Evolution of enzymatic activities in the enolase superfamily: crystal structure of (D)-glucarate dehydratase from Pseudomonas putida

The structure of (D)-glucarate dehydratase from Pseudomonas putida (GlucD) has been solved at 2.3 A resolution by multiple isomorphous replacement and refined to a final R-factor of 19.0%. The protein crystallizes in the space group I222 with one subunit in the asymmetric unit. The unit cell dimensions are a = 69.6 A, b = 108.8 A, and c = 122.6 A. The crystals were grown using the batch method where the primary precipitant was poly(ethylene glycol) 1000. The structure reveals that GlucD is a tetramer of four identical polypeptides, each containing 451 residues. The structure was determined without a bound substrate or substrate analogue. Three disordered regions are noted: the N-terminus through residue 11, a loop containing residues 99 through 110, and the C-terminus from residue 423. On the basis of primary sequence alignments, we previously concluded that GlucD is a member of the mandelate racemase (MR) subfamily of the enolase superfamily [Babbitt, P. C., Hasson, M. S., Wedekind, J. E., Palmer, D. R. J., Barrett, W. C., Reed, G. J., Rayment, I., Ringe, D., Kenyon, G. L., and Gerlt, J. A. (1996) Biochemistry 35, 16489-16501]. This prediction is now verified, since the overall fold of GlucD is strikingly similar to those of MR, muconate lactonizing enzyme I, and enolase. Also, many of the active site residues of GlucD can be superimposed on those found in the active site of MR. The implications of this structure on the evolution of catalysis in the enolase superfamily are discussed.     

Stress and strain in staphylococcal nuclease

Protein molecules generally adopt a tertiary structure in which all backbone and side chain conformations are arranged in local energy minima; however, in several well-refined protein structures examples of locally strained geometries, such as cis peptide bonds, have been observed. Staphylococcal nuclease A contains a single cis peptide bond between residues Lys 116 and Pro 117 within a type VIa beta-turn. Alternative native folded forms of nuclease A have been detected by NMR spectroscopy and attributed to a mixture of cis and trans isomers at the Lys 116-Pro 117 peptide bond. Analyses of nuclease variants K116G and K116A by NMR spectroscopy and X-ray crystallography are reported herein. The structure of K116A is indistinguishable from that of nuclease A, including a cis 116-117 peptide bond (92% populated in solution). The overall fold of K116G is also indistinguishable from nuclease A except in the region of the substitution (residues 112-117), which contains a predominantly trans Gly 116-Pro 117 peptide bond (80% populated in solution). Both Lys and Ala would be prohibited from adopting the backbone conformation of Gly 116 due to steric clashes between the beta-carbon and the surrounding residues. One explanation for these results is that the position of the ends of the residue 112-117 loop only allow trans conformations where the local backbone interactions associated with the phi and psi torsion angles are strained. When the 116-117 peptide bond is cis, less strained backbone conformations are available. Thus the relaxation of the backbone strain intrinsic to the trans conformation compensates for the energetically unfavorable cis X-Pro peptide bond. With the removal of the side chain from residue 116 (K116G), the backbone strain of the trans conformation is reduced to the point that the conformation associated with the cis peptide bond is no longer favorable.     

The X-ray structure of Escherichia coli enoyl reductase with bound NAD+ at 2.1 A resolution

