Calcium affinity of the NH2-terminal epidermal growth factor-like module of factor X. Effect of the gamma-carboxyglutamic acid-containing module.
The NH2-terminal epidermal growth factor (EGF)-like module of vitamin K-dependent coagulation factors IX and X and protein C each has one calcium binding site. This module (residues 45-86) from factor X has been isolated previously and found to bind calcium with a Kd of 2.2 mM at physiological pH and ionic strength. We have now demonstrated that it binds calcium with a Kd of 120 microM in a fragment that consists of the Gla module and the NH2-terminal EGF-like module. The presence of the Gla module (residues 1-44) increases the calcium affinity of the site in the EGF-like module approximately 20-fold, thus making it essentially saturated in vivo. Decarboxylation of the Gla residues to Glu has no significant effect on the calcium affinity of the EGF-like module. A proteolytic fragment of factor X (residues 29-86) and a synthetic peptide (residues 34-86), folded to a native conformation, were used to demonstrate that the contribution of the Gla module to the calcium affinity of the site in the EGF-like module is mediated by its 17 COOH-terminal residues, 12 of which form an alpha-helix in the intact Gla module. In the NMR structure of the NH2-terminal EGF-like module in factor X, five calcium ligating groups have been identified (Selander-Sunnerhagen, M., Ullner, M., Persson, E., Teleman, O., Stenflo, J., and Drakenberg, T. (1992) J. Biol. Chem. 267, 19642-19649). As calcium usually requires seven to eight oxygen ligands, there is reason to believe that the Gla module contributes ligands, or negative charge, to increase the calcium affinity. Our findings suggest that the calcium affinity of EGF-like modules in other proteins may also be influenced by neighboring modules.
Valcarce
C
C
Department of Clinical Chemistry, University of Lund, Malmö General Hospital, Sweden.
Selander-Sunnerhagen
M
M
Tämlitz
A M
AM
Drakenberg
T
T
Björk
I
I
Stenflo
J
J
D015055:1-Carboxyglutamic Acid / Q000032:analysis / Q000378:metabolism*; D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000378:metabolism*; D005170:Factor X / Q000737:chemistry / Q000378:metabolism*; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D010446:Peptide Fragments; D015055:1-Carboxyglutamic Acid; D004815:Epidermal Growth Factor; D005170:Factor X; D002118:Calcium