The mobility of an HIV-1 integrase active site loop is correlated with catalytic activity

Replication of HIV-1 requires the covalent integration of the viral cDNA in to the host chromosomal DNA directed by the virus-encoded integrase protein . Here we explore the importance of a protein surface loop near the integra se active site using protein engineering and X-ray crystallography. We have redetermined the structure of the integrase catalytic domain (residues 50- 212) using an independent phase set at 1.7 Angstrom resolution. The structu re extends helix alpha 4 on its N-terminal side (residues 149-154), thus de fining the position of the three conserved active site residues. Evident in this and in previous structures is a conformationally flexible loop compos ed of residues 141-148. To probe the role of flexibility in this loop, we r eplaced Gly 140 and Gly 149, residues that appear to act as conformational hinges, with Ala residues. X-ray structures of the catalytic domain mutants G149A and G140A/G149A show further rigidity of alpha 4 and the adjoining l oop. Activity assays in vitro revealed that these mutants are impaired in c atalysis. The DNA binding affinity, however, is minimally affected by these mutants as assayed by UV cross-linking. We propose that the conformational flexibility of this active site loop: is important for a postbinding catal ytic step.