THE EFFECT OF ASPARTATE HYDROXYLATION ON CALCIUM-BINDING TO EPIDERMALGROWTH FACTOR-LIKE MODULES IN COAGULATION FACTOR-IX AND FACTOR-X

Hydroxylation of aspartic acid to erythro-beta-aspartic acid (Hya) occ urs in epidermal growth factor (EGF)-like modules in numerous extracel lular proteins with diverse functions. Several EGF-like modules with t he consensus sequence for hydroxylation bind Ca2+, and it has therefor e been suggested that the hydroxyl group is essential for Ca2+ binding . To determine directly the influence of beta-hydroxylation on calcium binding in the EGF-like modules from coagulation factors IX and X, we have now measured calcium binding to both the fully beta-hydroxylated and the non-beta-hydroxylated modules of the two proteins. At low ion ic strength, the Hya-containing module of factor X has a slightly high er Ca2+ affinity, but at physiological salt concentrations this differ ence is no longer significant for either factor IX or X. Analysis of t he H-1 NMR chemical shift differences between the hydroxylated and non hydroxylated factor X modules show that hydroxylation has no effect on the domain fold. Furthermore, measurements on factor IX show that hyd roxylation has no effect on the Ca2+/Mg2+ specificity of the ion bindi ng site. We conclude that the hydroxyl group is not a direct ligand fo r the calcium ion in these EGF-like modules, nor is it essential for h igh-affinity Ca2+ binding.