THE STRUCTURAL STABILITY OF THE HIV-1 PROTEASE

The most common strategy in the development of HIV-1 protease inhibito rs has been the design of high affinity transition state analogs that effectively compete with natural substrates for the active site. A sec ond approach has been the development of compounds that inactivate the protease by destabilizing its quaternary or tertiary structure. A suc cessful optimization of these strategies requires an accurate knowledg e of the energetics of structural stabilization and binding, and the i dentification of those regions in the protease molecule that are criti cal to stability and function. Here the energetics of stabilization of the HIV-1 protease has been measured for the first time by high sensi tivity differential scanning calorimetry. These studies have permitted the evaluation of the different components of the Gibbs energy of sta bilization (the enthalpy, entropy and heat capacity changes). The stab ility of the protease is pH-dependent and due to its dimeric nature is also concentration-dependent. At pH 3.4 the Gibbs energy of stabiliza tion is close to 10 kcal/mol at 25 degrees C, consistent with a dissoc iation constant of 5 x 10(-8) M. The stability of the protease increas es at higher pH values. At pH 5, the Gibbs energy of stabilization is 14.5 kcal/mol at 25 degrees C, consistent with a dissociation constant of 2.3 x 10(-11) M. The pH dependence of the Gibbs energy of stabiliz ation indicates that between pH 3.4 and pH 5 an average of 3-4 ionizab le groups per dimer became protonated upon unfolding. A structure-base d thermodynamic analysis of the protease molecule indicates that most of the Gibbs energy of stabilization is provided by the dimerization i nterface and that the isolated subunits are intrinsically unstable. Th e Gibbs energy, however, is not uniformly distributed along the dimeri zation interface. The dimer interface is characterized by the presence of clusters of residues (hot spots) that contribute significantly and other regions that contribute very little to subunit association. At the dimerization interface, residues located at the carboxy and amino termini contribute dose to 75% of the total Gibbs energy (Cys95, Thr96 , Leu97, Asn98 and Phe99 and Pro1, Ile3, Leu5). Residues Thr26, Gly27 and Asp29 located at the base of the active site are also important, a nd to a lesser extent Gly49, Ile50, Gly51 located at the tip of the fl ap region. The structure-based thermodynamic analysis also predicts th e existence of regions of the protease with only marginal stability an d a high propensity to undergo independent local unfolding. In particu lar, the flap region occupies a very shallow energy minimum and its co nformation can easily be affected by relatively small perturbations, T his property of the protease can be related to. the ability of some mu tations to elicit resistance towards certain inhibitors. (C) 1998 Acad emic Press.