Probing the S1/S1‘ Substrate Binding Pocket Geometry of HIV-1 Protease with Modified Aspartic Acid Analogues

Abstract

Aspartates 25 and 125, the active site residues of HIV-1 protease, participate functionally in proteolysis by what is believed to be a general acid-general base mechanism. However, the structural role that these residues may play in the formation and maintenance of the neighboring S1/S1' substrate binding pockets remains largely unstudied. Because the active site aspartic acids are essential for catalysis, alteration of these residues to any other naturally occurring amino acid by conventional site-directed mutagenesis renders the protease inactive, and hence impossible to characterize functionally. To investigate whether Asp-25 and Asp-125 may also play a structural role that influences substrate processing, a series of active site protease mutants has been produced in a cell-free protein synthesizing system via readthrough of mRNA nonsense (UAG) codons by chemically misacylated suppressor 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 activity measurements of the mutants that were produced, the introduction of the beta-methyl moiety was found to alter protease function to varying extents depending upon its orientation. While a beta-methyl group in the erythro orientation was the least deleterious to the specific activity of the protease, a beta-methyl group in the threo orientation, present in the modified proteins containing threo-beta-methylaspartate and beta,beta-dimethylaspartate, 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 the 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 beta-substituted aspartates in position 25 of HIV-1 protease indicated that the threo-beta-methyl moiety may partially obstruct the adjacent S1' binding pocket, and also cause reorganization within the pocket, especially with regard to residues Val-82 and Ile-84. This finding, in conjunction with the biochemical studies, suggests that the active site aspartate residues are in proximity to the S1/S1' binding pocket and may be spatially influenced by the residues presented in these pockets upon substrate binding. It thus seems possible that the catalytic residues cooperatively interact with the residues that constitute the S1/S1' binding 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.

Transition states of native and drug-resistant HIV-1 protease are the same

“…The cycle initiates with water activation by hydrogen-bonding interactions with the active site aspartate residues (1) (6, 7). Bell-shaped pH-rate profiles (8)(9)(10)(11)(12) have revealed an acidic pH optimum, and structural studies (13,14) suggest that the reactant-bound enzyme contains a single protonated Asp (with a di-Asp, net charge of −1) facilitating a general acid-base mechanism, which is common among many Asp proteases (7,15). Nucleophilic attack by the water generates a reversible gem-diol intermediate, (3) which has been observed structurally (16)(17)(18) and identified experimentally (19,20).…”

In VitroSelection Combined with Ribosomal Translation Containing Non-proteinogenic Amino Acids

“…The tolerance of ribosomal translation toward α‐ N ‐methyl amino acids has been evaluated extensively . For example, Suga′s group and Green′s group independently conducted comprehensive evaluations of 19 α‐ N ‐methyl amino acids with proteinogenic side chains and found that a majority of them were compatible with translation .…”

Comparative study of some energetic and steric parameters of the wild type and mutants HIV‐1 protease: a way to explain the viral resistance

“…It was proved by mutagenesis [7] that the aspartic acids (25,125) are principally involved in maintenance of the active state of the enzyme, more than the residues Thr (26, 126) -Gly (27,127) [8,9]. Kinetic and computational studies [10][11][12] show that the viral protease presents a high mutagenesis rate, thus it is able to develop strong resistance to inhibitors [10][11][12][13].…”