FLEXIBILITY AND FUNCTION IN HIV PROTEASE - DYNAMICS OF THE HIV-1 PROTEASE BOUND TO THE ASYMMETRIC INHIBITOR KYNOSTATIN-272 (KNI-272)

The HIV-1 protease is a 22 kDa homodimeric protein essential-for funct ion of the AIDS virus, and protease inhibitors have been developed int o effective HN drugs. In order to better understand HIV-1 protease-inh ibitor interactions, we have investigated amide backbone dynamics by c orrelated H-1-N-15 NMR spectroscopy. To date, HIV-1 protease/inhibitor complexes Studied by NMR Spectroscopy have been limited to C-2 symmet ric structures, consisting of the protease bound to a symmetric inhibi tor. Herein we report studies of the dynamics of HIV-1 protease comple xed to KNI-272, a potent (K-i ca. 5 pM), asymmetric inhibitor which li fts the chemical shift degeneracy of the protease monomers and allows us to ascertain if the individual protease monomers have significantly different backbone motions. Using isotope filtered/edited spectra of N-15/C-13 protease complexed with unlabeled KNI-272, together with dis tances derived from the protease/KNI-272 X-ray structure, we obtained monomer specific NMR signal assignments. We derived information about monomer dynamics from a Lipari-Szabo analysis of amide N-15 T-1, T-2,a nd NOE values. Modeling the complex as an axially symmetric:rotor yiel ded an average overall correlation time of 9.65 ns and an anisotropy, D-parallel to/D-perpendicular to, of 1.27. Over 90% of the backbone am ide sites are highly ordered with the squared order parameter, average d over all measured residues, being 0.85. High amplitude internal moti ons are observed in several loops in the protease; especially those in the elbows of the flaps, while millisecond to microsecond time scale motion is observed at the flap-tips. While these results are similar t o those reported for complexes with symmetric inhibitors, we find diff erences in internal motions between several residues in the flap of on e monomer and the corresponding residues on the other monomer. Residue Gly 149 has a significantly larger-order parameter than Gly 49; in ad dition, the motions on the chemical exchange time scale contribute to the relaxation of Gly 152 and Phe 153 but not to the relaxation of Gly 52 and Phe 53. These differences inflexibility correlate with differe nces in interactions made-by these residues with KNI-272, as seed in t he crystal structure.; We also find that: the average of the order par ameters measured for residues in monomer 1 is less than for monomer 2, a result that correlates with the observation that average (B) over d ot factor for these residues is less in monomer 2 than in monomer 1.