Showing papers in "Proteins in 1997"

Pfam : a comprehensive database of protein domain families based on seed alignments

Abstract: Databases of multiple se- quence alignments are a valuable aid to protein sequence classification and analysis. One of the main challenges when constructing such a data- base is to simultaneously satisfy the conflicting demands of completeness on the one hand and quality of alignment and domain definitions on the other. The latter properties are best dealt with by manual approaches, whereas complete- ness in practice is only amenable to automatic methods. Herein we present a database based on hidden Markov model profiles (HMMs), which combines high quality and completeness. Our database, Pfam, consists of parts A and B. Pfam-Ais curated and contains well-character- ized protein domain families with high quality alignments, which are maintained by using manually checked seed alignments and HMMs to find and align all members. Pfam-B contains sequence families that were generated auto- matically by applying the Domainer algorithm to cluster and align the remaining protein sequences after removal of Pfam-A domains. By using Pfam, a large number of previously unannotated proteins from theCaenorhabditis elegans genome project were classified. We have also identified many novel family member- ships in known proteins, including new kazal, Fibronectin type III, and response regulator receiver domains. Pfam-Afamilies have perma- nent accession numbers and form a library of HMMs available for searching and automatic annotation of new protein sequences. Proteins: 28:405-420, 1997. r1997 Wiley-Liss, Inc.

An evolutionary treasure: unification of a broad set of amidohydrolases related to urease.

Abstract: The recent determination of the three-dimensional structure of urease re- vealed striking similarities of enzyme architec- ture to adenosine deaminase and phosphotries- terase, evidence of a distant evolutionary relationship that had gone undetected by one- dimensional sequence comparisons. Here, based on an analysis of conservation patterns in three dimensions, we report the discovery of the same active-site architecture in an even larger set of enzymes involved primarily in nucleotide metabolism. As a consequence, we predict the three-dimensional fold and details of the active site architecture for dihy- droorotases, allantoinases, hydantoinases, AMP-, adenine and cytosine deaminases, imid- azolonepropionase, aryldialkylphosphatase, chlorohydrolases, formylmethanofuran dehy- drogenases, and proteins involved in animal neuronal development. Two member families are common to archaea, eubacteria, and eu- karyota. Thirteen other functions supported by the same structural motif and conserved chemi- cal mechanism apparently represent later adap- tations for different substrate specificities in different cellular contexts. Proteins 28:72-82, 1997 r1997 Wiley-Liss, Inc.

Flexible protein-ligand docking by global energy optimization in internal coordinates.

Abstract: Eight protein-ligand complexes were simulated by using global optimization of a complex energy function, including solvation, surface tension, and side-chain entropy in the internal coordinate space of the flexible ligand and the receptor side chains [Abagyan, R.A., Totrov, M.M. J. Mol. Biol. 235: 983-1002, 1994]. The procedure uses two types of efficient random moves, a pseudobrownian positional move [Abagyan, R.A., Totrov, M.M., Kuznetsov, D.A. J. Comp. Chem. 15:488-506, 1994] and a Biased-Probability multitorsion move [Abagyan, R.A., Totrov, M.M. J. Mol. Biol. 235: 983-1002, 1994], each accompanied by full local energy minimization. The best docking solutions were further ranked according to the interaction energy, which included intramolecular deformation energies of both receptor and ligand, the interaction energy, surface tension, side-chain entropic contribution, and an electrostatic term evaluated as a boundary element solution of the Poisson equation with the molecular surface as a dielectric boundary. The geometrical accuracy of the docking solutions ranged from 30% to 70% according to the relative displacement error measure at a 1.5 A scale. Similar results were obtained when the explicit receptor atoms were replaced with a grid potential.

Seventy‐five percent accuracy in protein secondary structure prediction

Abstract: In this study we present an accurate secondary structure prediction procedure by using an query and related sequences. The most novel aspect of our approach is its reliance on local pairwise alignment of the sequence to be predicted with each related sequence rather than utilization of a multiple alignment. The residue-by-residue accuracy of the method is 75% in three structural states after jack-knife tests. The gain in prediction accuracy compared with the existing techniques, which are at best 72%, is achieved by secondary structure propensities based on both local and long-range effects, utilization of similar sequence information in the form of carefully selected pairwise alignment fragments, and reliance on a large collection of known protein primary structures. The method is especially appropriate for large-scale sequence analysis of efforts such as genome characterization, where precise and significant multiple sequence alignments are not available or achievable.

