title: Inhibitor-induced conformational shifts and ligand-exchange dynamics for HIV-1 protease measured by pulsed EPR and NMR spectroscopy.
abstract: Double electron-electron resonance (DEER) spectroscopy was utilized to investigate shifts in conformational sampling induced by nine FDA-approved protease inhibitors (PIs) and a nonhydrolyzable substrate mimic for human immunodeficiency virus type 1 protease (HIV-1 PR) subtype B, subtype C, and CRF_01 A/E. The ligand-bound subtype C protease has broader DEER distance profiles, but trends for inhibitor-induced conformational shifts are comparable to those previously reported for subtype B. Ritonavir, one of the strong-binding inhibitors for subtypes B and C, induces less of the closed conformation in CRF_01 A/E. (1)H-(15)N heteronuclear single-quantum coherence (HSQC) spectra were acquired for each protease construct titrated with the same set of inhibitors. NMR (1)H-(15)N HSQC titration data show that inhibitor residence time in the protein binding pocket, inferred from resonance exchange broadening, shifting or splitting correlates with the degree of ligand-induced flap closure measured by DEER spectroscopy. These parallel results show that the ligand-induced conformational shifts resulting from protein-ligand interactions characterized by DEER spectroscopy of HIV-1 PR obtained at the cryogenic temperature are consistent with more physiological solution protein-ligand interactions observed by solution NMR spectroscopy.
authors.affiliation: Department of Chemistry, University of Florida, P.O. Box 117200, Gainesville, Florida 32611, USA.
authors.forename: Xi
authors.initials: X
authors.lastname: Huang
authors.forename: Ian Mitchelle S
authors.initials: IM
authors.lastname: de Vera
authors.forename: Angelo M
authors.initials: AM
authors.lastname: Veloro
authors.forename: Mandy E
authors.initials: ME
authors.lastname: Blackburn
authors.forename: Jamie L
authors.initials: JL
authors.lastname: Kear
authors.forename: Jeffery D
authors.initials: JD
authors.lastname: Carter
authors.forename: James R
authors.initials: JR
authors.lastname: Rocca
authors.forename: Carlos
authors.initials: C
authors.lastname: Simmerling
authors.forename: Ben M
authors.initials: BM
authors.lastname: Dunn
authors.forename: Gail E
authors.initials: GE
authors.lastname: Fanucci
mesh_terms: D004578:Electron Spin Resonance Spectroscopy; D016333:HIV Protease / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D017320:HIV Protease Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D008024:Ligands; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013816:Thermodynamics
chemical_list: D017320:HIV Protease Inhibitors; D008024:Ligands; D016333:HIV Protease