Enoyl acyl carrier protein reductase catalyses the last reductive step of fatty acid biosynthesis, reducing an enoyl acyl carrier protein to an acyl-acyl carrier protein with NAD(P)H as the cofactor. The crystal structure of enoyl reductase (ENR) from Escherichia coli has been determined to 2.1 A resolution using a combination of molecular replacement and isomorphous replacement and refined using data from 10 A to 2.1 A to an R-factor of 0.16. The final model consists of the four subunits of the tetramer, wherein each subunit is composed of 247 of the expected 262 residues, and a NAD+ cofactor for each subunit of the tetramer contained in the asymmetric unit plus a total of 327 solvent molecules. There are ten disordered residues per subunit which form a loop near the nucleotide binding site which may become ordered upon substrate binding. Each monomer is composed of a seven-stranded parallel beta-sheet flanked on each side by three alpha-helices with a further helix lying at the C terminus of the beta-sheet. This fold is highly reminiscent of the Rossmann fold, found in many NAD(P)H-dependent enzymes. Analysis of the sequence and structure of ENR and comparisons with the family of short-chain alcohol dehydrogenases, identify a conserved tyrosine and lysine residue as important for catalytic activity. Modelling studies suggest that a region of the protein surface that contains a number of strongly conserved hydrophobic residues and lies adjacent to the nicotinamide ring, forms the binding site for the fatty acid substrate.     

Structure of the complex of Maclura pomifera agglutinin and the T-antigen disaccharide, Galbeta1,3GalNAc

Maclura pomifera agglutinin is a tetrameric plant seed lectin with high affinity for the tumor-associated T-antigen disaccharide, Galbeta1,3GalNAcalpha, and hence for many O-linked glycopeptide structures. Unlike members of most lectin families, it lacks both metal ions and Cys residues. The structure of its complex with Galbeta1,3GalNAc was determined to 2.2 by first using multiwavelength anomalous diffraction with a lead derivative of the native protein, and then using molecular replacement with the unrefined structure as a model to solve the structure of the complex. The subunits share the beta-prism architecture and three-fold pseudo-symmetry of the related lectin jacalin, with the 21-residue beta-chains in the center of the tetramer. Interactions with the GalNAc predominate in the binding of the disaccharide. It forms a network of H-bonds with only one side chain, from an Asp residue, the amino group of the N-terminal Gly of the alpha-chain, and peptide backbone atoms of two aromatic residues. The Gal moiety does not H-bond directly with residues in the same monomer, i.e. there is no true subsite for it, but there are interactions through two water molecules. In the crystal, it interacts with residues in the binding site of an adjacent tetramer. The minimum energy conformation expected for the disaccharide is retained, despite its mediating the tetramer-tetramer interactions in the crystal packing. The resulting lattice is comparable to those seen for complexes of other lectins with branched glycopeptides.     

Three-dimensional structure of Escherichia coli dihydrodipicolinate reductase in complex with NADH and the inhibitor 2,6-pyridinedicarboxylate

Dihydrodipicolinate reductase catalyzes the NAD(P)H-dependent reduction of the alpha,beta-unsaturated cyclic imine dihydrodipicolinate to form the cyclic imine tetrahydrodipicolinate. The enzyme is a component of the biosynthetic pathway that leads to diaminopimelate and lysine in bacteria and higher plants. Because these pathways are unique to microorganisms and plants, they may represent attractive targets for new antimicrobial or herbicidal compounds. The three-dimensional structure of the ternary complex of Escherichia coli dihydrodipicolinate reductase with NADH and the inhibitor 2,6-pyridinedicarboxylate has been solved using a combination of molecular replacement and noncrystallographic symmetry averaging procedures and refined against 2.6 A resolution data to a crystallographic R-factor of 21.4% (Rfree is 29.7%). The native enzyme is a 120 000 molecular weight tetramer of identical subunits. The refined crystallographic model contains a tetramer, three molecules of NADH, three molecules of inhibitor, one phosphate ion, and 186 water molecules per asymmetric unit. Each subunit consists of two domains connected by two flexible hinge regions. While three of the four subunits of the tetramer have a closed conformation, in which the nicotinamide ring of the cofactor bound to the N-terminal domain and the reducible carbon of the substrate bound to the substrate binding domain are about 3.5 A away, the fourth subunit is unliganded and shows an open conformation, suggesting that the enzyme undergoes a major conformational change upon binding of both substrates. The residues involved in binding of the inhibitor and the residues involved in catalysis have been identified on the basis of the three-dimensional structure. Site-directed mutants have been used to further characterize the role of these residues in binding and catalysis. A chemical mechanism for the enzyme, based on these and previously reported data, is proposed.     