Model‐free methods of analyzing domain motions in proteins from simulation: A comparison of normal mode analysis and molecular dynamics simulation of lysozyme

Abstract: Model-free methods are introduced to determine quantities pertaining to protein domain motions from normal mode analyses and molecular dynamics simulations. For the normal mode analysis, the methods are based on the assumption that in low frequency modes, domain motions can be well approximated by modes of motion external to the domains. To analyze the molecular dynamics trajectory, a principal component analysis tailored specifically to analyze interdomain motions is applied. A method based on the curl of the atomic displacements is described, which yields a sharp discrimination of domains, and which defines a unique interdomain screw-axis. Hinge axes are defined and classified as twist or closure axes depending on their direction. The methods have been tested on lysozyme. A remarkable correspondence was found between the first normal mode axis and the first principal mode axis, with both axes passing within 3 A of the alpha-carbon atoms of residues 2, 39, and 56 of human lysozyme, and near the interdomain helix. The axes of the first modes are overwhelmingly closure axes. A lesser degree of correspondence is found for the second modes, but in both cases they are more twist axes than closure axes. Both analyses reveal that the interdomain connections allow only these two degrees of freedom, one more than provided by a pure mechanical hinge.

Prediction of Protein Conformational Freedom From Distance Constraints

Abstract: A method is presented that generates random protein structures that fulfil a set of upper and lower interatomic distance limits. These limits depend on distances mea- sured in experimental structures and the strength of the interatomic interaction. Struc- tural differences between generated struc- tures are similar to those obtained from experi- ment and from MD simulation. Although detailed aspects of dynamical mechanisms are not covered and the extent of variations are only estimated in a relative sense, applications to an IgG-binding domain, an SH3 binding domain, HPr, calmodulin, and lysozyme are presented which illustrate the use of the method as a fast and simple way to predict structural variability in proteins. The method may be used to support the design of mutants, when structural fluctuations for a large num- ber of mutants are to be screened. The results suggest that motional freedom in proteins is ruled largely by a set of simple geometric constraints. Proteins 29:240-251, 1997. r 1997 Wiley-Liss, Inc.

Evaluation of comparative protein structure modeling by MODELLER-3.

Abstract: We evaluate homology-derived 3D models of dihydrofolate reductase (DFR1), phosphotransferase enzyme IIA domain (PTE2A3), and mouse/human UBC9 protein (UBC924) which were submitted to the second Meeting on the Critical Assessment of Techniques for Protein Structure Prediction (CASP). The DFR1 and PTE2A3 models, based on alignments without large errors, were slightly closer to their corresponding X-ray structures than the closest template structures. By contrast, the UBC924 model was slightly worse than the best template due to a misalignment of the N-terminal helix. Although the current models appear to be more accurate than the models submitted to the CASP meeting in 1994, the four major types of errors in side chain packing, position, and conformation of aligned segments, position and conformation of inserted segments, and in alignment still occur to almost the same degree. The modest improvement probably originates from the careful manual selection of the templates and editing of the alignment, as well as from the iterative realignment and model building guided by various model evaluation techniques. This iterative approach to comparative modeling is likely to overcome at least some initial alignment errors, as demonstrated by the correct final alignment of the C terminus of DFR1. Proteins, Suppl. 1:50–58, 1997. © 1998 Wiley-Liss, Inc.

Mechanisms of signaling and related enzymes

Abstract: Most enzymes involved in cell signaling, such as protein kinases, protein phosphatases, GTPases, and nucleotide cyclases catalyze nucleophilic substitutions at phosphorus When possible, the mechanisms of such enzymes are most clearly described quantitatively in terms of how associative or dissociative they are The mechanisms of cell signaling enzymes range from or = 10(5), general acid catalysis (approximately 10(3 +/- 1)), general base catalysis (approximately 10(3 +/- 1)), and transition-state stabilization by cationic and hydrogen bond donating residues (approximately 10(3 +/- 1))

Protein domain movements: detection of rigid domains and visualization of hinges in comparisons of atomic coordinates

Abstract: The activity of many proteins induces conformational transitions by hinge-bending, which involves the movement of relatively rigid parts of a protein about flexible joints. We present an algorithm to identify and visualize the movements of rigid domains about common hinges in proteins. In comparing two structures, the method partitions a protein into domains of preserved geometry. The domains are extracted by an adaptive selection procedure using least-squares fitting. The user can maintain the spatial connectivity of the domains and filter significant structural differences (domain movements) from noise in the compared sets of atomic coordinates. The algorithm subsequently characterizes the relative movements of the found domains by effective rotation axes (hinges). The method is applied to several known instances of domain movements in protein structures, namely, in lactoferrin, hexokinase, actin, the extracellular domains of human tissue factor, and the receptor of human growth factor. The results are visualized with the molecular graphics package VMD (Humphrey et al., J. Mol. Graphics 14(1):33-38, 1996). Applications of the algorithm to the analysis of conformational changes in proteins and to biomolecular docking are discussed.

