Structural determinants of metal-induced conformational changes in HIV-1 integrase

Human immunodeficiency virus type 1 (HIV-1) integrase (IN) undergoes a reve rsible metal-induced conformational change that activates the enzyme (Asant e-Appiah, E., and Skalka, A. M. (1997) J. Biol. Chem. 272, 16196-16205), In this report, key structural features that mediate this conformational chan ge have been identified by site-directed mutagenesis, limited proteolysis, and mass spectrometry studies. The results reveal two separable metal-induc ed effects, One depends on residues in the N-terminal domain (amino acids 1 -50) and a C-terminal tail (amino acids 274-288) and is detected by increas ed resistance of the full-length protein to proteolytic digestion. This eff ect appears to depend on metal binding at an undefined location distinct fr om the known sites in the N-terminal and catalytic core domains. The second conformational change depends on metal binding at the active site in the c atalytic core domain, Substitution of acidic residues Asp(64) Or Glu(152) i n the catalytic core D,D(35)E motif or truncation of the Src homology 3 (SH 3)-like domain in the C-terminal region of the enzyme abolishes this metal- induced change. Comparison of tryptic digests of an HIV-1 IN derivative com petent for metal-induced conformational change and a conformation-defective D64N derivative identified specific regions in HIV-1 IN that are affected by this second change. A region in the N terminus that spans Lys(14), an ex tended loop and the adjacent region in the core domain (including lysines 1 36, 156, and 160 and Arg(173)), and residues at the C terminus beyond the S H3-like domain all become less accessible to proteolysis in the conformatio n-competent protein. In contrast, a region that encompasses Lys(258) in the putative DNA binding groove of the SHE-like domain becomes more sensitive to proteolysis in the presence of Mn2+. The results are consistent with a m odel in which the binding of the metal ion by residues of the D,D(35)E moti f elicits specific changes in all three domains of HIV-1 IN, inducing the r estructuring of the enzyme for catalytic competence.