A Gly --> Ser change causes defective folding in vitro of calcium-binding epidermal growth factor-like domains from factor IX and fibrillin-1

The calcium-binding epidermal growth factor-like (cbEGF) domain is a common motif found in extracellular proteins. A mutation that changes a highly conserved Gly residue to Ser in this domain has been identified both in the factor IX (FIX) and fibrillin-1 genes, where it is associated with relatively mild variants of hemophilia B and Marfan syndrome, respectively. We have investigated the structural consequences in vitro of this amino acid change when introduced into single cbEGF domains from human FIX (G60S) and human fibrillin-1 (G1127S), and a covalently linked pair of cbEGF domains from fibrillin-1. High pressure liquid chromatography analysis, mass spectrometry, and 1H NMR analysis demonstrate that wild-type cbEGF domains purified in the reduced form and refolded in vitro adopt the native fold. In contrast, the Gly --> Ser change causes defective folding of FIX and fibrillin-1 cbEGF domains. However, in the case of the factor IX mutant domain, a Ca2+-dependent change in conformation, identified by NMR in a proportion of the refolded material, suggests that some material refolds to a native-like structure. This is consistent with enzyme-linked immunosorbent assay analysis of FIX G60S from a hemophilia B patient Oxford d2, which demonstrates that the mutant protein is partially recognized by a monoclonal antibody specific for this region of FIX. NMR analysis of a covalently linked pair of fibrillin cbEGF domains demonstrates that the C-terminal domain adopts the native epidermal growth factor fold, despite the fact that the adjacent mutant domain is misfolded. The implications of these results for disease pathogenesis are discussed.     

Refined X-ray structure of Dictyostelium discoideum nucleoside diphosphate kinase at 1.8 A resolution

The X-ray structure of the nucleoside diphosphate kinase (NDP kinase) from Dictyostelium discoideum has been refined at 1.8 A resolution from a hexagonal crystal form with a 17 kDa monomer in its asymmetric unit. The atomic model was derived from the previously determined structure of a point mutant of the protein. It contains 150 amino acid residues out of 155, and 95 solvent molecules. The R-factor is 0.196 and the estimated accuracy of the average atomic position, 0.25 A. The Dictyostelium structure is described in detail and compared to those of Drosophila and Myxococcus xanthus NDP kinases. The protein is a hexamer with D3 symmetry. Residues 8 to 138 of each subunit form a globular alpha/beta domain. The four-stranded beta-sheet is antiparallel; its topology is different from other phosphate transfer enzymes, and also from the HPr protein which, like NDP kinase, carries a phosphorylated histidine. The same topology is nevertheless found in several other proteins that bind mononucleotides, RNA or DNA. Strand connections in NDP kinase involve alpha-helices and a 20-residue segment called the Kpn loop. The beta-sheet is regular except for a beta-bulge in edge strand beta 2 and a gamma-turn at residue Ile120 just preceding strand beta 4. The latter may induce strain in the main chain near the active site His122. The alpha 1 beta 2 motif participates in forming dimers within the hexamer, helices alpha 1 and alpha 3, the Kpn loop and C terminus, in forming trimers. The subunit fold and dimer interactions found in Dictyostelium are conserved in other NDP kinases. Trimer interactions probably occur in all eukaryotic enzymes. They are absent in the bacterial Myxococcus xanthus enzyme which is a tetramer, even though the subunit structure is very similar. In Dictyostelium, contacts between Kpn loops near the 3-fold axis block access to a central cavity lined with polar residues and filled with well-defined solvent molecules. Biochemical data on point mutants highlight the contribution of the Kpn loop to protein stability. In Myxococcus, the Kpn loops are on the tetramer surface and their sequence is poorly conserved. Yet, their conformation is maintained and they make a similar contribution to the substrate binding site.     