Species-specificity of the cohesin-dockerin interaction between Clostridium thermocellum and Clostridium cellulolyticum: prediction of specificity determinants of the dockerin domain.

Abstract: The cross-species specificity of the cohesin–dockerin interaction, which defines the incorporation of the enzymatic subunits into the cellulosome complex, has been investigated. Cohesin-containing segments from the cellulosomes of two different species, Clostridium thermocellum and Clostridium cellulolyticum, were allowed to interact with cellulosomal (dockerin-containing) enzymes from each species. In both cases, the cohesin domain of one bacterium interacted with enzymes from its own cellulosome in a calcium-dependent manner, but the same cohesin failed to recognize enzymes from the other species. Thus, in the case of these two bacteria, the cohesin–dockerin interaction seems to be species-specific. Based on intra- and cross-species sequence comparisons among the different dockerins together with their known specificities, we tender a prediction as to the amino-acid residues critical to recognition of the cohesins. The suspected residues were narrowed down to only four, which comprise a repeated pair located within the calcium-binding motif of two duplicated sequences, characteristic of the dockerin domain. According to the proposed model, these four residues do not participate in the binding of calcium per se; instead, they appear to serve as recognition codes in promoting interaction with the cohesin surface. Proteins 29:517–527, 1997. © 1997 Wiley-Liss, Inc.

NADP-dependent enzymes. I: Conserved stereochemistry of cofactor binding.

Abstract: The ubiquitous redox cofactors nicotinamide adenine dinucleotides [NAD and NADP] are very similar molecules, despite their participation in substantially different biochemical processes. NADP differs from NAD in only the presence of an additional phosphate group esterified to the 2'-hydroxyl group of the ribose at the adenine end and yet NADP is confined with few exceptions to the reactions of reductive biosynthesis, whereas NAD is used almost exclusively in oxidative degradations. The discrimination between NAD and NADP is therefore an impressive example of the power of molecular recognition by proteins. The many known tertiary structures of NADP complexes affords the possibility for an analysis of their discrimination. A systematic analysis of several crystal structures of NAD(P)-protein complexes show that: 1) the NADP coenzymes are more flexible in conformation than those of NAD; 2) although the protein-cofactor interactions are largely conserved in the NAD complexes, they are quite variable in those of NADP; and 3) in both cases the pocket around the nicotinamide moiety is substrate dependent. The conserved and variable interactions between protein and cofactors in the respective binding pockets are reported in detail. Discrimination between NAD and NADP is essentially a consequence of the overall pocket and not of a few residues. A clear fingerprint in NAD complexes is a carboxylate side chain that chelates the diol group at the ribose near the adenine, whereas in NADP complexes an arginine side chain faces the adenine plane and interacts with the phosphomonoester. The latter type of interaction might be a general feature of recognition of nucleotides by proteins. Other features such as strand-like hydrogen bonding between the NADP diphosphate moieties and the protein are also significant. The NADP binding pocket properties should prove useful in protein engineering and design.

The kinetics of protein-protein recognition.

Abstract: We examine a simple kinetic model for association that incorporates the basic features of protein-protein recognition within the rigid body approximation, that is, when no large conformation change occurs. Association starts with random collision at the rate k(coll) predicted by the Einstein-Smoluchowski equation. This creates an encounter pair that can evolve into a stable complex if and only if the two molecules are correctly oriented and positioned, which has a probability p(r). In the absence of long-range interactions, the bimolecular rate of association is p(r) k(coll). Long-range electrostatic interactions affect both k(coll) and p(r). The collision rate is multiplied by q(t), a factor larger than 1 when the molecules carry net charges of opposite sign as coulombic attraction makes collisions more frequent, and less than 1 in the opposite case. The probability p(r) is multiplied by a factor q(r) that represents the steering effect of electric dipoles, which preorient the molecules before they collide. The model is applied to experimental data obtained by Schreiber and Fersht (Nat. Struct. Biol. 3:427-431, 1996) on the kinetics of barnase-barstar association. When long-range electrostatic interactions are fully screened or mutated away, q(t)q(r) approximately 1, and the observed rate of productive collision is p(r) k(coll) approximately 10(5) M(-1) x s(-1). Under these conditions, p(r) approximately 1.5 x 10(-5) is determined by geometric constraints corresponding to a loss of rotational freedom. Its value is compatible with computer docking simulations and implies a rotational entropy loss deltaS(rot) approximately 22 e.u. in the transition state. At low ionic strength, long-range electrostatic interactions accelerate barnase-barstar association by a factor q(t)q(r) of up to 10(5) as favorable charge-charge and charge-dipole interactions work together to make it much faster than free diffusion would allow.

