Probing the S1/S1 ' substrate binding pocket geometry of HIV-1 protease with modified aspartic acid analogues

Aspartates 25 and 125, the active site residues of HIV-1 protease, particip ate functionally in proteolysis by what is believed to be a general acid-ge neral base mechanism. However, the structural role that these residues may play in the formation and maintenance of the neighboring S1/S1' substrate b inding pockets remains largely unstudied. Because the active site aspartic acids are essential for catalysis, alteration of these residues to any othe r naturally occurring amino acid by conventional site-directed mutagenesis renders the protease inactive, and hence impossible to characterize functio nally. To investigate whether Asp-25 and Asp-125 may also play a structural role that influences substrate processing, a series of active site proteas e mutants has been produced in a cell-free protein synthesizing system via readthrough of mRNA nonsense (UAG) codons by chemically misacylated suppres sor tRNAs. The suppressor tRNAs were activated with the unnatural aspartic acid analogues erythro-beta-methylaspartic acid, threo-beta-methylaspartic acid, or beta,beta-dimethylaspartic acid. On the basis of the specific acti vity measurements of the mutants that were produced, the introduction of th e beta-methyl moiety was found to alter protease function to varying extent s depending upon its orientation. While a beta-methyl group in the erythro orientation was the least deleterious to the specific activity of the prote ase, a beta-methyl group in the threo orientation, present in the modified proteins containing threo-beta-methylaspartate and beta,beta-dimethylaspart ate, resulted in specific activities between 0 and 45% of that of the wild type depending upon the substrate and the substituted active site position. Titration studies of pH versus specific activity and inactivation studies, using an aspartyl protease specific suicide inhibitor, demonstrated that t he mutant proteases maintained bell-shaped pH profiles, as well as suicide- inhibitor susceptibilities that are characteristic of aspartyl proteases. A molecular dynamics simulation of the a-substituted aspartates in position 25 of HIV-1 protease indicated that the threo-beta-methyl moiety may partia lly obstruct the adjacent S1' binding pocket, and also cause reorganization within the pocket, especially with regard to residues Val-82 and Ile-84. T his finding, in conjunction with the biochemical studies, suggests that the active site aspartate residues are in proximity to the S1/S1' binding pock et and may be spatially influenced by the residues presented in these pocke ts upon substrate binding. It thus seems possible that the catalytic residu es cooperatively interact with the residues that constitute the S1/S1' bind ing pockets and can be repositioned during substrate binding to orient the active site carboxylates with respect to the scissile amide bond, a process that likely affects the facility of proteolysis.