文摘
Double electron鈥揺lectron 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. <sup>1sup>H鈥?sup>15sup>N heteronuclear single-quantum coherence (HSQC) spectra were acquired for each protease construct titrated with the same set of inhibitors. NMR <sup>1sup>H鈥?sup>15sup>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鈥搇igand interactions characterized by DEER spectroscopy of HIV-1 PR obtained at the cryogenic temperature are consistent with more physiological solution protein鈥搇igand interactions observed by solution NMR spectroscopy.