Dr. Stephen J. Everse received his Ph.D. from the University of California, San Diego in 1995. Both his graduate and postdoctoral studies were in the lab of Russell F. Doolittle where he focused on obtaining a structural understanding of the fibrinogen molecule. His work resulted in the crystallographic structures of the fibrinogen fragment D and the fibrin fragment double-D.

He joined the Department of Biochemistry in the Fall of 1998 as part of the HHMI Structural Biology Initiative at the University of Vermont. As part of the initiative, HHMI provided for a state-of-the-art x-ray facilty which includes a rotating anode generator, two MAR345 detectors, two cryo-cooling systems and the necessary computers for structure solution.

His laboratory takes a crystallographic approach to address five fundamental questions:

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.

Insights into protein cofactor modulation of coagulation enzymes

Life threatening hemorrhage is associated with deficiencies of blood coagulation factors V and VIII. These are large (~ 330 kDa), homologous, single chain plasma proteins which are proteolytically activated by other coagulation factors (serine proteases). Coagulation can be separated into three distinct phases: initiation, propagation and termination. Initiation begins with chemical or mechanical damage to tissue which either exposes cell borne tissue factor (TF) to the plasma circulation (e.g., pericytes) or induces TF expression (e.g., monocytes, activated endothelium). TF interacts with a small amount of circulating factor VIIa in the plasma, to form the extrinsic tenase. The membrane bound extrinsic tenase generates small amounts of factors IXa and Xa. A fraction of the factor Xa produced cleaves prothrombin, its substrate, to form alpha-thrombin. This initial small amount of alpha-thrombin can activate the coagulation cofactor proteins, factors V and VIII. Cofactor activation allows assembly of the intrinsic tenase which provides for rapid (70 fold extrinsic) factor Xa activation, overcoming the rapid inactivation of the TFVIIa complex by circulating tissue factor pathway inhibitor (TFPI). If enough factor Xa is produced, the prothrombinase complex forms which leads to the rapid propagation phase of the coagulation cascade in which thrombin formation is accelerated. The prothrombinase complex (factors Va & Xa with Ca+2 on a phospholipid surface) is responsible for the explosive alpha-thrombin generation which is 105 fold over the factor Xa rate alone (Figure 1).

Figure 1: The cofactor complexes involved in coagulation. The size and density of the arrows are representative of the physiological contribution of each complex towards the assembly of the following complex.

Once formed, alpha-fibrin, the structural component of the clot. The termination phase results from the formation of the alpha-thrombinthrombomodulin complex, the steady decay of the extrinsic tenase through spontaneous activity loss, the inactivation of factor Va by activated protein C (APC), and the continual inactivation of the TFVIIa complex, factor Xa and alpha-thrombin by TFPI and antithrombin III.

The successive activation of zymogen components in the coagulation cascade is dependent upon the formation of enzymatic complexes. These complexes (enzyme + cofactor) assemble on cellular surfaces and function in the activation of the subsequent zymogen substrates. Complex formation, in general, leads to large increases in catalytic efficiency (kcat/Km) for zymogen activation, usually as a consequence of a large turnover (kcat) effect with smaller changes observed in Km. While the exact mechanism(s) for these increases in enzymatic efficiency are not clear, we know that cofactor binding to the protease alters the active site and generates a more efficient enzyme. Significant active site alterations are observed in factors Xa, VIIa and alpha-thrombin upon cofactor binding. However, cofactor binding does not appear to alter small substrate hydrolysis rates. Therefore, we infer that the observed active site re-arrangements must reflect changes in either the overall enzymatic architecture or more subtle alterations in enzyme/cofactor structure which influence macromolecular substrate recognition. Also, it is known that, in general, a reduced Km for a substrate is observed with a two-dimensional condensation of the enzyme which increases the collision rate. However, the sum of these effects does not equal theincrease in enzymatic efficiency observed. It has been postulated that cofactor binding to the enzyme does not directly alter the enzymatic efficiency, but rather its binding influences orientation or topology of the substrate, thus providing the enzyme with a "better"substrate. The most definitive method to determine if complex assembly leads to changes in orientation or topology in these molecules is x-ray crystallography.

Factors V & VIII share 35-43% sequence homology and an identical domain structure of: A1-A2-B-A3-C1-C2 (Figure 2).

Figure 2. Domain structure of ceruloplasmin, factor V, and factor VIII.