Ventral subiculum regulates hypothalamo-pituitary-adrenocortical and behavioural responses to cognitive stressors

The high resolution crystal structure of Saccharomyces cerevisiae phosphoglycerate mutase has been determined. This structure shows important differences from the lower resolution structure deposited in 1982. The crystal used to determine the new structure was of a different form, having spacegroup P2(1). The model was refined to a crystallographic R-factor of 18.9% and a free R-factor of 28.4% using all data between 25 and 2.3 A and employing a bulk solvent correction. The enzyme is a tetramer of identical, 246 amino acid subunits, whose structure is revealed to be a dimer of dimers, with four independent active sites located well away from the subunit contacts. Each subunit contains two domains, the larger with a typical nucleotide binding fold, although phosphoglycerate mutase has no physiological requirement to bind nucleotides. The catalytic-site histidine residues are no longer in a "clapping-hands" conformation, but more resemble the conformation seen in the distantly related enzymes prostatic acid phosphatase and fructose-2,6-bisphosphatase. However, the catalytic histidine residues in the mutase are found to be much closer to each other than in the phosphatase structures, perhaps due to the absence of bound ligands in the mutase crystal. An intricate web of H-bonds is found around the catalytic histidine residues, high-lighting residues probably important for maintaining their correct orientation and charge. The positions of certain other residues, including some found near the catalytic site and some lining the catalytic-site cleft, have been changed by the correction of registration errors between sequence and electron density in the original structure. Electron density was apparent for a portion of the functionally important C-terminal tail, which was absent from the earlier structure, showing it to adopt a mainly helical conformation.     

Easily searched protein folding potentials

In order to calculate the tertiary structure of a protein from its amino acid sequence, the thermodynamic approach requires a potential function of sequence and conformation that has its global minimum at the native conformation for many different proteins. Here we study the behavior of such functions for the simplest model system that still has the essential features of the protein folding problem, namely two-dimensional square lattice chain configurations involving two residue types. First we demonstrate a method for accurately recovering the given contact potential from only a knowledge of which sequences fold to which structures and what the non-native structures are. Second, we show how to derive from the same information more general potential functions having much better positive correlations between potential function value and conformational deviation from the native. These functions consequently permit faster and more reliable searches for the native conformation, given the native sequence. Furthermore, the method for finding such potentials is easily applied to more realistic protein models.     

Immunogenicity and conformational properties of an N-linked glycosylated peptide epitope of human T-lymphotropic virus type 1 (HTLV-I)

The identification and characterization of epitopes of human T-lymphotropic virus type 1 (HTLV-I), which elicit an effective humoral or cell-mediated immune response, remains a central obstacle to the development of a peptide-based vaccine against the virus infection. The objective of the studies presented here was to examine the influence of N-linked glycosylation on peptide structure and immunogenicity. We engineered the 233-253 sequence of gp46 of HTLV-I to contain an N-acetylglucosamine (GlcNAc) residue at Asn244. Secondary structure prediction using computer algorithms indicated that this peptide may contain a beta-turn at residues 242-246. Recent work with model glycopeptides suggests that beta-turn conformation in peptides may be induced, and probably is stabilized, by the presence of even a single sugar residue. In the present study, the structures of the 233-253 peptide, SC1, and the 233-253(Asn244-GlcNAc) glycopeptide, SC2, were determined. Similar conformation was exhibited by both the glycosylated and nonglycosylated peptide displaying a beta-turn at residues 243-246 and extended-chain structure at the peptide/glycopeptide termini. Both peptides were engineered into chimeric constructs with a promiscuous T-cell epitope from measles virus and were used as immunogens in rabbits. Both chimeric peptides were highly immunogenic in rabbits, producing high-titered antibodies as early as primary + three weeks. The antibodies generated against either construct were able to bind to whole virus (ELISA) and to gp46 (radioimmunoprecipitation assay). Additionally, human sera of individuals known to be positive for HTLV-I recognized both the glycosylated and nonglycosylated constructs. It appears that the 233-253 peptide is able to adopt a conformation that mimics the structure in native gp46, and addition of a GlcNAc residue at Asn244 does not affect the conformational preference or stability of this construct; nor does glycosylation alter immunogenicity but instead appears to enhance immune recognition.     

