Flexibility and Function in HIV Protease:  Dynamics of the HIV-1 Protease Bound to the Asymmetric Inhibitor Kynostatin 272 (KNI-272)

• Published in: Journal of the American Chemical Society 1998-08, Vol.120 (31), p.7916-7923
• Main Authors: Freedberg, Darón I., Wang, Yun-Xing, Stahl, Stephen J., Kaufman, Joshua D., Wingfield, Paul T., Kiso, Yoshiaki, Torchia, Dennis A.
• Format: Article
• Language: English
• Subjects: Human immunodeficiency virus 1; kynostatin 272
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja981206r

The HIV-1 protease is a 22 kDa homodimeric protein essential for function of the AIDS virus, and protease inhibitors have been developed into effective HIV drugs. In order to better understand HIV-1 protease−inhibitor interactions, we have investigated amide backbone dynamics by correlated 1H−15N NMR spectroscopy. To date, HIV-1 protease/inhibitor complexes studied by NMR spectroscopy have been limited to C 2 symmetric structures, consisting of the protease bound to a symmetric inhibitor. Herein we report studies of the dynamics of HIV-1 protease complexed to KNI-272, a potent (K i ca. 5 pM), asymmetric inhibitor which lifts 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 15N/13C protease complexed with unlabeled KNI-272, together with distances 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 15N T 1, T 2, and NOE values. Modeling the complex as an axially symmetric rotor yielded an average overall correlation time of 9.65 ns and an anisotropy, D ||/D ⊥, of 1.27. Over 90% of the backbone amide sites are highly ordered with the squared order parameter, averaged over all measured residues, being 0.85. High amplitude internal motions 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 to those reported for complexes with symmetric inhibitors, we find differences in internal motions between several residues in the flap of one monomer and the corresponding residues on the other monomer. Residue Gly 149 has a significantly larger order parameter than Gly 49; in addition, 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 in flexibility correlate with differences in interactions made by these residues with KNI-272, as seen in the crystal structure. We also find that the average of the order parameters measured for residues in monomer 1 is less than for monomer 2, a result that correlates with the observation that average B factor for these residues is less in monomer 2 than in monomer 1.