This primary structure homology has been exploited in biochemical studies and has lead to a general understanding of their biochemical function and the processes central to their regulation. One glaring defect in our understanding of these proteins lies in the nearly complete lack of 3D structural data. Using computer modeling, the A1-A2 and A3 domains of both factor V and factor VIII have been modeled based on the Ceruloplasmin structure. However, these models are disappointing in that many of the regions of interest, i.e., cleavage sites, macromolecular interaction sites, and the entire carboxy-terminal half of the light chain (C1-C2), are either not modeled or are energy minimized loop structures. Obtaining the structures of these proteins in both their intact associated forms (which express biological activity) and in their cleaved dissociated forms (inactive) would represent a huge advance in our understanding of their mechanism of function. As a start to providing a real structural understanding of the cofactors we, in collaboration with Dr. Kenneth G. Mann (University of Vermont), have grown crystals of bovine factor Va which diffract to 2.8Å. We have also identified a derivative for MIRAS phase determination. This structure is the first of a number of planned structures designed to detail the role of factor Va in the prothrombinase complex. Future structures include: the prothrombinase complex (factors Va & Xa with phospholipid and Ca2+), the prothrombinase complex with prothrombin (substrate bound), and the complex of factor Va and APC (Figure 1).

Seemingly dissimilar diseases such as hemophilia and atherosclerotic heart disease are related because they reflect extremes of coagulation responsiveness, i.e., hemorrhage or thrombosis. The process of atherosclerosis appears to be driven, in part, by the chronic activation of the coagulation system; therefore, it is critical to provide a detailed structural understanding of each step in the process. The successful completion of these studies will provide tremendous insight into the functions of the prothrombinase complex and the intrinsic tenase complex as well as the molecular basis for the synergistic amplification of enzymatic activity. A detailed understanding of cofactor function will ultimately be used in the rational design of new classes of therapeutics for regulating coagulation. For example, a large database of factor VIII mutants exist in which the structural basis for the phenotype is unknown. The ability to map these mutants to the actual structure of factor VIIIa will generate new avenues of research directed at stabilizing activity in factor VIIIa. Also, one of the key reactions in the regulation of thrombin formation is the proteolytic inactivation of factor Va by APC. APC cleaves after Arg506 which allows for the spontaneous release of the A2 domain and complete inactivation of factor Va. A genetic polymorphism, termed factor VLEIDEN, has been identified in factor V that affects approximately two percent of the Caucasian population. This mutation, Arg506 -> Gln, removes the primary APC cleavage site, thereby slowing factor Va's inactivation. Since homozygous carriers of factor VLEIDEN show a 80 fold increased risk for venous thrombosis, screening for factor VLEIDEN has become routine in cases of familial thrombosis. Hopefully the structure of the individual components and the complex will give us insights into why there should be an associated risk for venous thrombosis.

Insights into iron binding and release

Iron is essential for a wide variety of metabolic processes; its reactivity, which accounts for its involvement in these processes, also makes it potentially dangerous. In mammals, most iron is found as heme, within heme proteins, or as ferritin, a storage protein. Only a small fraction of iron is actually absorbed during the digestive process; the majority is recycled from red blood cells following their breakdown. In normal individuals 0.1% of the iron can be found circulating in the plasma pool bound to transferrin.

Transferrin (80 kDa) is a glycoprotein consisting of 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 3).

Figure 3. Ribbon structure of the N-lobe of human serum transferrin.

Transferrin is an effective iron chelator, binding with a dissociation constant of 1022 mol/L. This chelation serves three purposes: (1) it renders the iron soluble under physiological conditions, (2) it prevents iron free radical formation and damage, and (3) it facilitates the transport of iron into the cells.

Circulating transferrin binds to the ubiquitously expressed transferrin receptor on the cell surface. The complex is endocytosed into an endosome where the acidic pH weakens the binding and produces conformational changes in both transferrin and the receptor thereby allowing for the release of the iron by a process which is still not well understood. The apo-transferrin-receptor complex is then recycled to the cell surface where the apo-transferrin is released to scavenge more iron.

In collaboration with Dr. Anne B. Mason (University of Vermont) 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. We believe that these structures, in combination with the physical chemical binding data of these mutants, will enable us to fully understand the iron uptake and release properties of transferrin.

A long term goal is to obtain the structure for the complex of transferrin with the transferrin receptor. The solution of this structure is critical to understanding the role of each in such diseases as hemochromatosis. Although the defective protein responsible for hemochromatosis has been discovered and its structure with the receptor is known, the mechanistic basis of the disease remains obscure. Understanding transferrin iron uptake and release from the complex seems essential to clarify the basis of the disease.

Questions, answers or comments, e-mail: Stephen