Systematic fold recognition analysis of the sequences encoded by the genome of Mycoplasma pneumoniae

A robust tool for fold recognition was applied to the systematic analysis of the sequences below 200 residues encoded by the genome of Mycoplasma pneumoniae. The goal was to determine the additional information gain achievable in genome analysis by fold recognition, beyond the intrinsic limits of homology studies. A list of 124 sequences encoding for soluble proteins or domains not homologous to each other, or to proteins with known three-dimensional structure, was analyzed, resulting in significant Z scores for the energy of the structural models in 12 of these cases. This result indicates that systematic application of fold recognition techniques to the analysis of structurally unassigned soluble proteins can lead to high-confidence structural predictions with an efficiency of about 10%, a relevant contribution besides the complementary approach of homology analysis. Four of the predictions presented include mapping of the putative active site of the target sequence and lead to the detection of probable catalytic and binding residues. The data are discussed with reference to the functional implications of the structural models and to the results reported for the homologous genome of Mycoplasma genitalium.     

Structures of native and complexed complement factor D: implications of the atypical His57 conformation and self-inhibitory loop in the regulation of specific serine protease activity

Factor D is a serine protease essential for the activation of the alternative pathway of complement. The structures of native factor D and a complex formed with isatoic anhydride inhibitor were determined at resolution of 2.3 and 1.5 A, respectively, in an isomorphous monoclinic crystal form containing one molecule per asymmetric unit. The native structure was compared with structures determined previously in a triclinic cell containing two molecules with different active site conformations. The current structure shows greater similarity with molecule B in the triclinic cell, suggesting that this may be the dominant factor D conformation in solution. The major conformational differences with molecule A in the triclinic cell are located in four regions, three of which are close to the active site and include some of the residues shown to be critical for factor D catalytic activity. The conformational flexibility associated with these regions is proposed to provide a structural basis for the previously proposed substrate-induced reversible conformational changes in factor D. The high-resolution structure of the factor D/isatoic anhydride complex reveals the binding mode of the mechanism-based inhibitor. The higher specificity towards factor D over trypsin and thrombin is based on hydrophobic interactions between the inhibitor benzyl ring and the aliphatic side-chain of Arg218 that is salt bridged with Asp189 at the bottom of the primary specificity (S1) pocket. Comparison of factor D structural variants with other serine protease structures revealed the presence of a unique "self-inhibitory loop". This loop (214-218) dictates the resting-state conformation of factor D by (1) preventing His57 from adopting active tautomer conformation, (2) preventing the P1 to P3 residues of the substrate from forming anti-parallel beta-sheets with the non-specific substrate binding loop, and (3) blocking the accessibility of Asp189 to the positive1y charged P1 residue of the substrate. The conformational switch from resting-state to active-state can only be induced by the single macromolecular substrate, C3b-bound factor B. This self-inhibitory mechanism is highly correlated with the unique functional properties of factor D, which include high specificity toward factor B, low esterolytic activity toward synthetic substrates, and absence of regulation by zymogen and serpin-like or other natural inhibitors in blood.     

