Multiple Routes and Milestones in the Folding of HIV-1 Protease Monomer

Abstract

Evolution has lead proteins to display funneled energy landscapes with small degrees of ruggedness. However, a funneled landscape does not preclude the presence of multiple kinetically relevant folding routes. Here we show that for an extremely relevant biological case, the monomer of HIV type 1 protease (HIV-1-PR), multiple pathways and milestones can coexist along the folding process.We provide a comprehensive picture of the folding mechanism of HIV-1-PR monomer using a variety of theoretical and computational techniques. These include all-atom molecular dynamics simulations in explicit solvent, an analysis of the network of structure clusters found in multiple high-temperature unfolding simulations and a complete characterization of the free energy surface carried out using an all-atom structure based potential and a combination of metadynamics and parallel tempering.Our results confirm that the monomer in solution is stable and show unambiguously that at least two (un)folding pathways exist. Moreover, we demonstrate how the formation of a hydrophobic core can be considered a milestone in the folding process which must occur along all the routes that lead towards the protein's native state. These results also provide a theoretical framework that is able to rationalize both the experimental evidences and the evolutionary data for HIV-1-PR monomer. Finally, our characterization of the ensemble of possible folding routes substantiates a rational drug design strategy based on inhibiting the folding of each of the subunits that build the HIV-1 protease homo-dimer.