The primary goal of this project is to determine the molecular structure of a full length HIV-1 integrase, to harness it to elucidate interactions with DNA, and to initiate structure-assisted drug design. The structure will be determined from crystals that derive from a series if mutations introduced in succession with the goal of improving solubility of full length integrase while preserving activity. Initially a low resolution the structure will be determined from current about 0.4 mm sized crystals (space group C2; unit cell dimension a-160A, b=245A, c=302A, and beta=94 degrees. Further mutations will be used to introduce unique labeling sites, for heavy metal isomorphous replacement and anomalous dispersion procedures. Molecular replacement will use the catalytic domains of HIV-1 integrase, ASV integrase, and Mu transposes.
The aim i s to reveal the relative positions of the N-terminal zinc-finger domain, the catalytic domain, and DNA binding C-terminal domain in the apoenzyme complex. Another aim is to improve resolution of diffraction. The ensuring strategy builds on the now routine crystal growth deriving from the intensive mutagenesis, expression, purification, characterization and crystallization efforts in the Pis laboratory. The first crystals provide the key assay, by means of x-ray diffraction, needed for improvement in resolution of diffraction. Screens of future conditions, and further diffraction, needed for improvement in resolution of diffraction. Screens of further conditions, and further mutagenesis will be used. Functional assessment of variants will be carried out in collaboration. Attempts will be made to cocrystalize our soluble integrase construct with already known inhibitors, and inhibitors under development. Cocrystallization of integrase with oligonucleotides will be attempted using a Y-shaped duplex previously constructed to mimic a reaction intermediate, and linear constructs. Further nucleic acid constructs will be designed in collaboration. The size and length of double stranded oligonucleotide constructs will be varied to obtain crystals of a complex suitable for structure determination.
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