Conformation of the sebacyl beta1Lys82-beta2Lys82 crosslink in T-state human hemoglobin

The crystal structure of human T state hemoglobin crosslinked with bis(3,5-dibromo-salicyl) sebacate has been determined at 1.9 A resolution. The final crystallographic R factor is 0.168 with root-mean-square deviations (RMSD) from ideal bond distance of 0.018 A. The 10-carbon sebacyl residue found in the beta cleft covalently links the two betaLys82 residues. The sebacyl residue assumes a zigzag conformation with cis amide bonds formed by the NZ atoms of betaLys82's and the sebacyl carbonyl oxygens. The atoms of the crosslink have an occupancy factor of 1.0 with an average temperature factor for all atoms of 34 A2. An RMSD of 0.27 for all CA's of the tetramer is observed when the crosslinked deoxyhemoglobin is compared with deoxyhemoglobin refined by using a similar protocol, 2HHD [Fronticelli et al. J. Biol. Chem. 269: 23965-23969, 1994]. Thus, no significant perturbations in the tertiary or quaternary structure are introduced by the presence of the sebacyl residue. However, the sebacyl residue does displace seven water molecules in the beta cleft and the conformations of the beta1Lys82 and beta2Lys82 are altered because of the crosslinking. The carbonyl oxygen that is part of the amide bond formed with the NZ of beta2Lys82 forms a hydrogen bond with side chain of beta2Asn139 that is in turn hydrogen-bonded to the side chain of beta2Arg104. A comparison of the observed conformation with that modeled [Bucci et al. Biochemistry 35:3418-3425, 1996] shows significant differences. The differences in the structures can be rationalized in terms of compensating changes in the estimated free-energy balance, based on differences in exposed surface areas and the observed shift in the side-chain hydrogen-bonding pattern involving beta2Arg104, beta2Asn139, and the associated sebacyl carbonyl group.     

Rapidly evolving hepatitis C virus-related cirrhosis in a human immunodeficiency virus-infected patient receiving triple antiretroviral therapy

The Tyr92-Pro93 peptide group of bovine pancreatic ribonuclease A (RNase A) exists in the cis conformation in the native state. From unfolding/refolding kinetic studies of the disulfide-intact wild-type protein and of a variant in which Pro93 had been replaced by Ala, it had been suggested that the Tyr92-Ala93 peptide group also exists in the cis conformation in the native state. Here, we report the crystal structure of the P93A variant. Although there is disorder in the region of residues 92 and 93, the best structural model contains a cis peptide at this position, lending support to the results of the kinetics experiments. We also report the crystal structure of the C[40, 95]A variant, which is an analog of the major rate-determining three-disulfide intermediate in the oxidative folding of RNase A, missing the 40-95 disulfide bond. As had been detected by NMR spectroscopy, the crystal structure of this analog shows disorder in the region surrounding the missing disulfide. However, the global chain fold of the remainder of the protein, including the disulfide bond between Cys65 and Cys72, appears to be unaffected by the mutation.     

Mechanism of heparin activation of antithrombin. Role of individual residues of the pentasaccharide activating sequence in the recognition of native and activated states of antithrombin

To determine the role of individual saccharide residues of a specific heparin pentasaccharide, denoted DEFGH, in the allosteric activation of the serpin, antithrombin, we studied the effect of deleting pentasaccharide residues on this activation. Binding, spectroscopic, and kinetic analyses demonstrated that deletion of reducing-end residues G and H or nonreducing-end residue D produced variable losses in pentasaccharide binding energy of approximately 15-75% but did not affect the oligosaccharide's ability to conformationally activate the serpin or to enhance the rate at which the serpin inhibited factor Xa. Rapid kinetic studies revealed that elimination of the reducing-end disaccharide marginally affected binding to the native low-heparin-affinity conformational state of antithrombin but greatly affected the conversion of the serpin to the activated high-heparin- affinity state, although the activated conformation was still favored. In contrast, removal of the nonreducing- end residue D drastically affected the initial low-heparin-affinity interaction so as to favor an alternative activation pathway wherein the oligosaccharide shifted a preexisiting equilibrium between native and activated serpin conformations in favor of the activated state. These results demonstrate that the nonreducing-end residues of the pentasaccharide function both to recognize the native low-heparin-affinity conformation of antithrombin and to induce and stabilize the activated high-heparin-affinity conformation. Residues at the reducing-end, however, poorly recognize the native conformation and instead function primarily to bind and stabilize the activated antithrombin conformation. Together, these findings establish an important role of the heparin pentasaccharide sequence in preferential binding and stabilization of the activated conformational state of the serpin.     

