This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We propose to study mammalian hormone-sensitive lipase (HSL), a key enzyme in fatty acid mobilization in adipose and other tissues, to shed light on its structural organization pertaining to its catalytic function. The activity of HSL is regulated through reversible phosphorylation at various sites located within a 'regulatory module'. HSL interacts with other cellular proteins, as well as self associating; dimeric HSL has almost 40 times higher activity than the monomers. Despite its importance in lipid metabolism, the structural properties of HSL remain largely unknown. HSL contains three major domains: an N-terminal protein-docking domain, a C-terminal domain that contains the reactive catalytic triad, and a regulatory module that is inserted in the middle of the C-terminal portion of HSL. The secondary structure organization of the C-terminal portion of HSL has been predicted based on the crystal structures of several fungal and bacterial lipases and esterases. Unfortunately, the predicted three-dimensional structure of HSL could only be developed for the C-terminal portion of HSL; the N-terminal 300 residues and the 150-residue 'regulatory module' within the C-terminal of HSL had to be eliminated due to lack of structural information. The accuracy of the model and the contribution of the N-terminal portion and the regulatory module to the structure and function of HSL remain unknown. Our project objectives are to 1) obtain low-resolution solution structure of the entire HSL, as well as the N- and C-terminal domains separately; 2) study its monomer-dimer conversion, which appears to be one mechanism controlling its catalytic activity; 3) compare the low-resolution C-terminal domain structure with those predicated on homology with the crystal structures determined for bacterial and fungal homologs in order to examine the structural role of the catalytic triad in the mammalian enzyme.
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