Molecular Modeling Studies Suggest That Zinc Ions Inhibit HIV-1 Protease by Binding at Catalytic Aspartates

Abstract

Human immunodeficiency virus type 1 protease (HIV-1 PR) is a 99-amino acid, virally encoded protein responsible for proteolytic cleavage of viral gag and gag-pol fusion polyproteins into functional products (1-3). The activity of the protease is required for viral infectivity in vitro (4). Consequently, HIV-1 PR is an attractive therapeutic target for rational drug design (5,6), and the focus of a tremendous amount of research.HIV-1 PR has been characterized as an aspartic acid protease based on sequence homology to related enzymes (7, catalytic pH studies (8), and inhibition by wellknown aspartyl protease inhibitors (9,10).The active form of the enzyme behaves as a dimer (11,12). These findings have been confirmed by the elucidation of X-ray crystallographic structures of the enzyme both unbound (13-15) and bound to synthetic inhibitors (16)(17)(18)(19)(20).One feature that distinguishes the HIV-1 protease from cellular aspartyl proteases is the pH dependence of its activity. The optimum pH range for activity of the HIV-1 protease (4.5-6.0) (8) is considerably higher than for cellular enzymes in vitro (typically 2.0-4.0) (21). A notable exception is human renin, which has a pH optimum in the range of 5.5-7.5 (22). The pH optima of these enzymes presumably reflect the catalytic mechanism generally accepted for aspartyl proteases which requires one of the catalytic aspartate residues to be protonated. Recently, Zhang et al. (23) reported that zinc ions are inhibitors of both renin and HIV-1 PR at neutral pH. Although the zinc binding site(s) that lead to inactivation have not been determined, evidence suggests that one site may occur at or near the catalytic aspartate residues. In this study, molecular dynamics methodology was used to explore the possibility of zinc binding in the active site of HIV-1 PR and predict the effects of this binding on the structure. Our results are consistent with the requirements of zinc binding reported by Zhang et al. (23) and give supporting evidence that this is the site that leads to inhibition of the protease. MethodsWe performed molecular mechanics and dynamics calculations using a modified version ofAMBER3.0 (Revision A) (24,25). The all-atom force field (26) was used for all standard residues, and solvent was treated explicitly using the TIP3P model (27). Parameters for chloride ions and zinc ions were taken from Lybrand et al. (28) and Bartolotti et al. (29), respectively. Electrostatic and van der Waals interactions were treated using a "twin range" (9/18 A) residue-based cutoff, updated every 20 steps. We performed simulations under constant temperature (300 K) and pressure (1 bar) conditions using a 1-fsec integration time step, carried out to 200 psec.We obtained the starting positions of the heavy atoms for the unbound and Zn2-bound dimers from the crystallographic structure of the synthetic (Aba6795) HIV-1 protease at pH 7.0 (14).The net charge of the unbound dimer was assumed to be +4, consistent with the normal protonation states of the component amino acids at ...

Metal Complexes as Enzyme Inhibitors

“…The earlier study, in fact, demonstrates that the inhibition by zinc is strongly pH dependent, estimating that zinc competes with a proton for a group with p K a = 7.0. Molecular dynamic simulations performed on protease in the presence of zinc indicate that zinc ions bind to the catalytic active site Asp25 and Asp25‘ residues without disrupting the structure of the enzyme …”