Bound Water Molecules at the Interface between the HIV-1 Protease and a Potent Inhibitor, KNI-272, Determined by NMR

KNI-272 is a peptidomimetic transition state analog inhibitor, having very high specificity and binding affinity for the HIV-1 protease. In order to understand the interactions that enhance drug binding to the protease, we recorded 2D water/NOESY and water/ROESY spectra to identify water molecules that bind tightly to the protease/KNI-272 complex. Well-ordered water molecules are observed at the protease/inhibitor interface in the crystal structure of the complex that have short interproton distances to the Ile50/150, Ala28/128, and Asp29/129 amide protons. The cross peaks between these protein protons and water protons, observed in water/NOESY and water/ROESY spectra, provide strong evidence that these water molecules are present in the solution structure of the complex. Analysis of measured NOE and ROE cross relaxation rates indicates that, in solution, these water molecules have long residence times, at least 1 ns and possibly greater than 7 ns. The presence of long-lived hydration water molecules at the protein/inhibitor interface suggests that interactions involving these water molecules contribute to the potency of the inhibitor. Hence, consideration of the potential role of hydration water molecules in stabilizing protein/inhibitor structures could contribute to improved drug design and to a better understanding of the mechanisms of drug resistance.