Recombination and positive selection contribute to evolution of Listeria monocytogenes inlA.

The surface molecule InlA interacts with E-cadherin to promote invasion of Listeria monocytogenes into selected host cells. DNA sequencing of inlA for 40 L. monocytogenes isolates revealed 107 synonymous and 45 nonsynonymous substitutions. A frameshift mutation in a homopolymeric tract encoding part of the InlA signal peptide was identified in three lineage II isolates, which also showed reduced ability to invade human intestinal epithelial cells. Phylogenies showed clear separation of inlA sequences into lineages I and II. Thirteen inlA recombination events, predominantly involving lineage II strains as recipients (12 events), were detected and a number of amino acid residues were shown to be under positive selection. Four of the 45 non-synonymous changes were found to be under positive selection with posterior probabilities >95 %. Mapping of polymorphic and positively selected amino acid sites on the partial crystal structure for InlA showed that the internalin surface of the leucine-rich repeat (LRR) region that faces the InlA receptor E-cadherin does not include any polymorphic sites; all polymorphic and positively selected amino acids mapped to the outer face of the LRR region or to other InlA regions. The data show that (i) inlA is highly polymorphic and evolution of inlA involved a considerable number of recombination events in lineage II isolates; (ii) positive selection at specific amino acid sites appears to contribute to evolution of inlA, including fixation of recombinant events; and (iii) single-nucleotide deletions in a lineage II-specific 3' homopolymeric tract in inlA lead to complete loss of InlA or to production of truncated InlA, which conveys reduced invasiveness. In conclusion, inlA has a complex evolutionary history, which is consistent with L. monocytogenes' natural history as an environmental pathogen with broad host-range, including its adaptation to environments and hosts where different inlA alleles may provide a selective advantage or where inlA may not be required.

Physics-based protein-structure prediction using a hierarchical protocol based on the UNRES force field: assessment in two blind tests.

Recent improvements in the protein-structure prediction method developed in our laboratory, based on the thermodynamic hypothesis, are described. The conformational space is searched extensively at the united-residue level by using our physics-based UNRES energy function and the conformational space annealing method of global optimization. The lowest-energy coarse-grained structures are then converted to an all-atom representation and energy-minimized with the ECEPP/3 force field. The procedure was assessed in two recent blind tests of protein-structure prediction. During the first blind test, we predicted large fragments of alpha and alpha+beta proteins [60-70 residues with C(alpha) rms deviation (rmsd) <6 A]. However, for alpha+beta proteins, significant topological errors occurred despite low rmsd values. In the second exercise, we predicted whole structures of five proteins (two alpha and three alpha+beta, with sizes of 53-235 residues) with remarkably good accuracy. In particular, for the genomic target TM0487 (a 102-residue alpha+beta protein from Thermotoga maritima), we predicted the complete, topologically correct structure with 7.3-A C(alpha) rmsd. So far this protein is the largest alpha+beta protein predicted based solely on the amino acid sequence and a physics-based potential-energy function and search procedure. For target T0198, a phosphate transport system regulator PhoU from T. maritima (a 235-residue mainly alpha-helical protein), we predicted the topology of the whole six-helix bundle correctly within 8 A rmsd, except the 32 C-terminal residues, most of which form a beta-hairpin. These and other examples described in this work demonstrate significant progress in physics-based protein-structure prediction.

Selection on Glycine beta-1,3-endoglucanase genes differentially inhibited by a Phytophthora glucanase inhibitor protein.