Crystal structures of the methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath): implications for substrate gating and component interactions.

Abstract: The crystal structure of the nonheme iron-containing hydroxylase component of methane monooxygenase hydroxylase (MMOH) from Methylococcus capsulatus (Bath) has been solved in two crystal forms, one of which was refined to 1.7 A resolution. The enzyme is composed of two copies each of three subunits (α2β2γ2), and all three subunits are almost completely α-helical, with the exception of two β hairpin structures in the α subunit. The active site of each α subunit contains one dinuclear iron center, housed in a four-helix bundle. The two iron atoms are octahedrally coordinated by 2 histidine and 4 glutamic acid residues as well as by a bridging hydroxide ion, a terminal water molecule, and at 4°C, a bridging acetate ion, which is replaced at −160°C with a bridging water molecule. Comparison of the results for two crystal forms demonstrates overall conservation and relative orientation of the domain structures. The most prominent structural difference identified between the two crystal forms is in an altered side chain conformation for Leu 110 at the active site cavity. We suggest that this residue serves as one component of a hydrophobic gate controlling access of substrates to and products from the active site. The leucine gate may be responsible for the effect of the B protein component on the reactivity of the reduced hydroxylase with dioxygen. A potential reductase binding site has been assigned based on an analysis of crystal packing in the two forms and corroborated by inhibition studies with a synthetic peptide corresponding to the proposed docking position. Proteins 29:141–152, 1997. © 1997 Wiley-Liss, Inc.

Predicting protein structure using hidden markov models

Abstract: We discuss how methods based on hidden Markov models performed in the fold recognition section of the CASP2 experiment. Hidden Markov models were built for a set of about a thousand structures from the PDB database, and each CASP2 target sequence was scored against this library of hidden Markov models. In addition, a hidden Markov model was built for each of the target sequences, and all of the sequences in PDB were scored against that target model. Having high scores from both methods was found to be highly indicative of the target and a structure being homologous. Predictions were made based on several criteria: the scores with the structure models, the scores with the target models, consistency between the secondary structure in the known structure and predictions for the target (using the program PhD), human examination of predicted alignments between target and structure (using RASMOL), and solvation preferences in the alignment of the target and structure. The method worked well in comparison to other methods used at CASP2 for targets of moderate difficulty, where the closest structure in PDB could be aligned to the target with at least 15% residue identity. There was no evidence for the method''s effectiveness for harder cases, where the residue identity was much lower than 15%.

Simulation of protein conformational freedom as a function of pH: constant-pH molecular dynamics using implicit titration.

Abstract: Solution pH is a determinant parameter on protein function and stability, and its inclusion in molecular dynamics simulations is attractive for studies at the molecular level. Current molecular dynamics simulations can consider pH only in a very limited way, through a somewhat arbitrary choice of a set of fixed charges on the titrable sites. Conversely, continuum electrostatic methods that explicitly treat pH effects assume a single protein conformation whose choice is not clearly defined. In this paper we describe a general method that combines both titration and conformational freedom. The method is based on a potential of mean force for implicit titration and combines both usual molecular dynamics and pH-dependent calculations based on continuum methods. A simple implementation of the method, using a mean field approximation, is presented and applied to the bovine pancreatic trypsin inhibitor. We believe that this constant-pH molecular dynamics method, by correctly sampling both charges and conformation, can become a valuable help in the understanding of the dependence of protein function and stability on pH. © 1997 Wiley-Liss Inc.

