title: Mutational analysis of the integral membrane methyltransferase isoprenylcysteine carboxyl methyltransferase (ICMT) reveals potential substrate binding sites.
abstract: The eukaryotic integral membrane enzyme isoprenylcysteine carboxyl methyltransferase (ICMT) methylates the carboxylate of a lipid-modified cysteine at the C terminus of its protein substrates. This is the final post-translational modification of proteins containing a CAAX motif, including the oncoprotein Ras, and therefore, ICMT may serve as a therapeutic target in cancer development. ICMT has no discernible sequence homology with soluble methyltransferases, and aspects of its catalytic mechanism are unknown. For example, how both the methyl donor S-adenosyl-l-methionine (AdoMet), which is water-soluble, and the methyl acceptor isoprenylcysteine, which is lipophilic, are recognized within the same active site is not clear. To identify regions of ICMT critical for activity, we combined scanning mutagenesis with methyltransferase assays. We mutated nearly half of the residues of the ortholog of human ICMT from Anopheles gambiae and observed reduced or undetectable catalytic activity for 62 of the mutants. The crystal structure of a distantly related prokaryotic methyltransferase (Ma Mtase), which has sequence similarity with ICMT in its AdoMet binding site but methylates different substrates, provides context for the mutational analysis. The data suggest that ICMT and Ma MTase bind AdoMet in a similar manner. With regard to residues potentially involved in isoprenylcysteine binding, we identified numerous amino acids within transmembrane regions of ICMT that dramatically reduced catalytic activity when mutated. Certain substitutions of these caused substrate inhibition by isoprenylcysteine, suggesting that they contribute to the isoprenylcysteine binding site. The data provide evidence that the active site of ICMT spans both cytosolic and membrane-embedded regions of the protein.
authors: Structural Biology Program, Sloan-Kettering Institute and Weill Cornell Graduate School of Medical Sciences of Cornell University, New York, New York 10065; Graduate Program in Biochemistry and Structural Biology, Cell and Developmental Biology, and Molecular Biology, Weill Cornell Graduate School of Medical Sciences of Cornell University, New York, New York 10065.
authors: Melinda M
authors: MM
authors: Diver
authors: Structural Biology Program, Sloan-Kettering Institute and Weill Cornell Graduate School of Medical Sciences of Cornell University, New York, New York 10065. Electronic address: longs@mskcc.org.
authors: Stephen B
authors: SB
authors: Long
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000852:Anopheles / Q000201:enzymology*; D020134:Catalytic Domain; D017124:Conserved Sequence; D003545:Cysteine / Q000031:analogs & derivatives* / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011496:Protein Methyltransferases / Q000737:chemistry / Q000235:genetics*; D012436:S-Adenosylmethionine / Q000737:chemistry; D040681:Structural Homology, Protein
chemical_list: D019476:Insect Proteins; D012436:S-Adenosylmethionine; D011496:Protein Methyltransferases; C056501:protein-S-isoprenylcysteine O-methyltransferase; D003545:Cysteine
keywords: Enzyme Mechanism; Isoprenylcysteine Carboxyl Methyltransferase (ICMT); Membrane Enzyme; Post-translational Modification (PTM); Protein Methylation; Ras Protein; Site-directed mutagenesis; Substrate Recognition