Structure & Function of Proteins
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.
The majority of projects in the laboratory use crystallography to address fundamental issues in biomedical science: How is protein function defined by structure? How do protein cofactors modulate enzymes? How does structure prescribe the binding affinity of a metal?
To begin to dissect the intricate relationship between a protein cofactor and its enzyme we selected to study the prothrombinase complex in collaboration with Dr. Kenneth Mann (Biochemistry). The prothrombinase complex is composed the coagulation co-factor factor Va, a proteolytically activated form of factor V (330 kDa) and the active enzyme, factor Xa. The prothrombinase complex is responsible for the tremendous burst of thrombin necessary for the generation of a successful clot. As a start to providing a real structural understanding of cofactors we solved the structure of bovine factor Vai (Figure 1). While efforts continue to crystallize the complex (factor Va + factor Xa +/- prothrombin) we have more recently focused on computer modeling (Figure 2) as a method to generate hypotheses that can be tested in the laboratory.
In a tremendously successful collaboration with Dr. Anne B. Mason (Biochemistry) we have engaged in structural studies of transferrin, the major plasma protein responsible for iron transport, and its specific receptor, the transferrin receptor. 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. We have been solving structures of single lobes (Figure 3), the entire transferrin molecule, as well as the transferrin/transferrin receptor complex (Figure 4) to understand the complex relationship between structure, pH and iron status (bound/not bound).
In collaboration with Dr. Robert Hondal (Biochemistry) we have been using structure to explore mechanism in thioredoxin reductases (TR). Our structure of Drosophila melanogaster TR allowed us to postulate why mammalian TRs requires selenocysteine but Drosophila TR can utilize cysteine (Figure 5).
In addition to the on-going structural work in the laboratory, we maintain an active collaboration in the mathematical modeling of coagulation with Kathleen Brummel-Ziedins (Biochemistry), Thomas Orfeo (Biochemistry), and Kenneth Mann (Biochemistry). Recent efforts have been directed at extending our “base” model (Figure 6).
Crystal structure of bovine factor Vai (missing the A2 domain). Domains present are indicated, carbohydrates shown in orange, the copper atom is pink, and the calcium atom is in grey. For more information please see Adams et al. PNAS. 101: 8918-8923, 2004.
Molecular model of the human prothrombinase complex (factor Va and factor Xa) on a hypothetical membrane. Factor Va is colored as shown in Figure 1 with the exception that the A2 domain is colored orange. Factor Xa is shown in cyan with its active site indicated. For more information please see Everse et al. “A molecular model of the human prothrombinase complex” (2008) Recent Advances in Thrombosis and Hemostasis 2008. K. Tanaka & E.W. Davie (Eds). Springer Japan: Chiyoda-ku, pp. 107-132.
Electron density (1.5 sigma, 2Fo-Fc map) of oxalate (orange) bound to the N-lobe of human serum transferrin (1RYO). The structure was determined at 1.2 Å. Iron depicted as a red and water as white spheres. For more information please see Halbrooks et al. JMB. 339: 217-226, 2004.
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
Brummel-Ziedins KE, Everse SJ, Mann KG, Orfeo T (2013) Modeling thrombin generation: plasma composition based approach. J Thromb Thrombolysis : .
Rumora AE, Wang SX, Ferris LA, Everse SJ, Kelm RJ Jr (2013) Structural basis of multisite single-stranded DNA recognition and ACTA2 repression by purine-rich element binding protein B (Purβ). Biochemistry 52(26): 4439-50.
Foley JH, Orfeo T, Undas A, McLean KC, Bernstein IM, Rivard GE, Mann KG, Everse SJ, Brummel-Ziedins KE (2013) From principle to practice: bridging the gap in patient profiling. PLoS One 8(1): e54728.
Bravo MC, Orfeo T, Mann KG, Everse SJ (2012) Modeling of human factor Va inactivation by activated protein C. BMC Syst Biol 6: 45.
Danforth CM, Orfeo T, Everse SJ, Mann KG, Brummel-Ziedins KE (2012) Defining the boundaries of normal thrombin generation: investigations into hemostasis. PLoS One 7(2): e30385.
Eckenroth BE, Steere AN, Chasteen ND, Everse SJ, Mason AB (2011) How the binding of human transferrin primes the transferrin receptor potentiating iron release at endosomal pH. Proc Natl Acad Sci U S A 108(32): 13089-94.
Jungck JR, Donovan SS, Weisstein AE, Khiripet N, Everse SJ (2010) Bioinformatics education dissemination with an evolutionary problem solving perspective. Brief Bioinform 11(6): 570-81.
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