EGF-like module pair 3-4 in vitamin K-dependent protein S: modulation of calcium affinity of module 4 by module 3, and interaction with factor X.
Calcium-binding epidermal growth factor (EGF)-like modules are found in numerous extracellular and membrane proteins involved in such diverse processes as blood coagulation, lipoprotein metabolism, determination of cell fate, and cell adhesion. Vitamin K-dependent protein S, a cofactor of the anticoagulant enzyme activated protein C, has four EGF-like modules in tandem with the three C-terminal modules each harbouring a Ca(2+)-binding consensus sequence. Recombinant fragments containing EGF modules 1-4 and 2-4 have two Ca(2+)-binding sites with dissociation constants ranging from 10(-8) to 10(-5) M. Module-module interactions that greatly influence the Ca(2+) affinity of individual modules have been identified. As a step towards an analysis of the structural basis of the high Ca(2+) affinity, we expressed the Ca(2+)-binding EGF pair 3-4 from human protein S. Correct folding was shown by (1)H NMR spectroscopy. Calcium-binding properties of the C-terminal module were determined by titration with chromophoric chelators; binding to the low-affinity N-terminal site was monitored by (1)H-(15)N NMR spectroscopy. At physiological pH and ionic strength, the dissociation constants for Ca(2+) binding were 1.0x10(-6) M and 4. 8x10(-3) M for modules 4 and 3, respectively, i.e. the calcium affinity of the C-terminal site was about 5000-fold higher than that of the N-terminal site. Moreover, the Ca(2+) affinity of EGF 4, in the pair 3-4, was about 9000-fold higher than that of synthetic EGF 4. The EGF modules in protein S are known to mediate the interaction with factor Xa. We have now found modules 3-4 to be involved in this interaction. However, the individual modules 3 and 4 manifested no measurable activity.
Stenberg
Y
Y
Department of Clinical Chemistry, Lund University, University Hospital, Malmö, Malmö, S-205 02, Sweden.
Muranyi
A
A
Steen
C
C
Thulin
E
E
Drakenberg
T
T
Stenflo
J
J
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism*; D002614:Chelating Agents; D016384:Consensus Sequence; D004815:Epidermal Growth Factor / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015951:Factor Xa / Q000378:metabolism*; D065427:Factor Xa Inhibitors; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding; D017293:Protein S / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013816:Thermodynamics; D017186:Titrimetry
D002614:Chelating Agents; D065427:Factor Xa Inhibitors; D010446:Peptide Fragments; D017293:Protein S; D011993:Recombinant Fusion Proteins; D004815:Epidermal Growth Factor; D015951:Factor Xa; D002118:Calcium