Conformational flexibility in the flap domains of ligand-free HIV protease.

The crystal structures of wild-type HIV protease (HIV PR) in the absence of substrate or inhibitor in two related crystal forms at 1.4 and 2.15 A resolution are reported. In one crystal form HIV PR adopts an 'open' conformation with a 7.7 A separation between the tips of the flaps in the homodimer. In the other crystal form the tips of the flaps are 'curled' towards the 80s loop, forming contacts across the local twofold axis. The 2.3 A resolution crystal structure of a sixfold mutant of HIV PR in the absence of substrate or inhibitor is also reported. The mutant HIV PR, which evolved in response to treatment with the potent inhibitor TL-3, contains six point mutations relative to the wild-type enzyme (L24I, M46I, F53L, L63P, V77I, V82A). In this structure the flaps also adopt a 'curled' conformation, but are separated and not in contact. Comparison of the apo structures to those with TL-3 bound demonstrates the extent of conformational change induced by inhibitor binding, which includes reorganization of the packing between twofold-related flaps. Further comparison with six other apo HIV PR structures reveals that the 'open' and 'curled' conformations define two distinct families in HIV PR. These conformational states include hinge motion of residues at either end of the flaps, opening and closing the entire beta-loop, and translational motion of the flap normal to the dimer twofold axis and relative to the 80s loop. The alternate conformations also entail changes in the beta-turn at the tip of the flap. These observations provide insight into the plasticity of the flap domains, the nature of their motions and their critical role in binding substrates and inhibitors.