PI: Forsén (LU)
Project: Solution structures of blood coagulation protein domains
Studies of a single and a pair of modules from mosaic blood coagulation proteins by nuclear magnetic resonance spectroscopy.
The blood coagulation- and anti coagulation system harbours a large number of proteins which take part in a finely tuned system which has the task of containing vessel damage without causing undesired intra-vessel coagulation which could lead to thrombosis. To this, the system needs to be ready to become activated rapidly following an injury or other unbalance. Many of the proteins are built up by homologous domains, or modules, which are believed to have spread by exon shuffling, i.e. nature has found a useful building block that it can reuse after limited modification. One of the modules occurring frequently in the proteins of this system is the epidermal growth factor-like (EGF) module. This is one of the most common modules in mosaic proteins, present in proteins with highly divergent function.
To better understand the role of EGF modules as building blocks of blood coagulation- and anti coagulation proteins we are studying them isolated and in oligomers of varying length. The use of oligomers allows us to better understand the cross-talk between the modules, where Ca2+ plays an important role. We also want to gain an understanding to whether general properties of this group of modules can be defined.
Solution structure of the N-terminal EGF-like domain of human factor VII
Blood coagulation is initiated by Ca2+-dependent binding of coagulation factor VIIa (FVIIa) to its cofactor, tissue factor (TF). FVIIa consists of an N-terminal Gla module followed by two EGF-like modules, the first of which can bind one Ca2+ ion (Kd~150 x 10-6 M) and a C-terminal serine protease module. Using 1H nuclear magnetic resonance spectroscopy we have determined the solution structure of a synthetic N-terminal EGF-like module (EGF1) of human FVII (residues 45-85) in the absence of Ca2+. The structure of apo EGF1 was compared with the Ca2+-bound EGF1 in the complex of FVIIa and TF [Banner, D. W. et al. (1996) Nature 380, 41-46]. Amino acid side chains that are crucial for the binding of FVIIa to TF show a similar conformation in the Ca2+ free and in the Ca2+ bound form. This observation is consistent with the idea that the Ca2+ dependence of this interaction is due to altered orientation between the Gla and EGF1 modules rather than local structural changes within EGF1.
Structural basis of high affinity Ca2+ binding site in EGF domains 3-4 of human protein S
Vitamin-K dependent protein S is a cofactor for the anti-coagulant activated protein C. It contains four sequential EGF-like modules, whereof the three C-terminal have a sequence motif associated with Ca2+ binding. It was concluded that EGF3 and EGF4 contain the strongest Ca2+ binding sites, but that the affinity was strongly dependent on the presence of neighbouring modules. For example, the dissociation constants (Kd) of the two strongest Ca2+ binding sites in a 1-4 construct are 2 x 10-6 and 2 x 10-8 M, while the isolated third and fourth modules have 103-105 times weaker binding affinities. We have used 1H-15N NMR spectroscopy to monitor shift changes resulting from Ca2+ titration to an EGF 3-4 oligomer, and could conclude that in this case the presence of EGF 4 only alters the affinity of the N-terminal module modestly. 1H, 15N, 13C NMR spectroscopy is in progress with the aim of determining the structure and elucidating the dynamic properties of the EGF 3-4 module pair. (Work in progress with Yvonne Stenberg, Johan Evenäs, Johan Stenflo, Sture Forsén, and Torbjörn Drakenberg)
Andreas Muranyi, Bryan E. Finn, Garry P. Gippert, Sture Forsén, Johan Stenflo, and Torbjörn Drakenberg (1998) Solution structure of the N-terminal EGF-like Domain from Human Factor VII. Biochemistry 37, 10605-10615