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Experiments were carried out to determine whether complexes between MHC class I molecules and synthetic peptides are representative of those formed under more physiologically relevant conditions, with peptides derived intracellularly from processed antigens. Lysis of cells sensitized with exogenously provided and endogenously generated peptide analogues of the optimal nonameric peptide 58–66 (GILGFVFTL; derived from the influenza virus matrix protein) was compared. Endogenous loading was accomplished by expressing minigene DNA coding for alanine-substituted analogues of peptide 58–66 in HLA-A2-positive cells. Susceptibility to lysis by HLA-A2-restricted, peptide-specific cytotoxic lymphocytes was compared with lysis of cells sensitized with the same synthetic peptides. Although results were quite comparable, differences were observed. The endogenously presented analogues 58–66L60A, G61A, T65A, and L66A were recognized more efficiently than the corresponding exogenously presented analogues. This difference in recognition was most striking for peptide 58–66G61A. These results indicate the need for caution in using synthetic peptides in defining peptide binding motifs. Additional experiments with endogenously expressed analogues of 58–66 with substitutions other than alanine were carried out to define the interaction between this peptide and HLA-A2. Results are compatible with the interpretation that residues 58, 59, and 60 interact with pockets A, B, and D, respectively, in the HLA-A2 binding groove and that these interactions contribute to peptide binding.

Motivation: Many bioinformatic approaches exist for finding novel genes within genomic sequence data. Traditionally, homology search-based methods are often the first approach employed in determining whether a novel gene exists that is similar to a known gene. Unfortunately, distantly related genes or motifs often are difficult to find using single query-based homology search algorithms against large sequence datasets such as the human genome. Therefore, the motivation behind this work was to develop an approach to enhance the sensitivity of traditional single query-based homology algorithms against genomic data without losing search selectivity. Results: We demonstrate that by searching against a genome fragmented into all possible reading frames, the sensitivity of homology-based searches is enhanced without degrading its selectivity. Using the ETS-domain, bromodomain and acetyl-CoA acetyltransferase gene as queries, we were able to demonstrate that direct protein–protein searches using BLAST2P or FASTA3 against a human genome segmented among all possible reading frames and translated was substantially more sensitive than traditional protein–DNA searches against a raw genomic sequence using an application such as TBLAST2N. Receiver operating characteristic analysis was employed to demonstrate that the algorithms remained selective, while comparisons of the algorithms showed that the protein–protein searches were more sensitive in identifying hits. Therefore, through the overprediction of reading frames by this method and the increased sensitivity of protein–protein based homology search algorithms, a genome can be deeply mined, potentially finding hits overlooked by protein–DNA searches against raw genomic data.

Various methods for deriving atomic partial charges from the quantum chemical electrostatic potential and moments have been tested for the sucrose molecule. We show that if no further information is used, the charges on some carbon atoms become large and charge patterns involving these atoms are badly determined and poorly transferable. Adding lone-pairs on the ether oxygen atoms or dividing the molecule into smaller fragments did not cure the instabilities. We develop a method, CHELP-BOW0, that restrains charges toward zero with different weights for different atoms. These harmonic restraints preserve the linear form of the least-squares equations, which are solved in a single step using singular-value decomposition. CHELP-BOW0 improves the chemical transferability of the charges compared to unrestrained methods, and slightly improves their conformational transferability. It introduces a modest degradation of the fit compared to unrestrained CHELP-BOW (mean average deviation of the potential 0.00016 vs. 0.00010 a.u.). A second new method, CHELP-BOWC, avoids the need for restraints by including several conformations in the fit, weighting each according to its estimated energy in solution. CHELP-BOWC charges are more transferable than CHELP-BOW or CHELP-BOW0 charges to conformations not included in the training set. Restraints to zero charge do not further improve transferability of the CHELP-BOWC charges. We, therefore, recommend CHELP-BOW charges for rigid molecules and CHELP-BOWC charges for flexible molecules. (C) 2002 John Wiley Sons, Inc.

