Project Description:
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? Three model systems are being studied to explore these basic questions:

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 Va_i (Figure 1). While attempting to crystallize both factor Va and the prothrombinase complex we have been building and refining models of the prothrombinase complex.

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 2A). In collaboration with Dr. Anne B. Mason (Biochemistry) we have been solving structures of mutants of the N-lobe of human serum transferrin (hTF) in order to understand the iron uptake and release properties of this protein. In addition we continue efforts to determine structures of diferric and monoferric hTFs.

Thioredoxin reductase is part of the thioredoxin system, an antioxidant system responsible for protecting the cell against oxidative stress. In collaboration with Dr. Robert Hondal (Biochemistry) we have been solving structures of thioredoxin reductases (Figure 2B). Our goal is to gain a structural insight into the mechanism and the role of selenocysteine.

Figure 1: Structure of bovine factor Va_i (Adams et al. 2004 PNAS 101:8918-23).

Figure 2: A:Oxalate (orange) in the iron binding cleft of the N-lobe of human serum transferrin (Halbrooks et al 2004 JMB 339:217-26).
Alignment of the tetrapeptide SCCS(ox) in the structure of TR from Drosophila. The tetrapeptide structures (B: C+ conformation and C: T- conformation determined by NMR spectroscopy) were modeled in the active site of TR providing a structural basis for our mechanism (for details see Eckenroth et al 2007 Biochemistry 46:4694-705).

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.