Evolution under Drug Pressure Remodels the Folding Free-Energy Landscape of Mature HIV-1 Protease

Abstract

Using high-pressure NMR spectroscopy and differential scanning calorimetry, we investigate the folding landscape of the mature HIV-1 protease homodimer. The cooperativity of unfolding was measured in the absence or presence of an active site inhibitor DMP323, for the active protease (PR), its inactive variant PRD25N and an extremely multi-drug resistant mutant, PR20. The individual fit of the pressure denaturation profiles gives rise to first order, ΔGNMR, and second order, ΔVNMR (the derivative of ΔGNMR with pressure) apparent thermodynamic parameters for each amide proton considered. Heterogeneity in the apparent ΔVNMR values reflects departure from an ideal cooperative unfolding transition. The narrow to broad distribution of ΔVNMR spanning the extremes from inhibitor-free PR20D25N to PR-DMP323 complex, and distinctively for PRD25N-DMP323 complex, indicated large variations in folding cooperativity. Consistent with this data, the shape of thermal unfolding transitions varies from asymmetric for PR to nearly symmetric for PR20, as dimer-inhibitor ternary complexes. Lack of structural cooperativity was observed between regions located close to the active site, including the hinge and tip of the glycine-rich flaps, and the rest of the protein. These results strongly suggest that inhibitor binding drastically decreases the cooperativity of unfolding by trapping the closed flap conformation in a deep energy minimum. To evade this conformational trap, PR20 evolves exhibiting a smoother folding landscape with nearly an ideal two-state (cooperative) unfolding transition. This study highlights the malleability of retroviral protease folding pathways by illustrating how the selection of mutations under drug pressure remodels the free-energy landscape as a primary mechanism.

Structural Studies of a Rationally Selected Multi-Drug Resistant HIV-1 Protease Reveal Synergistic Effect of Distal Mutations on Flap Dynamics

“…It has been suggested that selection for the open-flap conformation alters the folding landscape of highly drug resistant mutants, exemplified by PR20, to avoid a free-energy trap of inhibitor-bound enzyme [54]. This all-in-one mechanism is beneficial for viral survival since it is independent of substrate binding at the active site and possibly confers cross resistance to multiple drugs by providing an escape pathway.…”

Evolutionary trend toward kinetic stability in the folding trajectory of RNases H

“…Thus, the energy landscape defines a protein's ability to fold in a biologically reasonable time scale, avoid misfolding, and prevent frequent unfolding-all of which are likely important for the overall fitness of an organism. Indeed, numerous studies have proposed how protein folding might affect molecular evolution (2)(3)(4)(5)(6)(7)(8), and there is growing evidence that folding pathways and their associated kinetics and energetics are evolutionarily constrained. To date, however, there is little experimental evidence detailing how folding properties have changed throughout evolution.…”

Practical aspects of high-pressure NMR spectroscopy and its applications in protein biophysics and structural biology

“…These regions coincide with the so-called flap and hinge domains of HIV-1 protease, involved in the binding of inhibitor (d, highlighted in red). Reprinted from Louis and Roche [88] with permission from Elsevier Publishing.…”