Plant endo-beta-1,3-glucanases (EGases) degrade the cell wall polysaccharides of attacking pathogens and release elicitors of additional plant defenses. Isozymes EGaseA and EGaseB of soybean differ in susceptibility to a glucanase inhibitor protein (GIP1) produced by Phytophthora sojae, a major soybean pathogen. EGaseA, the major elicitor-releasing isozyme, is a high-affinity ligand for GIP1, which completely inhibits it, whereas EGaseB is unaffected by GIP1. We tested for departures from neutral evolution on the basis of partial sequences of EGaseA and EGaseB from 20 widespread accessions of Glycine soja (the wild progenitor of soybean), from 4 other Glycine species, and across dicotyledonous plants. G. soja exhibited little intraspecific variation at either locus. Phylogeny-based codon evolution models detected strong evidence of positive selection on Glycine EGaseA and weaker evidence for selection on dicot EGases and Glycine EGaseB. Positively selected peptide sites were identified and located on a structural model of EGase bound to GIP1. Positively selected sites and highly variable sites were found disproportionately within 4.5 angstroms of bound GIP1. Low variation within G. soja EGases, coupled with positive selection in both Glycine and dicot lineages and the proximity of rapidly evolving sites to GIP1, suggests an arms race involving repeated adaptation to pathogen attack and inhibition.

The protein folding problem: global optimization of the force fields.

The evolutionary development of a theoretical approach to the protein folding problem, in our laboratory, is traced. The theoretical foundations and the development of a suitable empirical all-atom potential energy function and a global optimization search are examined. Whereas the all-atom approach has thus far succeeded for relatively small molecules and for alpha-helical proteins containing up to 46 residues, it has been necessary to develop a hierarchical approach to treat larger proteins. In the hierarchical approach to single- and multiple-chain proteins, global optimization is carried out for a simplified united residue (UNRES) description of a polypeptide chain to locate the region in which the global minimum lies. Conversion of the UNRES structures in this region to all-atom structures is followed by a local search in this region. The performance of this approach in successive CASP blind tests for predicting protein structure by an ab initio physics-based method is described. Finally, a recent attempt to compute a folding pathway is discussed.

Recent improvements in prediction of protein structure by global optimization of a potential energy function.

Recent improvements of a hierarchical ab initio or de novo approach for predicting both alpha and beta structures of proteins are described. The united-residue energy function used in this procedure includes multibody interactions from a cumulant expansion of the free energy of polypeptide chains, with their relative weights determined by Z-score optimization. The critical initial stage of the hierarchical procedure involves a search of conformational space by the conformational space annealing (CSA) method, followed by optimization of an all-atom model. The procedure was assessed in a recent blind test of protein structure prediction (CASP4). The resulting lowest-energy structures of the target proteins (ranging in size from 70 to 244 residues) agreed with the experimental structures in many respects. The entire experimental structure of a cyclic alpha-helical protein of 70 residues was predicted to within 4.3 A alpha-carbon (C(alpha)) rms deviation (rmsd) whereas, for other alpha-helical proteins, fragments of roughly 60 residues were predicted to within 6.0 A C(alpha) rmsd. Whereas beta structures can now be predicted with the new procedure, the success rate for alpha/beta- and beta-proteins is lower than that for alpha-proteins at present. For the beta portions of alpha/beta structures, the C(alpha) rmsd's are less than 6.0 A for contiguous fragments of 30-40 residues; for one target, three fragments (of length 10, 23, and 28 residues, respectively) formed a compact part of the tertiary structure with a C(alpha) rmsd less than 6.0 A. Overall, these results constitute an important step toward the ab initio prediction of protein structure solely from the amino acid sequence.

Influence of lysine content and pH on the stability of alanine-based copolypeptides.

