A solution NMR study of the binding kinetics and the internal dynamics of an HIV-1 protease-substrate complex.
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 (15)N-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, 2 x 10(4) M(-1)s(-1) and 10 s(-1), respectively, at 20 degrees 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 (15)N 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-microsec. 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.
Katoh
Etsuko
E
Biochemistry Department, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan.
Louis
John M
JM
Yamazaki
Toshimasa
T
Gronenborn
Angela M
AM
Torchia
Dennis A
DA
Ishima
Rieko
R
D016333:HIV Protease / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D012996:Solutions; D013379:Substrate Specificity
D012996:Solutions; D016333:HIV Protease