Structural requirements for Ca2+ binding to the gamma-carboxyglutamic acid and epidermal growth factor-like regions of factor IX. Studies using intact domains isolated from controlled proteolytic digests of bovine factor IX.
Blood coagulation factor IX is composed of discrete domains with an NH2-terminal vitamin K-dependent gamma-carboxyglutamic acid (Gla)-containing region, followed by two domains that are homologous with the epidermal growth factor (EGF) precursor and a COOH-terminal serine protease part. Calcium ions bind to the Gla-containing region and to the NH2-terminal EGF-like domain. To be able to determine the structure and function of the Gla- and EGF-like domains, we have devised a method for cleaving factor IX under controlled conditions and isolating the intact domains in high yield, either separately or linked together. The Ca2+ and Mg2+ binding properties of these fragments were examined by monitoring the metal ion-induced changes in intrinsic protein fluorescence. A fragment, consisting of the Gla region linked to the two EGF-like domains, bound Ca2+ in a manner that was indistinguishable from that of the intact molecule, indicating a native conformation. The Ca2+ affinity of the isolated Gla region was lower, suggesting that the EGF-like domains function as a scaffold for the folding of the Gla region. The Gla-independent high affinity metal ion binding site in the NH2-terminal EGF-like domain was shown to bind Ca2+ but not Mg2+. A comparison with similar studies of factor X (Persson, E., Björk, I., and Stenflo, J. (1991) J. Biol. Chem. 266, 2444-2452) suggests that the Ca2(+)-induced fluorescence quenching is due to an altered environment primarily around the tryptophan residue in position 42.
Astermark
J
J
Department of Clinical Chemistry, University of Lund, Malmö General Hospital, Sweden.
Björk
I
I
Ohlin
A K
AK
Stenflo
J
J
D015055:1-Carboxyglutamic Acid / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000378:metabolism*; D005164:Factor IX / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000378:metabolism
D000596:Amino Acids; D010446:Peptide Fragments; D015055:1-Carboxyglutamic Acid; D004815:Epidermal Growth Factor; D014364:Tryptophan; D005164:Factor IX; D002918:Chymotrypsin; D008274:Magnesium; D002118:Calcium