Extent and nature of contacts between protein molecules in crystal lattices and between subunits of protein oligomers

Abstract: A survey was compiled of several characteristics of the intersubunit contacts in 58 oligomeric proteins, and of the intermolecular contracts in the lattice for 223 protein crystal structures. The total number of atoms in contact and the secondary structure elements involved are similar in the two types of interfaces. Crystal contact patches are frequently smaller than patches involved in oligomer interfaces. Crystal contacts result from more numerous interactions by polar residues, compared with a tendency toward nonpolar amino acids at oligomer interfaces. Arginine is the only amino acid prominent in both types of interfaces. Potentials of mean force for residue-residue contacts at both crystal and oligomer interfaces were derived from comparison of the number of observed residue-residue interactions with the number expected by mass action. They show that hydrophobic interactions at oligomer interfaces favor aromatic amino acids and methionine over aliphatic amino acids; and that crystal contacts form in such a way as to avoid inclusion of hydrophobic interactions. They also suggest that complex salt bridges with certain amino acid compositions might be important in oligomer formation. For a protein that is recalcitrant to crystallization, substitution of lysine residues with arginine or glutamine is a recommended strategy.

Loss of Translational Entropy in Binding, Folding, and Catalysis

Abstract: There is a loss of transla- tional entropy associated with the formation of a complex between two molecules in solu- tion. Estimation of this contribution is essen- tial for understanding binding, protein-pro- tein association, and catalysis. Based on the cell model of liquids, it is possible to estimate the loss of translational entropy in all these cases. The resulting formulas are straightfor- ward, and the calculations are easy to perform. Comparison of the results with experimental data suggests that the proposed method pro- vides estimates that are much more accurate than those obtained with existing methods. Proteins 28:144-149, 1997. r 1997 Wiley-Liss, Inc.

Structure of soybean lipoxygenase L3 and a comparison with its L1 isoenzyme

Abstract: Soybean lipoxygenase isoenzyme L3 represents a second example (after L1) of the X-ray structure (R = 17% at 2.6 A resolution) for a member of the large family of lipoxygenases. L1 and L3 have different characteristics in catalysis, although they share 72% sequence identity (the changes impact 255 amino acids) and similar folding (average C alpha rms deviation of 1 A). The critical nonheme iron site has the same features as for L1:3O and 3N in pseudo C3v orientation, with two oxygen atoms (from Asn713 and water) at a nonbinding distance. Asn713 and His518 are strategically located at the junction of three cavities connecting the iron site with the molecule surface. The most visible differences between L1 and L3 isoenzymes occur in and near these cavities, affecting their accessibility and volume. Among the L1/L3 substitutions Glu256/ Thr274, Tyr409/His429, and Ser747/Asp766 affect the salt bridges (L1: Glu256...His248 and Asp490...Arg707) that in L1 restrict the access to the iron site from two opposite directions. The L3 molecule has a passage going through the whole length of the helical domain, starting at the interface with the Nt-domain (near 25-27 and 254-278) and going to the opposite end of the Ct-domain (near 367, 749). The substrate binding and the role of His513, His266, His776 (and other residues nearby) are illustrated and discussed by using models of linoleic acid binding. These hypotheses provide a possible explanation for a stringent stereo-specificity of catalytic products in L1 (that produces predominantly 13-hydroperoxide) versus the lack of such specificity in L3 (that turns out a mixture of 9- and 13-hydroperoxides and their diastereoisomers).

The high-resolution crystal structure of a 24-kDa gyrase B fragment from E. coli complexed with one of the most potent coumarin inhibitors, clorobiocin

Abstract: Coumarin antibiotics, such as clorobiocin, novobiocin, and coumermycin A1, inhibit the supercoiling activity of gyrase by binding to the gyrase B (GyrB) subunit. Previous crystallographic studies of a 24-kDa N-terminal domain of GyrB from E. coli complexed with novobiocin and a cyclothialidine analogue have shown that both ligands act by binding at the ATP-binding site. Clorobiocin is a natural antibiotic isolated from several Streptomyces strains and differs from novobiocin in that the methyl group at the 8 position in the coumarin ring of novobiocin is replaced by a chlorine atom, and the carbamoyl at the 3' position of the noviose sugar is substituted by a 5-methyl-2-pyrrolylcarbonyl group. To understand the difference in affinity, in order that this information might be exploited in rational drug design, the crystal structure of the 24-kDa GyrB fragment in complex with clorobiocin was determined to high resolution. This structure was determined independently in two laboratories, which allowed the validation of equivalent interpretations. The clorobiocin complex structure is compared with the crystal structures of gyrase complexes with novobiocin and 5'-adenylyl-beta, gamma-imidodiphosphate, and with information on the bound conformation of novobiocin in the p24-novobiocin complex obtained by heteronuclear isotope-filtered NMR experiments in solution. Moreover, to understand the differences in energetics of binding of clorobiocin and novobiocin to the protein, the results from isothermal titration calorimetry are also presented.

