HIV-1 Protease Asp25Asn and Asp25Ser Mutants in Complexes with a HIV-1 gag Substrate. Abelardo M. Silva, Sergei Gulnik, Pavel Majer, Angela Lee, Walid Qoronfleh, Wei Shao, John W. Erickson. Structural Biochemistry Program / SAIC, National Cancer Institute, Frederick, Maryland 21702 .

The structures of two active-site mutants of the HIV-1 protease have been determined in complexes with a decapeptide that mimics the P17-P24 HIV-1 gag poly-protein processing site. The decapeptide, Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-Asn, is cleaved at the Try-Pro C-N bond by the wild- type enzyme. The mutations introduced in the protease replaced the catalytic Asp 25 for Asn in one case, and for Ser in the other, producing inactive enzymes in our assay. Diffraction data extended to 2.0å for the Asp25Asn mutant complex, and to 2.5Å for the Asp25Ser one. The refined models reveal that the overall conformation of the mutated molecules are identical to that of the active form. The decapeptide is bound in an extended conformation with a bend around the flap. The hydrogen-bonding pattern between main-chain decapeptide and protein atoms has a 2-fold symmetry character despite the intrinsic asymmetric nature of the peptide in itself. The enzyme breaks its internal 2-fold symmetry, most noticeably at the flap and some loops displaying relative movements of up to 2.0A, to maintain symmetric interaction with its substrate. The Asp25Asn complex provides a reliable model of the interaction of the scissile bond with the catalytic dyad. The plane of the peptide bond is perpendicular to the molecular 2-fold axis, and its carbonyl oxygen would be at hydrogen bonding distance from one of the aspartic acid side-chain oxygens. The carbonyl carbon is located at a ideal position from hydration by a "catalytic" water. The active-site/ substrate model also suggests that the flap-coordinate water together with the second catalytic aspartic acid, could be directly involved in the catalytic reaction.