To account for the relative contributions of lysine and alanine residues to the stability of alpha-helices of copolymers of these two residues, conformational energy calculations were carried out for several hexadecapeptides at several pHs. All the calculations considered explicitly the coupling between the conformation of the molecule and the ionization equilibria as a function of pH. The total free energy function used in these calculations included terms that account for the solvation free energy and free energy of ionization. These terms were evaluated by means of a fast multigrid boundary element method. Reasonable agreement with experimental values was obtained for the helix contents and vicinal coupling constants ((3)J(HNalpha)). The helix contents were found to depend strongly on the lysine content, in agreement with recent experimental results of Williams et al. (Journal of the American Chemical Society, 1998, Vol. 120, pp. 11033-11043) In the lowest energy conformation computed for a hexadecapeptide containing 3 lysine residues at pH 6, the lysine side chains are preferentially hydrated; this decreases the hydration of the backbone CO and NH groups, thereby forcing the latter to form hydrogen bonds with each other in the helical conformation. The lowest energy conformation computed for a hexadecapeptide containing 6 lysine residues at pH 6 shows a close proximity between the NH3(+) groups of the lysine side chains, a feature that was previously observed in calculations of short alanine-based oligopeptides. The calculation on a blocked 16-mer of alanine shows a 7% helix content based on the Boltzmann averaged vicinal coupling constants computed from the dihedral angles phi, consistent with previous experimental evidence on triblock copolymers containing a central block of alanines, and with earlier theoretical calculations.

Physical reasons for the unusual alpha-helix stabilization afforded by charged or neutral polar residues in alanine-rich peptides.

We have carried out conformational energy calculations on alanine-based copolymers with the sequence Ac-AAAAAXAAAA-NH(2) in water, where X stands for lysine or glutamine, to identify the underlying source of stability of alanine-based polypeptides containing charged or highly soluble polar residues in the absence of charge-charge interactions. The results indicate that ionizable or neutral polar residues introduced into the sequence to make them soluble sequester the water away from the CO and NH groups of the backbone, thereby enabling them to form internal hydrogen bonds. This solvation effect dictates the conformational preference and, hence, modifies the conformational propensity of alanine residues. Even though we carried out simulations for specific amino acid sequences, our results provide an understanding of some of the basic principles that govern the process of folding of these short sequences independently of the kind of residues introduced to make them soluble. In addition, we have investigated through simulations the effect of the bulk dielectric constant on the conformational preferences of these peptides. Extensive conformational Monte Carlo searches on terminally blocked 10-mer and 16-mer homopolymers of alanine in the absence of salt were carried out assuming values for the dielectric constant of the solvent epsilon of 80, 40, and 2. Our simulations show a clear tendency of these oligopeptides to augment the alpha-helix content as the bulk dielectric constant of the solvent is lowered. This behavior is due mainly to a loss of exposure of the CO and NH groups to the aqueous solvent. Experimental evidence indicates that the helical propensity of the amino acids in water shows a dramatic increase on addition of certain alcohols, such us trifluoroethanol. Our results provide a possible explanation of the mechanism by which alcohol/water mixtures affect the free energy of helical alanine oligopeptides relative to nonhelical ones.

Molecular simulation study of cooperativity in hydrophobic association.

To investigate the cooperativity of hydrophobic interactions, the potential of mean force of two- and three-molecule methane clusters in water was determined by molecular dynamics simulations using two methods: umbrella-sampling with the weighted histogram analysis method and thermodynamic integration. Two water models, TIP3P and TIP4P, were used, while each methane molecule was modeled as a united atom. It was found that the three-body potential of mean force is not additive, i.e., it cannot be calculated as a sum of two-body contributions, but requires an additional three-body cooperative term. The cooperative term, which amounts to only about 10% of the total hydrophobic association free energy, was found to increase the strength of hydrophobic association; this finding differs from the results of earlier Monte Carlo studies with the free energy perturbation method of Rank and Baker (1997). As in the work of Rank and Baker, the solvent contribution to the potential of mean force was found to be well approximated by the molecular surface of two methane molecules. Moreover, we also found that the cooperative term is well represented by the difference between the molecular surface of the three-methane cluster and those of all three pairs of methane molecules. In addition, it was found that, while there is a cooperative contribution to the hydrophobic association free energy albeit a small one, the errors associated with the use of pairwise potentials are comparable to or larger than this contribution.

Calculation of protein conformation by global optimization of a potential energy function.

