, small positional differences in framework/loop splice points. Intradomain structural variability and, to a lesser extent, domain-domain orientation appear to cause the observed loop divergences. The results suggest that the selection of framework regions for loop grafting procedures is more critical than previously thought. Immunoglobulin variable domains, VL 1 and VH, associate noncovalently to form the Fv, a dimer of antiparallel, eight-stranded β-sandwiches(1 2). The VL and VH β-sheets from different antibodies are nearly identical in three dimensions. However, the six complementarity determining region (CDR) loops (L1-L3, H1-H3), which connect the β-strands of the conserved framework and encode antigenic specificities, are much more variable in both sequence and conformation (2 3). Chothia, Lesk, and colleagues (4 5) identified sets of similar “canonical” conformations for all CDR loops except H3. Between 50 and 95% of antibody sequences are consistent with the classified canonical conformations(4), which are determined by conserved interactions of only a few key residues (“structural determinants”) within the loop and/or the framework regions. Some differences in the position of canonical CDR loops relative to superposed framework regions by comparing x-ray structures (4) or x-ray and modeled structures (5) were previously observed. However, these effects were observed in structures determined at medium resolution, generally considered minor(4 5), and not systematically explored. We have compared, via least-squares superpositions, the Fvs of seven x-ray structures refined to high resolution (better than 2 Å) available in the Brookhaven Protein Data Bank(6). It was anticipated that a systematic comparison of structures determined to such high precision would (i) shed light on the general relation between CDR loop conformation and position and (ii) help to assess the limitations of a procedure widely used in comparative model building, i.e. splicing of loops from a known x-ray structure onto the conserved structural scaffold of an antibody model(7 8). Fig. 1 shows a comparison (9) of the amino acid sequences of the seven antibodies, which include various heavy and light chains (both κ and ) from free as well as antigen-complexed antibodies. The 4-4-20 Fv, the highest resolution structure with κ light chain, was used as the template on which backbone segments of the other Fvs were superposed. Cumulative backbone root mean square (rms) deviations of the β-strands were determined after superposing each of these antibodies on the 4-4-20 Fv. Two alternative least-squares superpositions were used, employing different pairs of equivalence residues. The S1 set of residues consisted of only the most conserved regions of the Fvs, i.e. the four short 4-residue segments (10) of the central β-sheets. The S2 set consisted of a more extended set of residues and included the majority of the β-sheet framework (Fig. 1) akin to Stanfield et al.(11). As can be seen in Table 1 the cumulative backbone rms deviations were small and the results obtained with the two superposition sets were similar.

Hydrophobicity Profiles for Protein Sequence Analysis

Hydrophobic interactions are a major force in protein folding and numerous hydropathy scales have been developed to quantify the relative hydrophobicity of the amino acids. Hydropathy profiles can be used to examine the surface features of proteins in order to generate hypotheses that can be confirmed experimentally. This unit describes the application of hydrophobicity plots to typical problems and provides suggested uses for a few selected scales.

Structure of the adenovirus E4 Orf6 protein predicted by fold recognition and comparative protein modeling.

To facilitate investigation of the molecular and biochemical functions of the adenovirus E4 Orf6 protein, we sought to derive three‐dimensional structural information using computational methods, particularly threading and comparative protein modeling. The amino acid sequence of the protein was used for secondary structure and hidden Markov model (HMM) analyses, and for fold recognition by the ProCeryon program. Six alternative models were generated from the top‐scoring folds identified by threading. These models were examined by 3D–1D analysis and evaluated in the light of available experimental evidence. The final model of the E4 protein derived from these and additional threading calculations was a chimera, with the tertiary structure of its C‐terminal 226 residues derived from a TIM barrel template and a mainly α‐nonbundle topology for its poorly conserved N‐terminal 68 residues. To assess the accuracy of this model, additional threading calculations were performed with E4 Orf6 sequences altered as in previous experimental studies. The proposed structural model is consistent with the reported secondary structure of a functionally important C‐terminal sequence and can account for the properties of proteins carrying alterations in functionally important sequences or of those that disrupt an unusual zinc‐coordination motif. Proteins 2001;44:97–109.

Factors influencing accuracy of computer-built models: a study based on leucine zipper GCN4 structure.

A three-dimensional model of the leucine zipper GCN4 built from its amino acid sequence had been reported previously by us. When the two alternative x-ray structures of the GCN4 dimer became available, the root mean square (r.m.s.) shifts between our model and the structures were determined as approximately 2.7 A on all atoms. These values are similar to the r.m.s. shift of 2.8 A between the two GCN4 structures in the different crystal forms (C2 and P2(1)2(1)2(1)). CONGEN conformational searches were run to better understand the conditions that may determine the preference of different conformers in different environments and to test the sensitivity of our current modeling techniques. With a judicious choice of CONGEN search parameters, the backbone r.m.s. deviation improved to 0.8 A and 2.5 A on all atoms. The side-chain conformations of Val and Leu at the helical interface were well reproduced (1.2 A r.m.s.), and the large side-chain misplacements occurred with only a small number of charged amino acids and a tyrosine. Inclusion of the crystal environment (C2 symmetry), as a passive background, into the side-chain conformational search further improved the accuracy of the model to an r.m.s. deviation of 2.1 A. Conformational searches carried out in the two different crystal environments and employing the AMBER protein/DNA forcefield, as implemented in CONGEN, gave the r.m.s. values of 2.2 A (for the C2 symmetry) and 2.5 A (for the P2(1)2(1)2(1) symmetry). In the C2 symmetry crystal, as much as 40% of the surface of each dimer was involved in crystal contacts with other dimers, and the charged residues on the surface often interacted with immobilized water molecules. Thus, occasional large r.m.s. deviations between the model and the x-ray side chains were due to specific conditions that did not occur in solution.

Computational biochemistry of antibodies and T-cell receptors.

This chapter discusses the diverse computer-aided techniques that are applied for the analysis of the immune structure-function relationship, and put the main results obtained with these methods in a broader perspective. Structural data are the bedrock on which computational immunology stands, and the chapter starts with general comments on the nature of the data. The three-dimensional structures are as Cartesian coordinates of molecules determined by X-ray crystallography, or more recently, multidimensional (hetero) nuclear magnetic resonance (NMR) spectroscopy. These protein and DNA structures represent atomic models that best fit to electron density (X-ray) or interatomic distance (NMR) data. Nominal resolution, the crystallographic residual factor (R factor), and the B factor are values describing crystallographic accuracy and precision, which approach fractions of angstroms for well-resolved structures. Protocols for three dimensional modeling of binding sites, binding affinity and specificity, molecular basis of protein antigenicity, antibody engineering, and T-Cell receptor modeling and engineering are discussed in the chapter. One of the exciting developments of antibody engineering has been the advent of combinatorial libraries of immunoglobulin polypeptides, and their expression on the surface of filamentous phage vectors.