Protein structural requirements for Ca2+ binding to the light chain of factor X. Studies using isolated intact fragments containing the gamma-carboxyglutamic acid region and/or the epidermal growth factor-like domains.
Coagulation factor X is a multidomain proenzyme of a serine protease. Calcium ions bind to the vitamin K-dependent gamma-carboxyglutamic acid (Gla) residues and to a site in the NH2-terminal of two epidermal growth factor (EGF)-like domains. To study structure-function relationships in the NH2-terminal part of factor X and to determine the structure of isolated domains, we have developed methods that allow the subsequent isolation of the first or both EGF-like domains with or without an attached Gla domain from controlled proteolytic digests of the protein. The Ca2(+)-induced changes of the intrinsic protein fluorescence were measured to elucidate whether the isolated fragments retain their native conformation. Changes in the fluorescence caused by Ca2+ binding were found to result from perturbations of the environment of the Trp residue in position 41. Calcium ion binding to the Gla-containing region linked to the NH2-terminal EGF-like domain was identical with that to intact factor X, indicating a native orientation of the ligand binding groups in the fragment. In contrast, the isolated Gla peptide had a lower affinity for Ca2+, suggesting that the NH2-terminal EGF-like domain serves as a scaffold for the folding of the Gla region. Similarly, the presence of the Gla region was found to increase the affinity of the Gla-independent site in the first EGF-like domain for Ca2+. The metal ion-induced resistance against chymotryptic cleavage COOH-terminal of Tyr-44 in intact factor X is similar in the isolated fragment that contains the Gla region linked to one EGF-like domain, indicating a native conformation of the fragment in the presence of Ca2+. Furthermore, the Gla-independent metal ion binding site binds Ca2+ but does not appear to bind Mg2+.
Persson
E
E
Department of Clinical Chemistry, University of Lund, Malmö General Hospital, Sweden.
Björk
I
I
Stenflo
J
J
D015055:1-Carboxyglutamic Acid / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000378:metabolism*; D005170:Factor X / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000378:metabolism
D000596:Amino Acids; D010446:Peptide Fragments; D015055:1-Carboxyglutamic Acid; D004815:Epidermal Growth Factor; D005170:Factor X; D002918:Chymotrypsin; D014357:Trypsin; D002118:Calcium