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

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Abstract
NMR studies of the binding of a substrate to an inactive HIV‐1 protease construct, containing an active site mutation PRD25N, 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 PRD25N with an equilibrium dissociation constant, KD, of 0.27 ± 0.05 mM, and upper limits of the association and dissociation rate constants, 2×104 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.