The long-term research goal is to determine the three-dimensional structure, dynamic properties and function of human fatty acid synthase (FAS), an enzyme which catalyzes the synthesis of fatty acids, and whose high activity is closely related to many human malignant tumors including breast, prostate, colon and ovarian carcinoma. This goal requires a detailed mechanistic understanding of how the homodimeric animal FAS of approximately 0.54 million Dalton carries out seven consecutive reactions and channeling of substrates for the biosynthesis of long chain fatty acids. Current informatics indicate that FAS, not only consists of multi-catalytic domains, but also exhibits high mobility with multiple conformations. These features, combined with its large size, make FAS a uniquely attractive and challenging system for structure-function studies. To meet the challenge, a team of investigators with expertise in electron cryo-microscopy (cryo-EM), x-ray crystallography, computational biophysics and enzymology has been assembled. The team will undertake a systematic approach to elucidate the FAS structure at the highest possible resolution, by combining data obtained from the various techniques. Because of the existence of multi-conformers of FAS, a novel hybrid experimental and computational refinement procedure will be employed iteratively to merge structural and computational informatics at various resolutions, in order to generate three-dimensional structures of the enzyme at progressively greater detail. Furthermore, the precise atomic details of the crystal structures of individual or tandem functional domains in the presence of ligands will be invaluable in understanding catalytic mechanisms and in the future rational design of anti-cancer therapeutics.
The specific aims are: 1) To determine, ultimately, the three-dimensional structure of FAS to 10 A by cryo-EM, augmented by a novel computational refinement procedure. 2) To determine the atomic structures of catalytic/functional domains, individually or in tandem, of FAS by x-ray crystallography. 3) To map out the locations of the domains by antibodyFab fragments and/or gold labeling and fitting crystal structures of domains to cryo-EM maps of the intact enzyme. 4) To integrate all the structural, computational and biochemical informatics, in order to derive a molecular basis for the mode of action of FAS leading to the multi-step catalytic synthesis of long chain fatty acid.
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