A novel hierarchical approach to protein folding has been applied to compute the unknown structures of seven target proteins provided by CASP3. The approach is based exclusively on the global optimization of a potential energy function for a united-residue model by conformational space annealing, followed by energy refinement using an all-atom potential. Comparison of the submitted models for five globular proteins with the experimental structures shows that the conformations of large fragments (approximately 60 aa) were predicted with rmsds of 4.2-6.8 A for the C alpha atoms. Our lowest-energy models for targets T0056 and T0061 were particularly successful, producing the correct fold of approximately 52% and 80% of the structures, respectively. These results support the thermodynamic hypothesis that protein structure can be computed solely by global optimization of a potential energy function for a given amino acid sequence.

On the pH-conformational dependence of the unblocked SYPYD peptide.

Simulations were carried out for an unblocked pentapeptide with the sequence Ser-Tyr-Pro-Tyr-Asp (SYPYD) with explicit consideration of the coupling between the conformation of the molecule and the ionization equilibria at a given pH. The available NMR experimental data indicate a high preference for the cis isomeric turn-like form of Tyr-Pro at intermediate pH (approximately 6) and a destabilization of the cis form at both high (approximately 9) and low (approximately 3) pH. In order to identify the source of the stability of the conformation of this pentapeptide as a function of pH, Monte Carlo simulations were used to generate an ensemble of low-energy conformations at different pH values (viz. 3, 6 and 9). The total free energy function used in these calculations includes terms that account for the solvation free energy and free energy of ionization. These terms are evaluated by means of a fast multigrid boundary element (MBE) method. In good qualitative agreement with the experiments, our results indicate that the Boltzmann averaged population of the cis isomeric form of the pentapeptide has a maximum (45 %) at pH 6 and is significantly smaller (25 % and 23 %) for higher and lower pH values, respectively, following the trend of the experimental data. Also, the degree of charge for the lowest-energy conformations, as well as the contribution of electrostatic interactions to the stability of the preferred conformations, vary widely at the different pH values. Different kinds of packing of the aromatic side-chains of Tyr2 and Tyr4 against the proline ring are observed at different pH values, indicating that their contribution to the stability of the low-energy conformations is also pH-dependent. In summary, our results provide a basis for discussing the nature of the interactions that stabilize turn-like conformations of the peptide SYPYD as a function of pH.

Protein structure prediction by global optimization of a potential energy function.

An approach based exclusively on finding the global minimum of an appropriate potential energy function has been used to predict the unknown structures of five globular proteins with sizes ranging from 89 to 140 amino acid residues. Comparison of the computed lowest-energy structures of two of them (HDEA and MarA) with the crystal structures, released by the Protein Data Bank after the predictions were made, shows that large fragments (61 residues) of both proteins were predicted with rms deviations of 4.2 and 6.0 A for the Calpha atoms, for HDEA and MarA, respectively. This represents 80% and 53% of the observed structures of HDEA and MarA, respectively. Similar rms deviations were obtained for approximately 60-residue fragments of the other three proteins. These results constitute an important step toward the prediction of protein structure based solely on global optimization of a potential energy function for a given amino acid sequence.

Role of hydrophobicity and solvent-mediated charge-charge interactions in stabilizing alpha-helices.

A theoretical study to identify the conformational preferences of lysine-based oligopeptides has been carried out. The solvation free energy and free energy of ionization of the oligopeptides have been calculated by using a fast multigrid boundary element method that considers the coupling between the conformation of the molecule and the ionization equilibria explicitly, at a given pH value. It has been found experimentally that isolated alanine and lysine residues have somewhat small intrinsic helix-forming tendencies; however, results from these simulations indicate that conformations containing right-handed alpha-helical turns are energetically favorable at low values of pH for lysine-based oligopeptides. Also, unusual patterns of interactions among lysine side chains with large hydrophobic contacts and close proximity (5-6 A) between charged NH3+ groups are observed. Similar arrangements of charged groups have been seen for lysine and arginine residues in experimentally determined structures of proteins available from the Protein Data Bank. The lowest-free-energy conformation of the sequence Ac-(LYS)6-NMe from these simulations showed large pKalpha shifts for some of the NH3+ groups of the lysine residues. Such large effects are not observed in the lowest-energy conformations of oligopeptide sequences with two, three, or four lysine residues. Calculations on the sequence Ac-LYS-(ALA)4-LYS-NMe also reveal low-energy alpha-helical conformations with interactions of one of the LYS side chains with the helix backbone in an arrangement quite similar to the one described recently by (Proc. Natl. Acad. Sci. U.S.A. 93:4025-4029). The results of this study provide a sound basis with which to discuss the nature of the interactions, such as hydrophobicity, charge-charge interaction, and solvent polarization effects, that stabilize right-handed alpha-helical conformations.