Glucoamylase structural, functional, and evolutionary relationships

Abstract: To correlate structural features with glucoamylase properties, a structure-based multisequence alignment was constructed using information from catalytic and starch-binding domain models. The catalytic domain is composed of three hydrophobic folding units, the most labile and least hydrophobic of them being missing in the most stable glucoamylase. The role of O-glycosylation in stabilizing the most hydrophobic folding unit, the only one where thermostabilizing mutations with unchanged activity have been made, is described. Differences in both length and composition of interhelical loops are correlated with stability and selectivity characteristics. Two new glucoamylase subfamilies are defined by using homology criteria. Protein parsimony analysis suggests an ancient bacterial origin for the glucoamylase gene. Increases in length of the belt surrounding the active site, degree of O-glycosylation, and length of the linker probably correspond to evolutionary steps that increase stability and secretion levels of Aspergillus-related glucoamylases.

The family of the IL-6-type cytokines: specificity and promiscuity of the receptor complexes

Abstract: The cytokines IL-6, LIF, CNTF, OSM, IL-11, and CT-1 have been grouped into the family of IL-6-type cytokines, since they all require gp130 for signal transduction. Interestingly, gp130 binds directly to OSM, whereas complex formation with the other cytokines depends on additional receptor subunits. Only limited structural information on these cytokines and their receptors is available. X-ray structures have been solved for the cytokines LIF and CNTF, whose up-up-down-down four-helix bundle is common to all of these cytokines, and for the receptors of hGH and prolactin, which contain two domains with a fibronectin III-like fold. Since cocrystallization and x-ray analysis of the up to four different proteins forming the receptor complexes of the IL-6-type cytokines is unlikely to be achieved in the near future, model building based on the existing structural information is the only approach for the time being. Here we present model structures of the complexes of human and murine IL-6 with their receptors. Their validity can be deduced from the fact that published mutagenesis data and the different receptor specificity of human and murine IL-6 can be understood. It is now possible to predict the relative positions and contacts for all molecules in their respective complexes. Such information can be used for the rational design of cytokine and receptor antagonists, which may have a valuable therapeutic perspective.

Evaluation of gramm low-resolution docking methodology on the hemagglutinin-antibody complex

Abstract: A single protein-protein pair, the complex of the influenza virus hemagglutinin with an antibody (Fab BH151), was suggested for prediction at the second experiment on the Critical Assessment of Techniques for Protein Structure Prediction. To predict the structure of the complex, we applied our docking program GRAMM at a decreased resolution (to accommodate the conformational inaccuracies). The lowest-energy match showed a remarkable "low-resolution" surface complementarity between the molecular structures. After receiving the experimental structure of the complex we had a chance to verify our assumptions and results. The analysis of the hemagglutinin-antibody interface revealed several significant conformational changes in the side chains, which resulted in deep interpenetrations of the hemagglutinin and the antibody structures. This confirmed our initial assumption that the structural changes will be beyond the tolerance of high-resolution rigid-body docking. The comparison of the predicted low-resolution match, submitted as the solution, and the experimentally determined complex showed significant structural discrepancies in the orientation of the antibody, due to the low-resolution character of the docking. Because of the severe structural errors, no residue-residue contacts were predicted correctly. However, a significant part of the antigenic site was determined. This illustrates the practical value of the present methodology for the initial prediction of the binding site, as well as points out the problem of transition from the low-resolution predictions of protein-protein complexes to the accurate structure.

Understanding the recognition of protein structural classes by amino acid composition

Abstract: Knowledge of amino acid composition, alone, is verified here to be suffi- cient for recognizing the structural class, a, b, a1b ,o r a / bof a given protein with an accu- racy of 81%. This is supported by results from exhaustive enumerations of all conformations for all sequences of simple, compact lattice models consisting of two types (hydrophobic and polar) of residues. Different compositions exhibit strong affinities for certain folds. Within the limits of validity of the lattice models, two factors appear to determine the choice of particular folds: 1) the coordination numbers of individual sites and 2) the size and geometry of non-bonded clusters. These two properties, collectively termed the distribution of non- bonded contacts, are quantitatively assessed by an eigenvalue analysis of the so-called Kirch- hoff or adjacency matrices obtained by con- sidering the non-bonded interactions on a lat- tice. The analysis permits the identification of conformations that possess the same distri- bution of non-bonded contacts. Furthermore, some distributions of non-bonded contacts are favored entropically, due to their high degeneracies. Thus, a competition between enthalpic and entropic effects is effective in determining the choice of a distribution for a given composition. Based on these findings, an analysis of non-bonded contacts in protein structures was made. The analysis shows that proteins belonging to the four distinct folding classes exhibit significant differences in their distributions of non-bonded contacts, which more directly explains the success in predicting structural class from amino acid composition. Proteins 29:172-185,