Effect of thymine tract length on the structure and stability of model telomeric sequences

DNA from the telomeres at the ends of eukaryotic chromosomes contains a stretch of simple tandemly repeated sequences in which clusters of G residues alternate with clusters of T/A sequences along one DNA strand. Model telomeric G-clusters form four-stranded structures in Na+ or K+, stabilized by Hoogsteen pairing between G bases. DNA containing a single copy of the G-cluster can self-associate to form tetramers, with a parallel-stranded, right-handed helical structure. Two copies of the 3'-terminal G strand form a folded-back hairpin that dimerizes to create an antiparallel quadruplex structure. We show here that the tetrameric structure is strongly influenced by the T residue flanking either side of the G-cluster. The parallel tetraplex formed by single copies of the sequences dTnG4 is most stable for n = 1 and least stable for n = 8, the longest tract we have studied. At least two thymine residues are required to allow formation of antiparallel folded-back hairpin dimers from two-copy oligomers of sequence d(TnG4)2 in Na+; additional T's destabilize this structure. In K+, the predominant structure formed is the four-stranded parallel tetramer in all cases. Kinetic analysis indicates that the quadruplex structure formed by Oxytricha telomeric DNA overhangs in the presence of Na+ arises by dimerization of two Hoogsteen base-paired hairpins, with a relatively low energy barrier.     

Monte Carlo simulations of protein folding. II. Application to protein A, ROP, and crambin

The hierarchy of lattice Monte Carlo models described in the accompanying paper (Kolinski, A., Skolnick, J. Monte Carlo simulations of protein folding. I. Lattice model and interaction scheme. Proteins 18:338-352, 1994) is applied to the simulation of protein folding and the prediction of 3-dimensional structure. Using sequence information alone, three proteins have been successfully folded: the B domain of staphylococcal protein A, a 120 residue, monomeric version of ROP dimer, and crambin. Starting from a random expanded conformation, the model proteins fold along relatively well-defined folding pathways. These involve a collection of early intermediates, which are followed by the final (and rate-determining) transition from compact intermediates closely resembling the molten globule state to the native-like state. The predicted structures are rather unique, with native-like packing of the side chains. The accuracy of the predicted native conformations is better than those obtained in previous folding simulations. The best (but by no means atypical) folds of protein A have a coordinate rms of 2.25 A from the native C alpha trace, and the best coordinate rms from crambin is 3.18 A. For ROP monomer, the lowest coordinate rms from equivalent C alpha s of ROP dimer is 3.65 A. Thus, for two simple helical proteins and a small alpha/beta protein, the ability to predict protein structure from sequence has been demonstrated.     

Fold assembly of small proteins using monte carlo simulations driven by restraints derived from multiple sequence alignments

The feasibility of predicting the global fold of small proteins by incorporating predicted secondary and tertiary restraints into ab initio folding simulations has been demonstrated on a test set comprised of 20 non-homologous proteins, of which one was a blind prediction of target 42 in the recent CASP2 contest. These proteins contain from 37 to 100 residues and represent all secondary structural classes and a representative variety of global topologies. Secondary structure restraints are provided by the PHD secondary structure prediction algorithm that incorporates multiple sequence information. Predicted tertiary restraints are derived from multiple sequence alignments via a two-step process. First, seed side-chain contacts are identified from correlated mutation analysis, and then a threading-based algorithm is used to expand the number of these seed contacts. A lattice-based reduced protein model and a folding algorithm designed to incorporate these predicted restraints is described. Depending upon fold complexity, it is possible to assemble native-like topologies whose coordinate root-mean-square deviation from native is between 3.0 A and 6.5 A. The requisite level of accuracy in side-chain contact map prediction can be roughly 25% on average, provided that about 60% of the contact predictions are correct within +/-1 residue and 95% of the predictions are correct within +/-4 residues. Precision in tertiary contact prediction is more critical than absolute accuracy. Furthermore, only a subset of the tertiary contacts, on the order of 25% of the total, is sufficient for successful topology assembly. Overall, this study suggests that the use of restraints derived from multiple sequence alignments combined with a fold assembly algorithm holds considerable promise for the prediction of the global topology of small proteins.     

