The long-term objective of this research is to understand the complete vitamin K cycle in terms of structure and function. This series of reactions is important for post- translational modification of proteins involved in blood coagulation, bone formation, and a number of other physiological processes. The primary focus will be studying the structure and function of the enzyme, 3-glutamyl carboxylase, and its mechanism of action. The broader goal is to reproduce the carboxylation process, but in addition, the entire vitamin K cycle in vitro. This should help to understand the roles that carboxylase, vitamin K oxidoreductase, and any other essential components in the vitamin K cycle play in physiologic settings. Primarily, these studies will be designed to address the following: (1) determine the ways carboxylase recognizes and interacts with substrates and ligands;(2) identify and characterize catalytic residues crucial for 3-carboxylation;(3) elucidate the role of regulatory sites on the mechanism of 3-carboxylation;and (4) determine the three-dimensional structure of the 3-glutamyl carboxylase. In particular, the specific aims are as follows:
Specific Aim 1 : Investigate the role of substrate affinity in the control of vitamin K-dependent protein carboxylation in mammalian cells.
Specific Aim 2 : Identify residues important for substrate binding, propeptide binding, and the residues that mediate the allosteric interaction between these two sites;develop models compatible with our results to explain how carboxylase functions.
Specific Aim 3 : Identify the active site catalytic residues of carboxylase.
Specific Aim 4 : Investigate the three-dimensional structure of 3-glutamyl carboxylase. The pursuit of these goals will include creation of mutant carboxylases and biochemical characterization of these modified enzymes;use of cell culture and molecular biology techniques to investigate expression of pro-coagulation factors, with different affinities for carboxylase to study how carboxylation is controlled in a cellular milieu;and use electron crystallography to gain structural information that we will correlate with information from our functional studies to achieve a more complete understanding of the 3-glutamyl carboxylase.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL048318-18
Application #
7996544
Study Section
Hemostasis and Thrombosis Study Section (HT)
Program Officer
Link, Rebecca P
Project Start
1992-04-01
Project End
2011-11-30
Budget Start
2010-12-01
Budget End
2011-11-30
Support Year
18
Fiscal Year
2011
Total Cost
$361,899
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Parker, Christine H; Morgan, Christopher R; Rand, Kasper D et al. (2014) A conformational investigation of propeptide binding to the integral membrane protein γ-glutamyl carboxylase using nanodisc hydrogen exchange mass spectrometry. Biochemistry 53:1511-20
Tie, J-K; Jin, D-Y; Tie, K et al. (2013) Evaluation of warfarin resistance using transcription activator-like effector nucleases-mediated vitamin K epoxide reductase knockout HEK293 cells. J Thromb Haemost 11:1556-64
Tie, Jian-Ke; Jin, Da-Yun; Stafford, Darrel W (2012) Human vitamin K epoxide reductase and its bacterial homologue have different membrane topologies and reaction mechanisms. J Biol Chem 287:33945-55
Tie, Jian-Ke; Jin, Da-Yun; Straight, David L et al. (2011) Functional study of the vitamin K cycle in mammalian cells. Blood 117:2967-74
Morgan, Christopher R; Hebling, Christine M; Rand, Kasper D et al. (2011) Conformational transitions in the membrane scaffold protein of phospholipid bilayer nanodiscs. Mol Cell Proteomics 10:M111.010876
Higgins-Gruber, Shannon L; Mutucumarana, Vasantha P; Lin, Pen-Jen et al. (2010) Effect of vitamin K-dependent protein precursor propeptide, vitamin K hydroquinone, and glutamate substrate binding on the structure and function of {gamma}-glutamyl carboxylase. J Biol Chem 285:31502-8
Hebling, Christine M; Morgan, Christopher R; Stafford, Darrel W et al. (2010) Conformational analysis of membrane proteins in phospholipid bilayer nanodiscs by hydrogen exchange mass spectrometry. Anal Chem 82:5415-9
Tie, Jian-Ke; Zheng, Mei-Yan; Hsiao, Kuang-Ling N et al. (2008) 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. Biochemistry 47:6301-10
Gui, Tong; Reheman, Adili; Funkhouser, William K et al. (2007) In vivo response to vascular injury in the absence of factor IX: examination in factor IX knockout mice. Thromb Res 121:225-34
Davis, Charles H; Deerfield 2nd, David; Wymore, Troy et al. (2007) A quantum chemical study of the mechanism of action of Vitamin K carboxylase (VKC) III. Intermediates and transition states. J Mol Graph Model 26:409-14

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