DOMAIN FLEXIBILITY IN RETROVIRAL PROTEASES - STRUCTURAL IMPLICATIONS FOR DRUG-RESISTANT MUTATIONS

Rigid body rotation of five domains and movements within their interfa cial joints provide a rational context for understanding why HIV prote ase mutations that arise in drug resistant strains are often spatially removed from the drug or substrate binding sites, Domain motions asso ciated with substrate binding in the retroviral HIV-1 and SIV protease s are identified and characterized. These motions are in addition to c losure of the flaps and result from rotations of similar to 6-7 degree s at primarily hydrophobic interfaces, A crystal structure of unligand ed SIV protease (incorporating the point mutation Ser 4 His to stabili ze the protease against autolysis) was determined to 2.0 Angstrom reso lution in a new space group, P3(2)21. The structure is in the most ''o pen'' conformation of any retroviral protease so far examined, with si x residues of the flaps disordered. Comparison of this and unliganded HIV structures, with their respective liganded structures by differenc e distance matrixes identifies five domains of the protease dimer that move as rigid bodies against one another: one terminal domain encompa ssing the N- and C-terminal beta sheet of the dimer, two core domains containing the catalytic aspartic acids, and two flap domains. The two core domains rotate toward each other on substrate binding, reshaping the binding pocket. We therefore show that, for enzymes,:mutations at interdomain interfaces that favor the unliganded form of the target a ctive site will increase the off-rate of the inhibitor, allowing the s ubstrate greater access for catalysis. This offers a mechanism of resi stance to competitive inhibitors, especially when the forward enzymati c reaction rate exceeds the rate of substrate dissociation.