Calcium-binding
Calcium-binding EGF-like domains (cbEGF-like domains) play a seminal role in diseases such as the Marfan syndrome or the X-chromosome linked hemorrhagic disorder hemophilia B and are among the most abundant extracellular calcium-binding domains. Importantly, cbEGF- like domains impart specific functions to a variety of proteins in the blood clotting cascade. Examples include the coagulation factors VII, IX and X, protein C and its cofactor protein S.
Calcium-binding EGF-like domains are typically composed of 45 amino acids, arranged as two antiparallel beta sheets. Several cysteine residues within this sequence form disulfide bridges.
Interestingly, cbEGF-like domains show no significant structural deviations from EGF-like domains, however, as the name suggests, cbEGF-like domains bind a single calcium ion. The binding affinity to calcium varies widely and often depends on adjacent domains. The consensus motif for calcium binding is Asp-Leu/Ile-Asp-Gln-Cys. Coordination of calcium strongly correlates with an unusual posttranslational modification of cbEGF-like domains: either an asparagine or aspartate is beta-hydroxylated giving rise to erythro-beta-hydroxyasparagine (Hyn) or erythro-beta-hydroxyaspartic acid (Hya), respectively. Hya can be found in the N-terminal cbEGF module (see below) of factors IX, X, and protein C. The Hyn modification appears to be more prevalent than Hya and has been shown to occur in fibrillin-1, an extracellular matrix protein. Both modifications are catalyzed by the dioxygenase Asp/Asn-beta-hydroxylase, and are unique to EGF domains in eukaryotes.
Further posttranslational modifications have been reported. Glycosylation in the form of O-linked di- or trisaccharides may occur at a serine residue between the first two cysteines of blood coagulation factors VII and IX. Factor VII exhibits an O-linked fucose at Ser60.
Importantly, multiple cbEGF domains are often connected by one or two amino acids to form larger, repetitive arrays, here referred to as 'cbEGF modules'. In the blood clotting cascade, coagulation factors VII, IX and X, and protein C contain a tandem of two cbEGF modules, whereas protein S has four. Impressivley, in fibrillin-1 and fibrillin-2, 43 cbEGF modules have been found. The modularity of these proteins adds complexity to protein-protein but also module-module interaction. In factors VII, IX and X, the two cbEGF modules are preceded by an N-terminal gamma-carboxyglutamic acid (Gla) containing module (the Gla module). In vitro studies on the Gla-cbEGF tandem isolated from factor X revealed a Kd-value of 0.1 mM for calcium binding with the free calcium blood plasma concentrations being approximately 1.2 mM. Surprisingly, in the absence of the Gla module, the cbEGF module exhibits a Kd-value of 2.2 mM for calcium. Thus, the presence of the Gla module increases calcium affinity 20-fold. Similarly, the activity of Gla and Serine protease modules are modified by the cbEGF modules. In the absence of calcium, the Gla and cbEGF modules are highly mobile. As the cbEGF module associates with calcium, however, movement of the Gla module is significantly restricted because the cbEGF module now adopts a conformation that locks the neighboring Gla module in a fixed position. Therefore, calcium coordination induces conformational changes which, in turn, might modulate enzymatic activity.
Impaired coordination of calcium can result in serious disorders. Defective calcium binding to coagulation factor IX contributes to the development of hemophilia B. Individuals afflicted with this hereditary disease tend to develope hemorrhages, potentially leading to life-threatening conditions. The cause of hemophilia B is decreased activity or deficiency of blood coagulation factor IX. Point mutations resulting in decreased affinity of factor IX to calcium are thought to be implicated in this bleeding disorder. On a molecular basis, it appears that hemophilia B can be the result of an impaired ability to localize the Gla module efficiently, as it usually occurs after calcium coordination by the cbEGF module in fully functional factor IX. This defect is thought to impair the biological function of factor IX. A similar problem occurs in patients suffering from hemophilia B and carrying a mutation (Glu78Lys) in factor IX that prevents interaction of the two cbEGF modules with one another. Conversely, in healthy individuals, Glu78 in the first cbEGF-module contacts Arg94 in the second cbEGF module and thereby aligns both modules. Thus, domain-domain interactions (partially facilitated by calcium coordination) are crucial for the catalytic activity of proteins involved in the blood-clotting cascade.
Read more about this topic: EGF-like Domain