New developments of the electrostatically driven Monte Carlo method: test on the membrane-bound portion of melittin.

The electrostatically driven Monte Carlo (EDMC) method has been greatly improved by adding a series of new features, including a procedure for cluster analysis of the accepted conformations. This information is used to guide the search for the global energy minimum. Alternative procedures for generating perturbed conformations to sample the conformational space were also included. These procedures enhance the efficiency of the method by generating a larger number of low-energy conformations. The improved EDMC method has been used to explore the conformational space of a 20-residue polypeptide chain whose sequence corresponds to the membrane-bound portion of melittin. The ECEPP/3 (Empirical Conformational Energy Program for Peptides) algorithm was used to describe the conformational energy of the chain. After an exhaustive search involving 14 independent runs, the lowest energy conformation (LEC) (-91.0 kcal/mol) of the entire study was encountered in four of the runs, while conformations higher in energy by no more than 1.8 kcal/mol were found in the remaining runs with the exception of one of them (run 8). The LEC is identical to the conformation found recently by J. Lee, H. A. Scheraga, and S. Rackovsky [(1988) "Conformational Analysis of the 20-Residue Membrane-Bound Portion of Melittin by Conformational Space Annealing," Biopolymers, Vol. 46, pp. 103-115] as the lowest energy conformation obtained in their study using the conformational space annealing method. These results suggest that this conformation corresponds to the global energy minimum of the ECEPP/3 potential function for this specific sequence: it also appears to be the conformation of lowest free energy.

Calculated electrostatic gradients in recombinant human H-chain ferritin.

Calculations to determine the electrostatic potential of the iron storage protein ferritin, using the human H-chain homopolymer (HuHF), reveal novel aspects of the protein. Some of the charge density correlates well with regions previously identified as active sites in the protein. The three-fold channels, the putative ferroxidase sites, and the nucleation sites all show expectedly negative values of the electrostatic potential. However, the outer entrance to the three-fold channels are surrounded by regions of positive potential, creating an electrostatic field directed toward the interior cavity. This electrostatic gradient provides a guidance mechanism for cations entering the protein cavity, indicating the three-fold channel as the major entrance to the protein. Pathways from the three-fold channels, indicated by electrostatic gradients on the inner surface, lead to the ferroxidase center, the nucleation center and to the interior entrance to the four-fold channel. Six glutamic acid residues at the nucleation site give rise to a region of very negative potential, surrounding a small positively charged center due to the presence of two conserved arginine residues, R63, in close proximity (4.9 A), suggesting that electrostatic fields could also play a role in the nucleation process. A large gradient in the electrostatic potential at the 4-fold channel gives rise to a field directed outward from the internal cavity, indicating the possibility that this channel functions to expel cations from inside the protein. The 4-fold channel could therefore provide an exit pathway for protons during mineralization, or iron leaving the protein cavity during de-mineralization.

Backbone makes a significant contribution to the electrostatics of alpha/beta-barrel proteins.

