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Terminal Interface Conformations Modulate Dimer Stability Prior to Amino Terminal Autoprocessing of HIV-1 Protease

Agniswamy, Johnson, Sayer, Jane M., Weber, Irene T., Louis, John M.
Biochemistry 2012 v.51 no.5 pp. 1041-1050
Human immunodeficiency virus 1, RNA-directed DNA polymerase, catalytic activity, crystal structure, dissociation, hydrogen bonding, polyproteins, proteinases, thermal stability, viruses
The HIV-1 protease (PR) mediates its own release (autoprocessing) from the polyprotein precursor, Gag-Pol, flanked by the transframe region (TFR) and reverse transcriptase at its N- and C-termini, respectively. Autoprocessing at the N-terminus of PR mediates stable dimer formation essential for catalytic activity, leading to the formation of infectious virus. An antiparallel β-sheet interface formed by the four N- and C-terminal residues of each subunit is important for dimer stability. Here, we present the first high-resolution crystal structures of model protease precursor-clinical inhibitor (PI darunavir or saquinavir) complexes, revealing varying conformations of the N-terminal flanking (S–⁴FNF–¹) and interface residues (P¹QIT⁴). A 180° rotation of the T⁴–L⁵ peptide bond is accompanied by a new Q²–L⁵ hydrogen bond and complete disengagement of PQIT from the β-sheet dimer interface, which may be a feature for intramolecular autoprocessing. This result is consistent with drastically lower thermal stability by 14–20 °C of PI complexes of precursors and the mature PR lacking its PQIT residues (by 18.3 °C). Similar to the TFR-PR precursor, this deletion also results in a darunavir dissociation constant (2 × 10⁴)-fold higher and a markedly increased dimer dissociation constant relative to the mature PR. The terminal β-sheet perturbations of the dimeric structure likely account for the drastically poorer inhibition of autoprocessing of TFR-PR relative to the mature PR, even though significant differences in active site–PI interactions in these structures were not observed. The novel conformations of the dimer interface may be exploited to target selectively the protease precursor prior to its N-terminal cleavage.