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A solution NMR study of the binding kinetics and the internal dynamics of an HIV-1 protease-substrate complex

¹ Biochemistry Department, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan
² Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA
³ Molecular Structural Biology Unit, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892-4307, USA

Reprint requests to: Rieko Ishima, Molecular Structural Biology Unit, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892-4307, USA; e-mail: rishima{at}dir.nidcr.nih.gov; fax: 301-502-5321.

NMR studies of the binding of a substrate to an inactive HIV-1 protease construct, containing an active site mutation PR_D25N, are reported. Substrate titration measurements monitored by HSQC spectra and a ¹⁵N-edited NOESY experiment show that the chromogenic substrate analog of the capsid/p2 cleavage site binds to PR_D25N with an equilibrium dissociation constant, K_D, of 0.27 ± 0.05 mM, and upper limits of the association and dissociation rate constants, 2x10⁴ M^-1s^-1 and 10 s^-1, respectively, at 20°C, pH 5.8. This association rate constant is not in the diffusion limit, suggesting that association is controlled by a rare event, such as opening of the protease flaps. Analysis of ¹⁵N relaxation experiments reveals a slight reduction of S² values in the flap region, indicating a small increase in the amplitude of internal motion on the sub-nsec timescale. In addition, several residues in the flap region are mobile on the conformational exchange timescale, msec–µsec. Flap dynamics of the protease-substrate complex are compared with those of protease-inhibitor complexes, and the implications of these results for substrate-binding models are discussed.

Keywords: Aspartic protease; enzyme; protein; AIDS; relaxation; slow exchange

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