LEDGF-Integrase Structural Biology

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DESCRIPTION (provided by applicant): Integration of HIV-1 cDNA into a cell chromosome is an essential step in the virus replication cycle. This makes the integrase an attractive drug target, and anti-integrase drugs are in US clinical trials. Rational drug design benefits from visualizing three-dimensional enzyme structures, and numerous structures of the HIV-1 integrase catalytic core domain have been solved. Although two domain N-terminal/core and core/C-terminal structures were more recently solved, there is no structure for the intact integrase, which in large part may be due to poor protein solubility. Indeed, all previous crystals contained one or more solubilizing mutations in the catalytic core domain, typified by F185K. In cells, integrase is likely to interact with normal human proteins, and the interaction between integrase and the recently identified human lens epithelium-derived growth factor (LEDGF) forms the basis for this application. We will use LEDGF protein to improve the solubility of HIV-1 integrase, and will crystallize LEDGF-integrase complexes. Based on its high solubility limit in physiologically-relevant buffer conditions, we conclude that unlike the integrase, LEDGF is well behaved in solution. The scientific basis for this proposal is defined by preliminary results demonstrating that purified LEDGF protein significantly improved the solubility of purified HIV-1 integrase in physiologically- relevant buffer conditions. Additional probing of the LEDGF structure by limited proteolysis identified a trypsin-resistant subdomain important for integrase binding and solubilization activities. We propose to solve the structure of the integrase binding subdomain of LEDGF by NMR spectroscopy, and crystallize the subdomain or full-length LEDGF in complex with HIV-1 integrase. The results of these experiments will define the structure of the region of an important cellular interactor for the integrase, as well as form the basis for solving the three dimensional structure of the intact HIV-1 enzyme.