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

被引:81
作者
Katoh, E
Louis, JM
Yamazaki, T
Gronenborn, AM
Torchia, DA
Ishima, R [1 ]
机构
[1] Natl Inst Dent & Craniofacial Res, Struct Mol Biol Unit, NIH, Bethesda, MD 20892 USA
[2] Natl Inst Agrobiol Sci, Dept Biochem, Tsukuba, Ibaraki 3058602, Japan
[3] NIDDKD, Chem Phys Lab, NIH, Bethesda, MD 20892 USA
关键词
aspartic protease; enzyme; protein; AIDS; relaxation; slow exchange;
D O I
10.1110/ps.0300703
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 N-15-edited NOESY experiment show that the chromogenic substrate analog of the capsid/p2 cleavage site binds to PRD25N with an equilibrium dissociation constant, K-D, of 0.27 +/- 0.05 mM, and upper limits of the association and dissociation rate constants, 2 x 10(4) M-1 s(-1) and 10 s(-1), respectively, at 20degreesC, 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 N-15 relaxation experiments reveals a slight reduction of S-2 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-musec. 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.
引用
收藏
页码:1376 / 1385
页数:10
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