Crystallizing Integrases: The Iterative Use of Molecular Biology and Screening to Improve Solubility and Order. Fred Dyda, Alison Burgess Hickman, Tim Jenkins, Shani Waninger*, John Scocca*, Robert Craigie, David R. Davies Laboratory of Molecular Biology, NIDDK, Bethesda MD. Department of Biochemistry, The John Hopkins University School of Hygiene and Public Health, Baltimore MD(*)

Proteins involved in genetic recombination present a challenge for crystallography: they tend to have low solubility and they also tend to display significant degrees of disorder.

HIV-1 integrase with the viral amino acid sequence had very low solubility, and even at low protein concentrations formed large aggregates. Domain definition by limited protease digests followed by multiple runs of single site point mutations proved to be extremely useful to obtain a version of the catalytic domain which was active and crystallizable in the same time. Success was also dependent on the availability of a rapid solubility assay to analyze the mutants. This assay also makes the method feasible for even larger proteins or protein fragments. After the structure determination has been completed, an additional structure-based mutant was used to obtain a different crystal form, which was solved by molecular replacement. In this form, the previously disordered parts of the active site became ordered with interpretable electron density.

HP1 integrase is a member of the tyrosine recombinase family. Although the full length protein is insoluble, limited papain digest identified subdomains with improved solution properties. The fragment containing the active site was crystallizable, but the crystals were not of diffraction quality. A second iteration of limited proteolysis on this fragment marked a less sensitive protease site, resulting in a slightly smaller fragment. Additionally, careful analysis of crystal screen experiments identified buffer components to be included in a second round of screening. These experiments resulted in new set of conditions, that eventually led to well diffracting crystals and to a successful structure determination. The structure revealed the pivotal role of one of the identified components in helping to prevent disorder.