HIV Protease Hinge Region Insertions at Codon 38 Affect Enzyme Kinetics, Conformational Stability and Dynamics

Abstract

HIV-1 protease is essential for the production of mature, infectious virions and is a major target in antiretroviral therapy. We successfully purified a HIV-1 subtype C variant, L38↑N↑L− 4, containing an insertion of asparagine and leucine at position 38 without the four background mutations - K20R, E35D, R57K, V82I using a modified purification protocol. Isothermal titration calorimetry indicated that 50% of the variant protease sample was in the active conformation compared to 62% of the wild type protease. The secondary structure composition of the variant protease was unaffected by the double insertion. The specific activity and kcat values of the variant protease were approximately 50% lower than the wild type protease values. The variant protease also exhibited a 1.6-fold increase in kcat/KM when compared to the wild type protease. Differential scanning calorimetry showed a 5 °C increase in Tm of the variant protease, indicating the variant was more stable than the wild type. Molecular dynamics simulations indicated the variant was more stable and compact than the wild type protease. A 3–4% increase in the flexibility of the hinge regions of the variant protease was observed. In addition, increased flexibility of the flaps, cantilever and fulcrum regions of the variant protease B chain was observed. The variant protease sampled only the closed flap conformation indicating a potential mechanism for drug resistance. The present study highlights the direct impact of a double amino acid insertion in hinge region on enzyme kinetics, conformational stability and dynamics of an HIV-1 subtype C variant protease.

Natural Polymorphisms D60E and I62V Stabilize a Closed Conformation in HIV-1 Protease in the Absence of an Inhibitor or Substrate

“…Although HIV-1 PR NPs only moderately impact enzyme activity and viral fitness, their presence has been shown to give rise to enhanced drug resistance [ 49 , 50 , 51 , 52 ]. Thus, by obtaining a better understanding of these alternate conformations as well as “hot spots” that can help induce a closed-like state, this work adds to a growing field of novel ways to target HIV-1 PR and drug resistance [ 38 , 48 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 ]. These concepts are also being explored in drug development against other proteases in emerging diseases [ 66 , 67 , 68 ].…”