W0040

HIV Integrase-Improving Crystal Quality Through Molecular Biology Techniques. Anne M. Hassell and Lisa M. Shewchuk, Glaxo Wellcome, Inc., Structural Chemistry Dept., Research Triangle Park, NC 27709 USA

HIV integrase catalyzes the insertion of viral DNA into the host genome through a distinct set of DNA cutting and joining reactions. In the 3' processing step, two nucleotides are removed from each 3' end of the double-stranded, blunt-ended DNA produced from reverse transcription. The recessed ends of the viral DNA are then covalently joined to the 5' ends of the target DNA, completing the integration process and permanently infecting the host cell. The fact that HIV integrase has no known counterpart in human cells makes it a very attractive target for the design of anti-HIV agents. However, structure-based drug design efforts were hindered by the insolubility of the enzyme. Dyda et al (Science 266, 1981-1986 [1994]) overcame the solubility difficulties encountered in their crystallization efforts by substituting LYS for PHE185. Although this mutation resulted in a three-dimensional structure of the HIV integrase core domain (50-212), the active site loop of that structure was disordered. We will describe how changing the expression plasmid and the NH2-terminal histidine tag improved the crystal quality, enabling us to obtain a three-dimensional structure with an ordered active site loop.