The electrostatic properties of seven alpha/beta-barrel enzymes selected from different evolutionary families were studied: triose phosphate isomerase, fructose-1,6-bisphosphate aldolase, pyruvate kinase, mandelate racemase, trimethylamine dehydrogenase, glycolate oxidase, and narbonin, a protein without any known enzymatic activity. The backbone of the alpha/beta-barrel has a distinct electrostatic field pattern, which is dipolar along the barrel axis. When the side chains are included in the calculations the general effect is to modulate the electrostatic pattern so that the electrostatic field is generally enhanced and is focused into a specific area near the active site. We use the electrostatic flux through a square surface near the active site to gauge the functionally relevant magnitude of the electrostatic field. The calculations reveal that in six out of the seven cases the backbone itself contributes greater than 45% of the total flux. The substantial electrostatic contribution of the backbone correlates with the known preference of alpha/beta-barrel enzymes for negatively charged substrates.

A molecular switch for biochemical logic gates: conformational studies.

This report presents the computer-assisted design of a molecular switching element, in which a molecular switch regulates the enzymatic activity of Ribonuclease A (RNase A). The molecular switch, an appropriately modified amino acid residue, is constructed with an electron donor group and an electron acceptor group, connected to one another with a conjugated double bond bridge. The switching mechanism is based on the azonium-hydrazo tautomerization, by which a charge separation induced in the excited state causes a rearrangement of the molecular electronic structure, resulting in the exchange of locations of single and double bonds. This rearrangement of bonds leads to different three-dimensional conformations of the switch. Using the electrostatically driven Monte Carlo (EDMC) method and the empirical conformational energy program for peptides (ECEPP/3) potential energy function, we carried out an exhaustive search of the conformational space of the switching element. The results of these calculations reveal two sets of conformations: in one set the access to the active site of the enzyme is preferentially blocked, while in the other set the active site is preferentially accessible. Integration of the designed element into biochemical logic gates operating under the rules of threshold value, and experimental implementation of this system, are considered.

Coupling between folding and ionization equilibria: effects of pH on the conformational preferences of polypeptides.

A new approach to the conformational study of polypeptides is presented. It considers explicitly the coupling between the conformation of the molecule and the ionization equilibria at a given pH value. Calculations of the solvation free energy and free energy of ionization of a 17-residue polypeptide are carried out using a fast multigrid boundary element method (MBE). The MBE method uses an adaptive tessellation of the molecular surface by boundary elements with non-regular size to solve the Poisson equation rapidly, and with a high degree of accuracy. The MBE method is integrated into the ECEPP (Empirical Conformational Energy Program for Peptides) algorithm to compute the coupling between the ionization state and the conformation of the molecule. This approach has been applied to study the conformational preference of a short polypeptide for which the available NMR and CD experimental data indicate that conformations containing a right-handed alpha-helical segment are energetically more favorable at low values of pH. The results of calculations using the present method agree quite well with experiments, in contrast to previous applications with standard techniques (using pre-assigned charges at each pH) that were not able to reproduce the experimental findings. Also, it is shown how the coupling to the conformation leads to different degrees of ionization of a given type of residue, for example glutamic acid, at different positions in the amino acid sequence, at any given pH. The results of this study provide a sound basis to discuss the origin of the stability of polypeptide conformations, and its dependence on the environmental conditions.

Stabilization of an isolated helical capping box in solution by hydrophobic interactions: evidence from the NMR study of bioactive peptides from the C-terminus of human C5a anaphylatoxin.

