Issue 8, 2016

Pulsed EPR characterization of HIV-1 protease conformational sampling and inhibitor-induced population shifts

Abstract

The conformational landscape of HIV-1 protease (PR) can be experimentally characterized by pulsed-EPR double electron–electron resonance (DEER). For this characterization, nitroxide spin labels are attached to an engineered cysteine residue in the flap region of HIV-1 PR. DEER distance measurements from spin-labels contained within each flap of the homodimer provide a detailed description of the conformational sampling of apo-enzyme as well as induced conformational shifts as a function of inhibitor binding. The distance distribution profiles are further interpreted in terms of a conformational ensemble scheme that consists of four unique states termed “curled/tucked”, “closed”, “semi-open” and “wide-open” conformations. Reported here are the DEER results for a drug-resistant variant clinical isolate sequence, V6, in the presence of FDA approved protease inhibitors (PIs) as well as a non-hydrolyzable substrate mimic, CaP2. Results are interpreted in the context of the current understanding of the relationship between conformational sampling, drug resistance, and kinetic efficiency of HIV-1PR as derived from previous DEER and kinetic data for a series of HIV-1PR constructs that contain drug-pressure selected mutations or natural polymorphisms. Specifically, these collective results support the notion that inhibitor-induced closure of the flaps correlates with inhibitor efficiency and drug resistance. This body of work also suggests DEER as a tool for studying conformational sampling in flexible enzymes as it relates to function.

Graphical abstract: Pulsed EPR characterization of HIV-1 protease conformational sampling and inhibitor-induced population shifts

Supplementary files

Article information

Article type
Paper
Submitted
01 Aug. 2015
Accepted
09 Okt. 2015
First published
09 Okt. 2015

Phys. Chem. Chem. Phys., 2016,18, 5819-5831

Author version available

Pulsed EPR characterization of HIV-1 protease conformational sampling and inhibitor-induced population shifts

Z. Liu, T. M. Casey, M. E. Blackburn, X. Huang, L. Pham, I. M. S. de Vera, J. D. Carter, J. L. Kear-Scott, A. M. Veloro, L. Galiano and G. E. Fanucci, Phys. Chem. Chem. Phys., 2016, 18, 5819 DOI: 10.1039/C5CP04556H

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