CALCIUM-DEPENDENT INTERACTION BETWEEN GAMMA-CARBOXYGLUTAMIC ACID-CONTAINING AND N-TERMINAL EPIDERMAL GROWTH FACTOR-LIKE MODULES IN FACTOR-X

The N-terminal epidermal growth factor (EGF)-like module in factor X b inds a single ca(2+) with low affinity (K-d = 2.2 mM). When it is link ed to the gamma-carboxyglutamic acid (Gla)-containing module, however, the affinity increases approximately 20-fold (K-d = 120 mu M), indica ting an interaction between the two modules and making the site in the N-terminal EGF-like module essentially saturated at physiological Ca2 + concentrations. We have now used the thioredoxin system to probe Ca2 +-induced conformational changes and interaction between modules in th e light chain of factor X. Thioredoxin, in conjunction with thioredoxi n reductase and NADPH, allows direct measurements of the rate and exte nt of disulfide bond reduction. Most disulfide bonds accessible to the reducing agent were found to be located in the light chain of the pro tein. Moreover, those disulfide bonds that were resistant to reduction by thioredoxin in the presence of Ca2+, but were readily reduced in t he absence of the metal ion, were located in the N-terminal EGF-like m odule and in the Gla module, whereas disulfide bonds in the C-terminal EGF-like module appeared to be equally accessible whether Ca2+ was pr esent or not. Comparison of the rate of disulfide bond reduction in th e isolated modules with that in mixtures of modules indicated that a C a2+-dependent interaction occurred between the Gla and the N-terminal EGF-like module. This interaction was mediated by the C-terminal alpha -helical part of the Gla module. The affinity between the modules was low and could not be determined accurately owing to competing equilibr ia, presumably Ca2+ dependent aggregation of the isolated Gla module. By comparing the rates of disulfide bond reduction in Gla module conta ining fragments before and after decarboxylation of Gla, we could demo nstrate that Ca2+ binding to sites in the Gla module as well as to the single site in the EGF-like module contribute to the interaction betw een the two modules.