Structural, kinetic, and thermodynamic studies of specificity designed HIV‐1 protease

Abstract

HIV-1 protease recognizes and cleaves more than 12 different substrates leading to viral maturation. While these substrates share no conserved motif, they are specifically selected for and cleaved by protease during viral life cycle. Drug resistant mutations evolve within the protease that compromise inhibitor binding but allow the continued recognition of all these substrates. While the substrate envelope defines a general shape for substrate recognition, successfully predicting the determinants of substrate binding specificity would provide additional insights into the mechanism of altered molecular recognition in resistant proteases. We designed a variant of HIV protease with altered specificity using positive computational design methods and validated the design using X-ray crystallography and enzyme biochemistry. The engineered variant, Pr3 (A28S/D30F/G48R), was designed to preferentially bind to one out of three of HIV protease's natural substrates; RT-RH over p2-NC and CA-p2. In kinetic assays, RT-RH binding specificity for Pr3 increased threefold compared to the wild-type (WT), which was further confirmed by isothermal titration calorimetry. Crystal structures of WT protease and the designed variant in complex with RT-RH, CA-p2, and p2-NC were determined. Structural analysis of the designed complexes revealed that one of the engineered substitutions (G48R) potentially stabilized heterogeneous flap conformations, thereby facilitating alternate modes of substrate binding. Our results demonstrate that while substrate specificity could be engineered in HIV protease, the structural pliability of protease restricted the propagation of interactions as predicted. These results offer new insights into the plasticity and structural determinants of substrate binding specificity of the HIV-1 protease.

Fullerene Derivatives Strongly Inhibit HIV-1 Replication by Affecting Virus Maturation without Impairing Protease Activity

“…Protease-mediated processing of Gag and Gag-Pol occurs in a strictly organized manner, and interfering with this highly ordered process results in immature virions (41,42); hence, local changes in the protein conformation could globally impact its processing. Importantly, the physical parameters that regulate the interaction of protease with its substrates are unclear (45,46); perhaps gaining an understanding of fullerene activity will shed light on this phenomenon.…”

A Direct Interaction with RNA Dramatically Enhances the Catalytic Activity of the HIV-1 Protease In Vitro

“…The purified protein was refolded by rapid dilution into a 10-fold volume of 0.05 M sodium acetate buffer at pH 5.5, containing 10% glycerol, 5% ethylene glycol, and 5 mM dithiothreitol (refolding buffer). The tethered dimer gene construct coded for two copies of the HIV-1 monomer linked by the nucleotide sequence that codes for Gly-Gly-Ser-Ser-Gly with unique nucleotide sequences for each monomer [82, 83]. The HIV-2 PR [64] was a generous gift from Dr. John M. Louis (NIH).…”

In Silico Prediction of Mutant HIV-1 Proteases Cleaving a Target Sequence

“…However, A28 was located by Alvizo et al . for the Pr3 mutant [18]. We envision Strategy2 also as a tool to locate those residues most involved in binding a given substrate peptide.…”