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
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.