A soluble active mutant of HIV-1 integrase: involvement of both the core and carboxyl-terminal domains in multimerization.
Structural studies of human immunodeficiency virus type 1 (HIV-1) integrase have been impeded by the low solubility of the protein. By systematic replacement of hydrophobic residues, we previously identified a single amino acid change (F185K) that dramatically improved the solubility of the catalytic domain of HIV-1 integrase and enabled the structure to be determined by x-ray crystallography. We have introduced the same mutation into full-length HIV-1 integrase. The resulting recombinant protein is soluble and fully active in vitro, whereas, HIV-1 carrying the mutation is replication-defective due to improper virus assembly. Analysis of the recombinant protein by gel filtration and sedimentation equilibrium demonstrate a dimer-tetramer self-association. We find that the regions involved in multimerization map to both the catalytic core and carboxyl-terminal domains. The dramatically improved solubility of this protein make it a good candidate for structural studies.
Jenkins
T M
TM
Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-0560, USA.
Engelman
A
A
Ghirlando
R
R
Craigie
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D004254:DNA Nucleotidyltransferases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D006801:Humans; D019426:Integrases; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D013379:Substrate Specificity; D014461:Ultracentrifugation; D016662:Virus Integration; D014779:Virus Replication
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D004254:DNA Nucleotidyltransferases; D019426:Integrases