1H nuclear magnetic resonance study on equilibrium between two four-stranded solution conformations of short d(CnT)

NMR analysis of d(C4T) showed the slow exchange between two distinct tetramers (each fully symmetric) in solution. For one tetramer, NOE cross-peak patterns characteristic of an i-motif structure (H1'-H1' and H6-H1'/H1'-H6) were observed between C1 and T5, indicating that this tetramer takes a completely intercalated conformation where the T5 residue is stacked on the C1.C1(+) pair of the other duplex (S-form). The other was found to be a tetramer in which one of the duplexes is shifted by one nucleotide unit (R-form), resulting in nonstacking 3' end thymidine residues and an equal number of stacked C.C+ pairs to that of the S-form. The same spectral features were observed for d(C3T) but neither for d(TC3) nor d(TC4), indicative of the critical role of the position of the thymidine residue in the tetrad isomerization. From NMR denaturation profiles, the S-forms were found to be more stable than the R-forms, and the linear relationship between the logarithm of the equilibrium constant (K = [tetramer]/[single]4) and the inverse of temperature (1/T) was confirmed for both forms, indicating conformity to the two-state transition model. Both enthalpy and entropy values of the formation of the S-form from four single strands were more negative than those of the R-form. The enthalpy term should contribute to the stabilization of the S-forms at low temperatures. The difference of the free energy values [DeltaG degrees(S-form) - DeltaG degrees(R-form)] was found to be -2.1 and -2.7 kJ.mol-1 at 20 degreesC for d(C4T) and d(C3T), respectively, explaining the higher stability of the S-forms. With increasing temperature, these two topologies were found to comparably exist at equilibrium in solution with slow exchange via dissociation to the single strands. A biological role of this topological isomerization is also suggested.     

Three-dimensional structure of an Fab-peptide complex: structural basis of HIV-1 protease inhibition by a monoclonal antibody

F11.2.32, a monoclonal antibody raised against HIV-1 protease (Kd = 5 nM), which inhibits proteolytic activity of the enzyme (K(inh) = 35(+/-3)nM), has been studied by crystallographic methods. The three-dimensional structure of the complex between the Fab fragment and a synthetic peptide, spanning residues 36 to 46 of the protease, has been determined at 2.2 A resolution, and that of the Fab in the free state has been determined at 2.6 A resolution. The refined model of the complex reveals ten well-ordered residues of the peptide (P36 to P45) bound in a hydrophobic cavity at the centre of the antigen-binding site. The peptide adopts a beta hairpin-like structure in which residues P38 to P42 form a type II beta-turn conformation. An intermolecular antiparallel beta-sheet is formed between the peptide and the CDR3-H loop of the antibody; additional polar interactions occur between main-chain atoms of the peptide and hydroxyl groups from tyrosine residues protruding from CDR1-L and CDR3-H. Three water molecules, located at the antigen-antibody interface, mediate polar interactions between the peptide and the most buried hypervariable loops, CDR3-L and CDR1-H. A comparison between the free and complexed Fab fragments shows that significant conformational changes occur in the long hypervariable regions, CDR1-L and CDR3-H, upon binding the peptide. The conformation of the bound peptide, which shows no overall structural similarity to the corresponding segment in HIV-1 protease, suggests that F11.2.32 might inhibit proteolysis by distorting the native structure of the enzyme.