The three-dimensional structure of the catalytic domain of stromelysin-1 complexed with an N-carboxyl alkyl inhibitor has been determined by NMR methods. The global fold consists of three helices, a five stranded beta-sheet and a methionine located in a turn near the catalytic histidines, classifying stromelysin-1 as a metzincin. Stromelysin-1 is unique in having two independent zinc binding sites: a catalytic site and a structural site. The inhibitor binds in an extended conformation. The S1' subsite is a deep hydrophobic pocket, whereas S2' appears shallow and S3' open.

The clinical efficacy of converting enzyme inhibitors1 for reducing blood pressure in a large percentage of hypertensive patients has aroused considerable interest in developing agents that interrupt the renin-angiotensin system at other points, for example by blockade of the angiotensin II receptor2 or by inhibition of the aspartic proteinase, renin. Substrate based design of renin inhibitors in which the scissile P1-P1′ dipeptide is replaced with a non-hydrolyzable group, often a mimetic of a tetrahedral transition state for amide bond hydrolysis, has provided a useful approach for obtaining a variety of inhibitor structure types,3 many with Ki’s of better than 10−9 M. A clinically useful renin inhibitor, however, has not yet emerged due to poor pharmacokinetics (i.e., metabolism or rapid clearance) or poor oral absorption, problems often encountered with peptidic drug targets. An example of this is seen with 1, a “tetrapeptide” inhibitor4 that spans the P4 through P3′ sites of the renin substrate and which utilizes the statine analog, ACHPA5, as a P1-P1′ dipeptide replacement (Figure 1). Although 1 is quite potent in vitro, very low levels of drug are found in the blood after oral administration to the rhesus monkey at 50 mg/kg, and the half life after intravenous administration is short (< 1 h). Rapid biliary excretion of intact drug has been demonstrated for a number of other renin inhibitors.6

As an outgrowth of our small molecule modeling effort, we have begun to develop a large molecule modeling system. This new effort centers around a VAX 11/780 coupled to an Evans and Sutherland Color Multi-Picture system. The software currently available is A. Jones' FRODO program for display and manipulation, M. Connolly's SURFACE program, A. Hagler's VFF energy calculation program, and a variety of small programs written in-house. The structure and capabilities of this system will be described. An application of the system to studying the mechanism of peptide cleavage by the zinc endopeptidase thermolysin will be presented. This investigation has led us to suggest a number of modifications to the previously proposed mechanism and offers a rationalization for published substrate data.

A quantitative function equivalent to the "molecular" surface proposed by F. M. Richards [(1977) Annu. Rev. Biophys. Bioeng. 6, 151--176] is defined by the closest approach of solvent spheres to a macromolecule. The function can be used to visualize surface topography, polarity, and charge either as a three-dimensional net or by mapping onto a plane; to calculate surface areas; and to demarcate complementary sites in contacts between subunits. Applications to shape-specific recognition in protein structure and aggregation are discussed.

Merck Sharp & Dohme Research Laboratories has developed enzyme-inhibitor modeling procedures based on the convenient display and manipulation features of the FRODO crystallographic modeling program (Jones, 1978, 1982). After implementing the program on a VAX computer controlling an Evans & Sutherland Multi Picture System, we have added useful techniques such as multiple superpositions, color-coded labels and dot surfaces, and a novel real-time atomic contact display (“CLARIFI”) which emphasizes crucial repulsions while showing nonspecific binding.A synthetic chemist in Merck's New Leads Discovery Department has used these tools to develop a revised picture of the transition state and substrate specificity of the zinc protease thermolysin (Hangauer et al., 1983a). A movie illustrating the model was produced with the aid of GRAMPS (O'Donnell & Olson, 1981).Our enzyme-inhibitor modeling also makes use of links to the existing small-molecule Merck Molecular Modeling System, and to empirical energetics programs. These techniques are complementary and should be seen as tools with which a scientist can generate and test structural ideas.