Short-range conformational energies, secondary structure propensities, and recognition of correct sequence-structure matches

Abstract: A statistical analysis of known structures is made for an assessment of the utility of short-range energy considerations. For each type of amino acid, the potentials governing (1) the torsions and bond angle changes of virtual C alpha-C alpha bonds and (2) the coupling between torsion and bond angle changes are derived. These contribute approximately -2 RT per residue to the stability of native proteins, approximately half of which is due to coupling effects. The torsional potentials for the alpha-helical states of different residues are verified to be strongly correlated with the free-energy change measurements made upon single-site mutations at solvent-exposed regions. Likewise, a satisfactory correlation is shown between the beta-sheet potentials of different amino acids and the scales from free-energy measurements, despite the role of tertiary context in stabilizing beta-sheets. Furthermore, there is excellent agreement between our residue-specific potentials for alpha-helical state and other thermodynamic based scales. Threading experiments performed by using an inverse folding protocol show that 50 of 62 test structures correctly recognize their native sequence on the basis of short-range potentials. The performance is improved to 55, upon simultaneous consideration of short-range potentials and the nonbonded interaction potentials between sequentially distant residues. Interactions between near residues along the primary structure, i.e., the local or short-range interactions, are known to be insufficient, alone, for understanding the tertiary structural preferences of proteins alone. Yet, knowledge of short-range conformational potentials permits rationalizing the secondary structure propensities and aids in the discrimination between correct and incorrect tertiary folds.

Hydrophobic patches on protein subunit interfaces: characteristics and prediction.

Abstract: Hydrophobic patches, defined as clusters of neighboring apolar atoms deemed accessible on a given protein surface, have been investigated on protein subunit interfaces. The data were taken from known tertiary structures of multimeric protein complexes. Amino acid composition and preference, patch size distribution, and patch contact complementarity across associating subunits were examined and compared with hydrophobic patches found on the solvent-accessible surface of the multimeric complexes. The largest or second largest patch on the accessible surface of the entire subunit was involved in multimeric interfaces in 90% of the cases. These results should prove useful for subunit design and engineering as well as for prediction of subunit interface regions.

Structural basis for thermostability and identification of potential active site residues for adenylate kinases from the archaeal genus Methanococcus.

Abstract: Sequence comparisons of highly related archaeal adenylate kinases (AKs) from the mesophilic Methanococcus voltae, the moderate thermophile Methanococcus thermolithotrophicus, and two extreme thermophiles Methanococcus igneus and Methanococcus jannaschii, allow identification of interactions responsible for the large variation in temperatures for optimal catalytic activity and thermostabilities observed for these proteins. The tertiary structures of the methanococcal AKs have been predicted by using homology modeling to further investigate the potential role of specific interactions on thermal stability and activity. The alignments for the methanococcal AKs have been generated by using an energy-based sequence–structure threading procedure against high-resolution crystal structures of eukaryotic, eubacterial, and mitochondrial adenylate and uridylate (UK) kinases. From these alignments, full atomic model structures have been produced using the program MODELLER. The final structures allow identification of potential active site interactions and place a polyproline region near the active site, both of which are unique to the archaeal AKs. Based on these model structures, the additional polar residues present in the thermophiles could contribute four additional salt bridges and a higher negative surface charge. Since only one of these possible salt bridges is interior, they do not appear significantly to the thermal stability. Instead, our model structures indicate that a larger and more hydrophobic core, due to a specific increase in aliphatic amino acid content and aliphatic side chain volume, in the thermophilic AKs is responsible for increased thermal stability. © 1997 Wiley-Liss Inc.