Synthetic analogues of the C-terminal portion of C5a were designed and found to be agonists of the C5a receptor [J. A. Ember et al. (1992) Jounral of Immunology, Vol. 148, p. 3165]. Nuclear magnetic resonance experiments were carried out to determine the solution conformation of the most potent analogue, the peptide C5a 65-74 (Tyr65, Phe67) (Tyr65-Ser66-Phe67-Lys68-Asp69-Met70 -Gln71- Leu72-Gly73-Arg74). Medium-range nuclear Overhauser effects (NOEs) were observed for residues 65-70 of this C5a peptide, suggesting that this region adopts a folded conformation in a significant population of the solution conformational ensemble. Quantitative analyses of (3)J(NH-alphaH) coupling constants and sequential NOE cross peaks gave an estimated helical population of 65% in the region Ser66-Met70. Additional evidence supporting the presence of a helical turn includes reduced amide-proton temperature coefficients and lowered (3)J(NH-alphaH) coupling constants in the region of Phe67-Met70. Conformational behavior of this C5a analogue peptide was studied using molecular modeling incorporating observed NOEs as constraints. The side chains of Tyr65, Phe67, and Met70 consistently form a hydrophobic cluster in all the model structures. The side chains of residues Ser66 and Asp69 can form reciprocal hydrogen bonds with the backbone NH groups of these two residues, indicating that residues Ser66-Phe67-Lys68-Asp69 (or SFKD) form a helix-stabilizing capping box (E. T. Harper and G. D. Rose (1993) Biochemistry, Vol. 32, p. 7605; H. X. Zhou et al. (1994) Proteins: Structure, Function and Genetics, Vol. 18, p. 1] even within the single turn of helical structure found in the analogue C5a peptide. A comparison of nmr results obtained for the analogue peptide and the natural decapeptide C5a 65-74 (Ile65-Ser66-His67-Lys68-Asp-69- Met70-Gln71-Leu72-Gly73-Arg74) indicated that incorporation of residues Tyr65 and Phe67 helps stabilize an isolated capping box involving residues Ser66-Asp69 in the C5a peptides through more extensive hydrophobic/aromatic interactions between residues Tyr65, Phe67, and Met70 in the analogue peptide C5a 65-74 (Tyr65, Phe67). These results constitute the first experimental demonstration of hydrophobic determinants in helical capping-box interactions, proposed recently by a statistical analysis of protein structures [J. W. Seale et al. (1994) Protein Science, Vol. 3, pp. 1741-1745]. The stabilized helical turn may also account for the greater potency of the analogue peptide C5a65-74 (Tyr65, Phe67) in receptor-binding assays.

Thrombin-bound conformation of a cyclic anticoagulant peptide using transferred nuclear Overhauser effect (NOE), distance geometry, and NOE simulations.

A cyclic hirudin C-terminal fragment, even truncated to eight residues, retains higher activity in binding to thrombin compared with its linear analogue of the same peptide length. In this paper, we report the results of a conformational study of a cyclic hirudin peptide 55-62 ([sequence: see text]) bound to the exosite of thrombin. Based on transferred NOE data and molecular modeling, conformation of the 18-membered ring was well defined with little mobility in the loop region. The backbone conformations of residues Glu (58) to Lys (61) are very similar to those in a native 11-residue linear peptide in the thrombin-bound state [Ni et al. (1990) Biochemistry, vol. 29, pp. 4479-4489; Skrzypczak-Jankun et al. (1991) Journal of Molecular Biology, vol. 221, pp. 1379-1393]. The side chain of Ile (59), the most critical residue, assumes a gauche- conformation, as observed in linear hirudin peptides. The bound conformations of residues Phe(56) and Glu (57) are defined by an iterative procedure involving the matching of computed and experimental transferred NOE intensities. The exosite of thrombin appears to be very specific to both the backbone and the side-chain conformations of the hirudin C-terminal peptides, especially those of residues Phe(56) and Ile(59).

Open "back door" in a molecular dynamics simulation of acetylcholinesterase.

The enzyme acetylcholinesterase generates a strong electrostatic field that can attract the cationic substrate acetylcholine to the active site. However, the long and narrow active site gorge seems inconsistent with the enzyme's high catalytic rate. A molecular dynamics simulation of acetylcholinesterase in water reveals the transient opening of a short channel, large enough to pass a water molecule, through a thin wall of the active site near tryptophan-84. This simulation suggests that substrate, products, or solvent could move through this "back door," in addition to the entrance revealed by the crystallographic structure. Electrostatic calculations show a strong field at the back door, oriented to attract the substrate and the reaction product choline and to repel the other reaction product, acetate. Analysis of the open back door conformation suggests a mutation that could seal the back door and thus test the hypothesis that thermal motion of this enzyme may open multiple routes of access to its active site.