Abstract
DHHC3 belongs to a family of DHHC palmitoyltransferase, which catalyzes the S-palmitoylation of target proteins by attaching a fatty acyl group to a cysteine. Recently, DHHC3 has been demonstrated to be a promising antitumor target in cancer therapeutics. However, the detailed structure and catalysis mechanism of DHHC3 remain elusive, considering its sequence diversity from the DHHC homologues with known crystal structures. Here, we described the expression and purification of human DHHC3 (hDHHC3) and truncated hDHHC3 with the flexible N-terminal domain (NTD) removed. Purified hDHHC3 proteins were used under various conditions for protein crystallization. LAMTOR1, one of the interacting proteins of hDHHC3 to facilitate the crystallization, was further identified by mass spectrometry and co-immunoprecipitation assay. The structural exploration using cryogenic electronic microscopy (cryo-EM) on the inactive hDHHS3 mutant showed a typical sideview of membrane proteins. These results provide a preliminary guidance for the structural determination of DHHC3.
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