Pulsed EPR characterization of HIV-1 protease conformational sampling and inhibitor-induced population shifts.
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.
Department of Chemistry, University of Florida, PO BOX 117200, Gainesville, FL 32611-7200, USA. fanucci@chem.ufl.edu.
Zhanglong
Z
Liu
Department of Chemistry, University of Florida, PO BOX 117200, Gainesville, FL 32611-7200, USA. fanucci@chem.ufl.edu.
Thomas M
TM
Casey
Department of Chemistry, University of Florida, PO BOX 117200, Gainesville, FL 32611-7200, USA. fanucci@chem.ufl.edu.
Mandy E
ME
Blackburn
Department of Chemistry, University of Florida, PO BOX 117200, Gainesville, FL 32611-7200, USA. fanucci@chem.ufl.edu.
Xi
X
Huang
Department of Chemistry, University of Florida, PO BOX 117200, Gainesville, FL 32611-7200, USA. fanucci@chem.ufl.edu.
Linh
L
Pham
Department of Chemistry, University of Florida, PO BOX 117200, Gainesville, FL 32611-7200, USA. fanucci@chem.ufl.edu.
Ian Mitchelle S
IM
de Vera
Department of Chemistry, University of Florida, PO BOX 117200, Gainesville, FL 32611-7200, USA. fanucci@chem.ufl.edu.
Jeffrey D
JD
Carter
Department of Chemistry, University of Florida, PO BOX 117200, Gainesville, FL 32611-7200, USA. fanucci@chem.ufl.edu.
Jamie L
JL
Kear-Scott
Department of Chemistry, University of Florida, PO BOX 117200, Gainesville, FL 32611-7200, USA. fanucci@chem.ufl.edu.
Angelo M
AM
Veloro
Department of Chemistry, University of Florida, PO BOX 117200, Gainesville, FL 32611-7200, USA. fanucci@chem.ufl.edu.
Luis
L
Galiano
Department of Chemistry, University of Florida, PO BOX 117200, Gainesville, FL 32611-7200, USA. fanucci@chem.ufl.edu.
Gail E
GE
Fanucci
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004351:Drug Resistance; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016333:HIV Protease / Q000737:chemistry* / Q000235:genetics; D015497:HIV-1 / Q000737:chemistry* / Q000187:drug effects; D006801:Humans; D008958:Models, Molecular; D011487:Protein Conformation
D004791:Enzyme Inhibitors; D016333:HIV Protease; C514803:p16 protease, Human immunodeficiency virus 1