Conformational changes in activated protein C caused by binding of the first epidermal growth factor-like module of protein S

Citation
Tm. Hackeng et al., Conformational changes in activated protein C caused by binding of the first epidermal growth factor-like module of protein S, BIOCHEM J, 349, 2000, pp. 757-764
Citations number
35
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMICAL JOURNAL
ISSN journal
02646021 → ACNP
Volume
349
Year of publication
2000
Part
3
Pages
757 - 764
Database
ISI
SICI code
0264-6021(20000801)349:<757:CCIAPC>2.0.ZU;2-I
Abstract
The first epidermal growth factor-like module of human plasma protein S (EG F1, residues 76-116) was chemically synthesized and tested for its ability to inhibit the anticoagulant cofactor activity of protein S for the anticoa gulant protease, activated protein C (APC). EGF1 completely inhibited the s timulation of APC activity by protein S in plasma coagulation assays, with 50 % inhibition at approx. 1 mu M EGF1, suggesting direct binding of EGF1 t o APC. To investigate a direct interaction between EGFI and APC, fluorescen ce resonance energy transfer (FRET) experiments were employed. APC labelled in the active site with fluorescein as the donor, and phospholipid Vesicle s containing octadecylrhodamine as the acceptor, showed that EGF1 associati on with APC caused an increase in energy transfer consistent with a relocat ion of the active site of APC from 94 Angstrom (9.4 nm) to 85 Angstrom abov e the phospholipid surface (assuming kappa(2) = 2/3). An identical increase in energy transfer between the APC active site-bound fluorescein and phosp holipid-bound rhodamine was obtained upon association of protein S or prote in S-C4b-binding protein complex with APC. The latter suggests the presence of a ternary complex of protein S-C4b-binding protein with APC on the phos pholipid surface. To cofirm a direct interaction of EGF1 with APC, rhodamin e was covalently attached to the alpha-N-terminus of EGF1, and binding of t he labelled EGF1 to APC was directly demonstrated using FRET. The data sugg ested a separation between the active site of APC and the alpha-N-terminus of EGF1 of 76 Angstrom (kappa(2) = 2/3), placing the APC-bound protein S-EG F1 close to, but above, the phospholipid surface and near the two EGF domai ns of APC. Thus we provide direct evidence for binding of protein S-EGF1 to APC and show that it induces a conformational change in APC.