Catalytic domain of human immunodeficiency virus type 1 integrase: identification of a soluble mutant by systematic replacement of hydrophobic residues.
The integrase protein of human immunodeficiency virus type 1 is necessary for the stable integration of the viral genome into host DNA. Integrase catalyzes the 3' processing of the linear viral DNA and the subsequent DNA strand transfer reaction that inserts the viral DNA ends into host DNA. Although full-length integrase is required for 3' processing and DNA strand transfer activities in vitro, the central core domain of integrase is sufficient to catalyze an apparent reversal of the DNA strand transfer reaction, termed disintegration. This catalytic core domain, as well as the full-length integrase, has been refractory to structural studies by x-ray crystallography or NMR because of its low solubility and propensity to aggregate. In an attempt to improve protein solubility, we used site-directed mutagenesis to replace hydrophobic residues within the core domain with either alanine or lysine. The single substitution of lysine for phenylalanine at position 185 resulted in a core domain that was highly soluble, monodisperse in solution, and retained catalytic activity. This amino acid change has enabled the catalytic domain of integrase to be crystallized and the structure has been solved to 2.5-A resolution [Dyda, F., Hickman, A. B., Jenkins, T. M., Engelman, A., Craigie, R. & Davies, D. R. (1994) Science 266, 1981-1986]. Systematic replacement of hydrophobic residues may be a useful strategy to improve the solubility of other proteins to facilitate structural and biochemical studies.
Jenkins
T M
TM
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD 20892-0560, USA.
Hickman
A B
AB
Dyda
F
F
Ghirlando
R
R
Davies
D R
DR
Craigie
R
R
D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D002850:Chromatography, Gel; D004254:DNA Nucleotidyltransferases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D006639:Histidine; D019426:Integrases; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016324:Sequence Tagged Sites; D012995:Solubility; D016662:Virus Integration
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D006639:Histidine; D004254:DNA Nucleotidyltransferases; D019426:Integrases