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

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 Kd 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 (kex -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

Solution Structure of the Ca(2+)-Binding EGF3-4 Pair from Vitamin K-Dependent Protein S: Identification of an Unusual Fold in EGF3(,).

Vitamin K-dependent protein S is a cofactor of activated protein C, a serine protease that regulates blood coagulation. Deficiency of protein S can cause venous thrombosis. Protein S has four EGF domains in tandem; domains 2-4 bind calcium with high affinity whereas domains 1-2 mediate interaction with activated protein C. We have now solved the solution structure of the EGF3-4 fragment of protein S. The linker between the two domains is similar to what has been observed in other calcium-binding EGF domains where it provides an extended conformation. Interestingly, a disagreement between NOE and RDC data revealed a conformational heterogeneity within EGF3 due to a hinge-like motion around Glu186 in the Cys-Glu-Cys sequence, the only point in the domain where flexibility is allowed. The dominant, bent conformation of EGF3 in the pair has no precedent among calcium-binding EGF domains. It is characterized by a change in the angle of Glu186 from 160 ± 40, as seen in ten other EGF domains, to 0 ± 15. NOESY data suggest that Tyr193, a residue not conserved in other calcium-binding EGF domains (except in the homologue Gas6), induces the unique fold of EGF3. However, SAXS data, obtained on EGF1-4 and EGF2-4, showed a dominant, extended conformation in these fragments. This may be due to a counterproductive domain-domain interaction between EGF2 and EGF4 if EGF3 is in a bent conformation. We speculate that the ability of EGF3 to adopt different conformations may be of functional significance in protein-protein interactions involving protein S

Structural and functional properties of the human Notch-1 ligand binding region

We present NMR structural and dynamics analysis of the putative ligand binding region of human Notch-1, comprising EGF-like domains 11–13. Functional integrity of an unglycosylated, recombinant fragment was confirmed by calcium-dependent binding of tetrameric complexes to ligand-expressing cells. EGF modules 11 and 12 adopt a well-defined, rod-like orientation rigidified by calcium. The interdomain tilt is similar to that found in previously studied calcium binding EGF pairs, but the angle of twist is significantly different. This leads to an extended double-stranded ? sheet structure, spanning the two EGF modules. Based on the conservation of residues involved in interdomain hydrophobic packing, we propose this arrangement to be prototypical of a distinct class of EGF linkages. On this premise, we have constructed a model of the 36 EGF modules of the Notch extracellular domain that enables predictions to be made about the general role of calcium binding to this region