The Folding Free-Energy Surface of HIV-1 Protease: Insights into the Thermodynamic Basis for Resistance to Inhibitors

Abstract

Spontaneous mutations at numerous sites distant from the active site of HIV-1 protease enable resistance to inhibitors while retaining enzymatic activity. As a benchmark for probing the effects of these mutations on the conformational adaptability of this dimeric β-barrel protein, the folding free energy surface of a pseudo wild-type variant, HIV-PR*, was determined by a combination of equilibrium and kinetic experiments on the urea-induced unfolding/refolding reactions. The equilibrium unfolding reaction was well-described by a two-state model involving only the native dimeric form and the unfolded monomer. The global analysis of the kinetic folding mechanism reveals the presence of a fully-folded monomeric intermediate that associates to form the native dimeric structure. Independent analysis of a stable monomeric version of the protease demonstrated that a small amplitude fluorescence phase in refolding and unfolding, not included in the global analysis of the dimeric protein, reflects the presence of a transient intermediate in the monomer folding reaction. The partially-folded and fully-folded monomers are only marginally stable with respect to the unfolded state, and the dimerization reaction provides a modest driving force at micromolar concentrations of protein. The thermodynamic properties of this system are such that mutations can readily shift the equilibrium from the dimeric native state towards weakly-folded states that have a lower affinity for inhibitors, but that could be induced to bind to their target proteolytic sites. Presumably, subsequent secondary mutations increase the stability of the native dimeric state in these variants and, thereby, optimize the catalytic properties of the resistant HIV-1 protease. Keywords thermodynamics; kinetics; dimer folding mechanisms; jelly-roll β-barrel motif; global analysis Human Immunodeficiency Virus (HIV), the viral infection that causes AIDS, has come to the forefront as a global public health crisis since the initial identification of this disease twentyfive years ago. 1,2 HIV-1 protease, the protein responsible for viral maturation through multiple cleavages of the Gag and Gag-Pol polyproteins, has been a therapeutic target in the treatment of AIDS for a number of years. 3 Structure-based drug design guided the development of the first generation of HIV-1 protease inhibitors. 4 Though these inhibitors initially showed great promise, the high frequency of mutations in the viral genome resulted in multiple HIV-1 protease variants that maintain activity yet are drug-resistant. 5 The presence of both active site and non-active site mutations in these variants 6 suggests that the retention of activity for the non-active site variants may arise from an altered free energy landscape that provides access to alternative HIV-1 protease conformations. 7 The proposal that differential perturbations in NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript the conformational ensemble of protease may be responsible for resistance is cons...

Protein intrinsic disorder as a flexible armor and a weapon of HIV-1

“…Each 99-residue monomer contributes a catalytically essential aspartic acid (Asp25) to the enzyme active site, which contains a conserved triad sequence, Asp-Thr-Gly, and resembles that of other aspartyl proteases [73]. The folding of this enzyme is a three-state process in which two monomers first fold independently and then dock in the dimer native state [216][217][218].…”

Pressure induced structural changes and dimer destabilization of HIV-1 protease studied by molecular dynamics simulations

“…Unfolding of HIV-1 PR monomer facilitated by denaturing agents was studied by Noel et al 10 who showed that certain mutations in the protease sequence increase its tendency to unfold. Moleculardynamics simulations were carried out in order to explain the mechanism of monomer folding and characterize its intermediates 11,12 .…”

The State of the Science: A 5 - Year Review on the Computer - Aided Design for Global Anti - AIDS Drug Development

“…HIV-1 PT is a homodimer with 99 residues per subunit (Fig. 9), and each subunit is composed of nine -strands and one -helix (Noel et al, 2009). -Strands 2 to 8 are involved in the formation of a jelly-roll -barrel topology within each subunit.…”

“…-Strands 2 to 8 are involved in the formation of a jelly-roll -barrel topology within each subunit. The dimeric interface contains an antiparallel -sheet formed by the interdigitation of the N-and Cterminal -strands in each subunit and by an interlocking and balanced pair of threonines, Thr26, in the active site (Noel et al, 2009). The active site-needing proteolysis is embed in the flap tips and crossing the subunit interface.…”