Kinetic characterization of the critical step in HIV-1 protease maturation

Abstract

HIV maturation requires multiple cleavage of long polyprotein chains into functional proteins that include the viral protease itself. Initial cleavage by the protease dimer occurs from within these precursors, and yet only a single protease monomer is embedded in each polyprotein chain. Self-activation has been proposed to start from a partially dimerized protease formed from monomers of different chains binding its own N termini by self-association to the active site, but a complete structural understanding of this critical step in HIV maturation is missing. Here, we captured the critical self-association of immature HIV-1 protease to its extended aminoterminal recognition motif using large-scale molecular dynamics simulations, thus confirming the postulated intramolecular mechanism in atomic detail. We show that self-association to a catalytically viable state requires structural cooperativity of the flexible β-hairpin "flap" regions of the enzyme and that the major transition pathway is first via self-association in the semiopen/open enzyme states, followed by enzyme conformational transition into a catalytically viable closed state. Furthermore, partial N-terminal threading can play a role in self-association, whereas wide opening of the flaps in concert with self-association is not observed. We estimate the association rate constant (k on ) to be on the order of ∼1 × 10 4 s −1 , suggesting that N-terminal self-association is not the rate-limiting step in the process. The shown mechanism also provides an interesting example of molecular conformational transitions along the association pathway.conformational kinetics | Markov state model | high-throughput molecular dynamics H IV, along with all retroviruses, achieves infectious maturation of nascent virus particles through cleavage of polyprotein precursors by the viral protease. In particular, the GagPol chains contain several covalently linked proteins including the protease itself (Fig. 1A). Thus, maturation of the virus is initiated by autocatalysis of viral protease initially embedded in GagPol precursors. HIV-1 protease is functional only in dimeric form (1, 2) because activity of monomeric protease precursor is three orders of magnitude less than the mature dimer (3), and yet only a single monomer is embedded within each precursor. Two individual monomers in different GagPol chains must, therefore, come together to form an embedded dimeric protease, which ultimately cleaves itself into a mature form (Fig. 1B).Experiments indicate initial cleavage by a precursor protease still embedded in the GagPol chain occurs through an intramolecular, concentration-independent mechanism with the precursor protease cleaving its own terminus (4) and critically modulated by the N-terminal region (5-8). Mutations that block N-terminal cleavage result in severe loss of efficiency in catalytic activity. Cleavage at the protease (PR), reverse transcriptase (RT) junction at the C-terminal end of the protease by the precursor protease occurs via an intermolecular, concentrat...

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