The Structural Biology Research Of Sfh3and APT1from Saccharomyces Cerevisiae

Phosphorylated derivatives of phosphatidylinositol (Ptdlns), or named phosphoinositides (PIPs), are elementary components of the membrane-associated signaling systems that maintain and regulate the compartmentalization of membranes. In recent study, the major PITP (PtdIns/phosphatidylcholine (PtdCho) transfer protein) in Saccharomyces cerevisiae, Sec14p is considered to be instructive components in the regulation of Ptdlns4-OH kinase activities for PIP signaling. In this regulation, Sec14p plays a cross role as a PtdCho sensor and a PtdIns-presenting nanoreactor, which follows a front-loaded strategy to provide PtdCho metabolic information to PIP signaling synthesis.Sfh3p shares with24%homology to Sec14p. Unlike Secl4p, the Sfh3does not display significant PtdCho-transfer activity in vivo. In this study, the crystal structure of Sfh3p has been analyzed as a homodimer, which is first reported comparing with the known structures of See14-like proteins as monomers. In this structure, three novel points are observed.1) The hydrophobic pocket for lipid binding is much larger than other known See14structures, which is capable of containing larger lipid molecule or number of lip molecules;2) the interaction between two molecules affects the forming of hydrophobic pocket profoundly, which leads surprising changes to the molecular status of apo/substrate binding;3) except the conserved areas for binding PtdIns, the microenvironment of the pocket interior is so hydrophobic that there are no other significant "polar points" for interaction with other phospholipid polarity group. The novel structure of sfh3suggests a possible binding pocket for new lipids, which is consistent with the hypothesis of Sec14like proteins in structure.Protein modification with fatty acids is a marked feature of many proteins in eukaryotic cells. Palmitoylation is the only one post-translational reversible process in lipid modification, which plays important roles in the dynamic subcellular localization, protein activity and stability, and complex assembly. Palmitoylation is autoacylated or catalyzed by protein acyltransferase (PAT) and depalmitoylation is catalyzed by acyl protein thioesterase (APT). In Homo sapiens, two kinds of APTs have been reported. While in Saccharomyces cerevisiae, there is only one protein, yAPT1catalyzing the depalmitoylaiton.yAPT1exhibits both acyl-thioesterase activity and acyl-esterase activity. However, yAptl significantly prefer protein substrate at least several hundredfold higher apparent affinity to small molecule substrates in vitro, Furthermore, comparing with other mammalian APT enzymes,yAPT1displays the marked specificity of different protein substrates. The structure relevance of this substrate preference is still unclear. In order to elucidate the structure differences and catalyze mechanism, a set of X-ray diffraction data was collected to2.30A and the crystal structure of full length yAPT1was analyzed in this study.