X-Ray Crystallography of Blood Coagulation Proteins and Transferrins
Dr. Everse received his Ph.D. in Chemistry from the University of California, San Diego in 1995. His postdoctoral work at UCSD focused on obtaining a structural understanding of the fibrinogen molecule. His work resulted in the X-ray crystallographic structures of the fibrinogen fragment D and the fibrin fragment double-D. He joined the Biochemistry department in the Fall of 1998 as part of the HHMI structural biology initiative at UVM. HHMI provided for a state-of-the-art X-ray facility including a rotating anode generator, two MAR345 detectors, two cryo-cooling systems and the necessary computer resources for structure determination.
His laboratory takes a crystallographic approach to address five fundamental questions: How is protein function defined by structure? What determines and mediates protein-protein and protein-membrane interactions? How does information get passed from one protein to another? How do protein cofactors modulate enzymes? How does structure prescribe the binding affinity of a metal? Two model systems are being studied to explore these basic questions. Both involve synergistic interactions: first, proteins which either amplify, propagate or terminate the blood coagulation response; and second, transferrin, the major plasma protein responsible for iron transport, interacting with its specific receptor.
Factor V is a large (330 kDa), single chain, coagulation cofactor which is proteolytically activated by other coagulation factors. As a start to providing a real structural understanding of the cofactors we, in collaboration with Dr. Kenneth G. Mann (Biochemistry), have solved the structure of bovine factor Vai (Figure 1).
Transferrin (80 kDa) is a glycoprotein with two homologous lobes. Each lobe consists of two domains that form a deep cleft which bind a single Fe (III) ion in conjunction with the concomitant binding of a carbonate ion in a pH dependent manner (Figure 2). In collaboration with Dr. Anne B. Mason (Biochemistry) we have begun crystallizing mutants of the N-lobe of human serum transferrin in order to understand the iron uptake and release properties of this protein.
Oxalate bound to the N-lobe of human transferrin.
Closeup image of Oxalate bound to the N-lobe of human transferrin.
Evolutionary conservancy in factor V.
Mason AB, Byrne SL, Everse SJ, Roberts SE, Chasteen ND, Smith VC, MacGillivray RT, Kandemir B, Bou-Abdallah F.A loop in the N-lobe of human serum transferrin is critical for binding to the transferrin receptor as revealed by mutagenesis, isothermal titration calorimetry, and epitope mapping. J Mol Recognit. 2009 22(6):521-9.
Danforth CM, Orfeo T, Mann KG, Brummel-Ziedins KE and Everse SJ. The impact of uncertainty in a blood coagulation model. Math Med Biol. 2009
Mason AB, Halbrooks PJ, James NG, Byrne SL, Grady JK, Chasteen ND, Bobst CE, Kaltashov IA, Smith VC, MacGillivray RT and Everse SJ. Structural and functional consequences of the substitution of glycine 65 with arginine in the N-lobe of human transferrin. Biochemistry. 2009 48(9):1945-53.
Lee CJ, Lin P, Chandrasekaran V, Duke RE, Everse SJ, Perera L and Pedersen LG. Proposed structural models of human factor Va and prothrombinase. J Thromb Haemost. 2008 6(1):83-9.
Eckenroth BE, Rould MA, Hondal RJ and Everse SJ. Structural and biochemical studies reveal differences in the catalytic mechanisms of mammalian and Drosophila melanogaster thioredoxin reductases. Biochemistry. 2007 46(16):4694-705
Wally J*, Halbrooks PJ*, Vonrhein C, Rould MA, Everse SJ, Mason AB and Buchanan SK. The crystal structure of iron-free human serum transferrin provides insight into inter-lobe communication and receptor binding. J Biol Chem. 2006 281(34):24934-44.
View the structure at PDB.
* indicates equal contribution
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