title: Orphan macrodomain protein (human C6orf130) is an O-acyl-ADP-ribose deacylase: solution structure and catalytic properties.
abstract: Post-translational modification of proteins/histones by lysine acylation has profound effects on the physiological function of modified proteins. Deacylation by NAD(+)-dependent sirtuin reactions yields as a product O-acyl-ADP-ribose, which has been implicated as a signaling molecule in modulating cellular processes. Macrodomain-containing proteins are reported to bind NAD(+)-derived metabolites. Here, we describe the structure and function of an orphan macrodomain protein, human C6orf130. This unique 17-kDa protein is a stand-alone macrodomain protein that occupies a distinct branch in the phylogenic tree. We demonstrate that C6orf130 catalyzes the efficient deacylation of O-acetyl-ADP-ribose, O-propionyl-ADP-ribose, and O-butyryl-ADP-ribose to produce ADP-ribose (ADPr) and acetate, propionate, and butyrate, respectively. Using NMR spectroscopy, we solved the structure of C6orf130 in the presence and absence of ADPr. The structures showed a canonical fold with a deep ligand (ADPr)-binding cleft. Structural comparisons of apo-C6orf130 and the ADPr-C6orf130 complex revealed fluctuations of the β(5)-α(4) loop that covers the bound ADPr, suggesting that the β(5)-α(4) loop functions as a gate to sequester substrate and offer flexibility to accommodate alternative substrates. The ADPr-C6orf130 complex identified amino acid residues involved in substrate binding and suggested residues that function in catalysis. Site-specific mutagenesis and steady-state kinetic analyses revealed two critical catalytic residues, Ser-35 and Asp-125. We propose a catalytic mechanism for deacylation of O-acyl-ADP-ribose by C6orf130 and discuss the biological implications in the context of reversible protein acylation at lysine residues.
authors.affiliation: Department of Biochemistry and Center for Eukaryotic Structural Genomics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226.
authors.forename: Francis C
authors.initials: FC
authors.lastname: Peterson
authors.affiliation: Department of Biomolecular Chemistry and Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, Wisconsin 53715.
authors.forename: Dawei
authors.initials: D
authors.lastname: Chen
authors.affiliation: Department of Biochemistry and Center for Eukaryotic Structural Genomics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226.
authors.forename: Betsy L
authors.initials: BL
authors.lastname: Lytle
authors.affiliation: DNA Damage Response Group, Paterson Institute for Cancer Research, University of Manchester, Manchester M20 4BX, United Kingdom.
authors.forename: Marianna N
authors.initials: MN
authors.lastname: Rossi
authors.affiliation: DNA Damage Response Group, Paterson Institute for Cancer Research, University of Manchester, Manchester M20 4BX, United Kingdom.
authors.forename: Ivan
authors.initials: I
authors.lastname: Ahel
authors.affiliation: Department of Biomolecular Chemistry and Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, Wisconsin 53715. Electronic address: jmdenu@wisc.edu.
authors.forename: John M
authors.initials: JM
authors.lastname: Denu
authors.affiliation: Department of Biochemistry and Center for Eukaryotic Structural Genomics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226. Electronic address: bvolkman@mcw.edu.
authors.forename: Brian F
authors.initials: BF
authors.lastname: Volkman
mesh_terms: D000215:Acylation; D002384:Catalysis; D004950:Esterases / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: D004950:Esterases