Assessment of comparative modeling in CASP2

Abstract: An assessment is presented for all submissions to the comparative modeling challenge in the 1996 Critical Assessment of Structure Prediction (CASP2). Of the original 12 target structures, 9 were solved prior to the meeting: 8 by X-ray crystallography and 1 by NMR spectroscopy. These targets varied over a large range of difficulty, as assessed by the percentage sequence identity with the principal parent structure, which ranged from 20% up to 85%. The overall quality of the models reflected the similarity of the principal parent. As expected, when the sequence alignment was correct, the core was accurately modeled, with the largest deviations occurring in the loops. Models were built which gave C alpha root-mean-square deviations (RMSDs) compared with the observed structure of 18 A. Compared with CASP1, the geometry of the models was significantly improved with no D-amino acids. By far the major contribution to RMSD error was the alignment accuracy, which varied from 100% down to 7% over the range of targets. In the structurally variable regions, global shifts, caused by hinge bending, were the major source of error, giving significantly lower local RMSDs than global RMSDs. In over 50% of these noncore regions, the difference between global and local RMSDs was more than 3 A, and was as high as 10 A for one structurally variable region. For the side chains, the chi 1 RMSDs are strongly correlated with the C alpha RMSDs. For models with C alpha deviations less than 1 A, on average 78.5% of side chains are placed in the correct rotamer, although the chi 1 RMSDs, though clearly better than random, were disappointing at around 46 degrees. As the backbone deviations increased, the side chain placement became less accurate, with an average chi 1 RMSD of 75 degrees on a 1.5-2.5 A C alpha backbone (average 51.4% correct rotamer). Refinement by energy minimization or molecular dynamics made only minor adjustments to improve local geometry and generally made small, but not significant, improvements to the RMSD. In total, 19 groups submitted 62 models (89 coordinate sets) that could be assessed. Most modelers used manual adjustments to sequence alignments and, in general, good alignments were obtained down to 25% sequence identity. The modeling methods ranged from "classical" modeling, involving core building followed by loop and side chain addition, to more sophisticated approaches based on probability distributions, Monte Carlo sampling or distance geometry. For each target, several groups produced equally good models, given the expected errors in the structures (about 0.5 A). No one method came out as clearly superior, although the approaches that inherit directly from the parents generally performed better than the more radical techniques. However, for each target there were some poor models, usually reflecting a poor sequence alignment, and the range of accuracy for each target is therefore large. Fully automated methods are able to perform very well for "easy" targets (85% sequence identity with parent), but when modeling using a distantly related parent, care and expertise, especially in performing the alignment, still appear to be important factors in generating accurate models.

Evaluation of the CASP2 docking section

Abstract: The docking section of CASP2 is reviewed. Seven small molecule ligand–protein targets and one protein–protein target were available for predictions. Many of the small molecule ligand complexes involved serine proteases. Overall results for the small molecule targets were good, with at least one prediction for each target being within 3 A root-mean-square deviation (RMSD) for nearly all targets and within 2 A RMSD for over half the targets. However, no single docking method seemed to consistently perform best. In addition, the predictions closest to the experimental results were not always those ranked the highest, pointing out that the evaluation (scoring) of potential solutions is still an area that needs improvement. The protein–protein target proved more difficult. None of the predictions did well in reproducing the geometry of the complex, although in many cases the interacting surfaces of the two proteins were predicted with reasonable accuracy. This target consisted of two large proteins and, therefore was a demanding target for docking methods. Proteins, Suppl. 1:198–204, 1997. © 1998 Wiley-Liss, Inc.

Effects of organic solvents on protein structures: observation of a structured helical core in hen egg-white lysozyme in aqueous dimethylsulfoxide.

Abstract: A partly folded state of hen egg-white lysozyme has been characterized in 50% DMSO. Low concentrations of DMSO ( 10%) a cooperative transition of the structure to a new, partially folded state is observed. This transition is essentially complete by appx.50% DMSO. NMR studies show an overall decrease in chemical shift dispersion with marked broadening of many resonances. A substantial number of backbone and side chain–side chain NOEs suggests the presence of secondary and tertiary interactions in the intermediate state. Tertiary organization of the aromatic residues is also demonstrated by enhanced near-UV circular dichroism and limited exposure of tryptophans as monitored by iodide quenching of fluorescence. The intermediate state exhibits enhanced binding to hydrophobic dyes. Further, the structural transition from this state to a largely unfolded conformation is cooperative. H/D exchange rates of several amide protons and four indole protons of tryptophans (W28, W108, W111, and W123), measured by refolding from 50% DMSO at different time intervals reveal that protection factors are high for the helical domain, whereas NH groups in the triple stranded antiparallel beta-sheet domain are largely solventexposed. An ordered hydrophobic core in the intermediate state comprising of helix A, helix B, and helix D is consistent with the high protection factors observed. The structured intermediate in 50% DMSO resembles the early kinetic intermediate observed in the refolding of hen egg white lysozyme, as well as a molten globule state of equine lysozyme at low pH. The results demonstrate the potential use of nonaqueous structure perturbing solvents like DMSO to stabilize partially folded conformations of proteins.