title: Transmembrane domain interactions and residue proline 378 are essential for proper structure, especially disulfide bond formation, in the human vitamin K-dependent gamma-glutamyl carboxylase.
abstract: We used recombinant techniques to create a two-chain form (residues 1-345 and residues 346-758) of the vitamin K-dependent gamma-glutamyl carboxylase, a glycoprotein located in the endoplasmic reticulum containing five transmembrane domains. The two-chain carboxylase had carboxylase and epoxidase activities similar to those of one-chain carboxylase. In addition, it had normal affinity for the propeptide of factor IX. We employed this molecule to investigate formation of the one disulfide bond in carboxylase, the transmembrane structure of carboxylase, and the potential interactions among the carboxylase's transmembrane domains. Our results indicate that the two peptides of the two-chain carboxylase are joined by a disulfide bond. Proline 378 is important for the structure necessary for disulfide formation. Results with the P378L carboxylase indicate that noncovalent bonds maintain the two-chain structure even when the disulfide bond is disrupted. As we had previously proposed, the fifth transmembrane domain of carboxylase is the last and only transmembrane domain in the C-terminal peptide of the two-chain carboxylase. We show that the noncovalent association between the two chains of carboxylase involves an interaction between the fifth transmembrane domain and the second transmembrane domain. Results of a homology model of transmembrane domains 2 and 5 suggest that not only do these two domains associate but that transmembrane domain 2 may interact with another transmembrane domain. This latter interaction may be mediated at least in part by a motif of glycine residues in the second transmembrane domain.
authors.affiliation: Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA. jktie@email.unc.edu
authors.forename: Jian-Ke
authors.initials: JK
authors.lastname: Tie
authors.forename: Mei-Yan
authors.initials: MY
authors.lastname: Zheng
authors.forename: Kuang-Ling N
authors.initials: KL
authors.lastname: Hsiao
authors.forename: Lalith
authors.initials: L
authors.lastname: Perera
authors.forename: Darrel W
authors.initials: DW
authors.lastname: Stafford
authors.forename: David L
authors.initials: DL
authors.lastname: Straight
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D019735:Carbon-Carbon Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology*; D004220:Disulfides / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011392:Proline / Q000737:chemistry* / Q000235:genetics; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014812:Vitamin K / Q000737:chemistry
chemical_list: D004220:Disulfides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014812:Vitamin K; D011392:Proline; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase