EGF-like modules in blood coagulation proteins : Ca²+ binding, module interactions, structure and dynamics as studied by NMR spectroscopy

Sammanfattning: Modules are independently folding protein domains defined on the gene level. The epidermal growth factor-like (EGF) modules are involved in protein-protein interactions and are found in numerous membrane proteins and extracellular proteins, including many proteins of the blood coagulation system. A subset of the EGF modules binds one Ca²⁺ with an affinity that is often influenced by the neighbouring module on the N-terminal side. The complex of factor VIIa (FVIIa) and tissue factor (TF) is important for the initiation of blood coagulation. Reports in the literature suggest that Ca²⁺ binding to the N-terminal EGF module (EGF 1) of FVIIa is essential for the complex to form. We determined the structure of the Ca²⁺-free EGF 1 using NMR spectroscopy. Comparison of this structure with that of the Ca²⁺-bound EGF 1 in the structure of the TF:FVIIa complex shows that only small conformational changes take place as a result of Ca²⁺ binding. These observations are consistent with the view that the Ca²⁺ binding to EGF 1 is crucial for a well-defined, relative orientation of the Gla and EGF 1 modules, which enables the formation of a high-affinity TF:FVIIa complex. Anticoagulant protein S has four EGF modules. The three C-terminal bind Ca²⁺ with high affinity. The fragment constituting the EGF 3-4 module pair from protein S (pS EGF 3-4) is the smallest fragment that retains high-affinity Ca²⁺ binding. The K_d for Ca²⁺ binding was determined to be 4.8 millimolar and 1.0 micromolar for EGF 3 and EGF 4, respectively. Thus the Ca²⁺ affinity of the N-terminal site was similar to that of the isolated EGF 3, while the affinity of EGF 4 in pS EGF 3-4 was approximately 9000-fold higher than that of the isolated EGF 4. The ¹H, ¹⁵N, ¹³CA and ¹³CB resonances of the module pair were assigned using multidimensional heteronuclear NMR spectroscopy. The effect of Ca²⁺ binding on individual resonances was studied. Apart from extensive shift effects of resonances close to the Ca²⁺-binding site, we observed shift effects far from the expected location of the binding site in each of the modules. Extensive spectral heterogeneity revealed cis-trans isomerisation at a very slow rate (k_ex< 0.2 s^-1) of the Lys 167-Pro 168 peptide bond or, possibly, trapping of the two conformers in the folding process. Both conformers have similar Ca²⁺ affinities and backbone dynamics. ¹⁵N spin relaxation data suggested that the module pair with one Ca²⁺ bound in EGF 4 has a well-defined relative orientation between EGF modules 3 and 4. In the absence of Ca²⁺, broadening of several resonances in EGF 4 suggests that chemical exchange is taking place. This is probably not consistent with a well-defined module interface. A comparison of residual dipolar couplings measured on a partly aligned pS EGF 3-4 sample with couplings calculated from the known structure of an EGF module pair from fibrillin-1 suggested that the two EGF modules of pS EGF 3-4 are oriented at an angle of approximately 90 º rather than being oriented in a rod-like arrangement (180 º) as in the fibrillin-1 EGF module pair.