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

Abstract

The HIV-1 Protease (PR) mediates its own release and promotes the proteolysis of the Gag-Pol polyprotein into mature structural and functional forms indispensable for viral maturation. Due to its central role in the virus life cycle, the HIV-1 PR has been a prominent target for clinical protease inhibitors for almost 25 years. However the short replication cycle of the virus together with the error-prone reverse transcriptase lead to the rapidly evolving selection of inhibitor-resistant mutations in HIV-1 PR. We use here high-pressure NMR spectroscopy to characterize the cooperativity of unfolding, in the absence and in the presence of a symmetric inhibitor, of the active mature PR, the inactive variant PRD25N and of the clinical multi-drug resistant PR20. We found that binding of the inhibitor drastically decreases the cooperativity of unfolding by trapping the closed flap conformation in a deep free-energy minimum. We show that in order to avoid this conformational trap, PR20 has evolved to exhibit a nearly ideal two-state unfolding transition. Our study highlights the malleability of the HIV-1 PR folding pathways and illustrates how selection for drug resistant mutations can lead to a major remodeling of the free-energy landscape.