Diacylglycerol acyltransferase 1 (DGAT1) is a microsomal membrane enzyme responsible for the final step in the synthesis of triacylglycerides. Although DGATs from a wide range of organisms have nearly identical sequences, there is little structural information available for these enzymes. The substrate binding sites of DGAT1 are predicted to be in its large luminal extramembranous loop and to include common motifs with acyl-CoA cholesterol acyltransferase enzymes and the diacylglycerol binding domain found in protein kinases.In this study, synthetic peptides corresponding to the predicted binding sites of DGAT1 enzyme were examined using synchrotron radiation circular dichroism spectroscopy, fluorescence emission and adsorption onto lipid monolayers to determine their interactions with substrates associated with triacylglyceride synthesis (oleoyl-CoA and dioleoylglycerol). One of the peptides, Sit1, which includes the FYxDWWN motif common to both DGAT1 and acyl-CoA cholesterol acyltransferase, changes its conformation in the presence of both substrates, suggesting its capability to bind their acyl chains. The other peptide (Sit2), which includes the putative diacylglycerol binding domain HKWCIRHFYKP found in protein kinase C and diacylglycerol kinases, appears to interact with the charged headgroup region of the substrates. Moreover, in an extended-peptide which contains Sit1 and Sit2 sequences separated by a flexible linker, larger conformational changes were induced by both substrates, suggesting that the two binding sites may bring the substrates into close proximity within the membrane, thus catalyzing the formation of the triacylglyceride product. (C) 2014 Elsevier B.V. All Lights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)UK Biotechnology and Biological Sciences Research Council (BBSRC)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)BBSRCSRCD measurements at the ISA (Denmark), Soleil (France)ANKA (Germany)EU Integrated Infrastructure InitiativeEuropean Light Sources Activities (ELISA)Univ Sao Paulo, Inst Fis Sao Carlos, Sao Carlos, SP, BrazilUniv London Birkbeck Coll, Inst Struct & Mol Biol, London WC1E 7HX, EnglandUniv Estadual Paulista, Inst Quim, Araraquara, BrazilUniv Estadual Paulista, Inst Quim, Araraquara, BrazilFAPESP: 09/17698-5UK Biotechnology and Biological Sciences Research Council (BBSRC)H023852UK Biotechnology and Biological Sciences Research Council (BBSRC)J019135CNPq: 553056/2011-5CNPq: 407337/2013-0BBSRCJ019747EU Integrated Infrastructure Initiative13European Light Sources Activities (ELISA)226716