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.