Protein S Alters the Active Site Location of Activated Protein C above the Membrane Surface

Abstract

The location of the active site of membrane-bound activated protein C (APC) relative to the phospholipid surface was determined both in the presence and absence of its cofactor, protein S, using fluorescence resonance energy transfer (FRET). APC was chemically modified to create the FRET donor species, Fl-FPR-APC, with a fluorescein dye (Fl) covalently attached to the active site via a D-Phe-Pro-Arg (FPR) tether and located in the active site near S 4 . FRET was observed when Fl-FPR-APC was titrated in the presence of Ca 2؉ ions with phosphatidylcholine/phosphatidylserine (4:1) vesicles containing the FRET acceptor, octadecylrhodamine (OR). Assuming a random orientation of transition dipoles ( 2 ‫؍‬ 2/3), the average distance of closest approach between the fluorescein in the active site of the membrane-bound APC and the OR at the membrane surface is 94 Å. The same calcium-dependent distance was obtained for both small and large unilamellar vesicles and for vesicles that contained phosphatidylethanolamine. The active site of membrane-bound APC is therefore located far above the phospholipid surface. Upon addition of protein S, the efficiency of Fl-FPR-APC to OR energy transfer increased due to a protein S-dependent rotational and/or translational movement of the APC protease domain relative to the surface. If this movement were solely translational, then the average height of the fluorescein in the membrane-bound APC⅐protein S complex would be 84 Å above the surface. The extent of Fl-FPR-APC to OR energy transfer was unaltered by the addition of thrombin-inactivated protein S. The protein S effect was also specific for APC, since the addition of protein S to similarly-labeled derivatives of factor Xa, factor IXa, or factor VIIa did not alter the locations of their active sites. This direct measurement demonstrates that the binding of the protein S cofactor to its cognate enzyme elicits a relocation of the active site of APC relative to the membrane surface and thereby provides a structural explanation for the recently observed protein S-dependent change in the site of factor Va cleavage by APC.

Relocating the Active Site of Activated Protein C Eliminates the Need for Its Protein S Cofactor

“…FRET Measurements-FRET experiments were performed as before (12), except that the D (donor-containing) and DA (containing donor and acceptor) microcells initially received 15 nM Fl-FPR-APC-PTGla (the donor), whereas microcells B (blank) and A (acceptor-containing) received 15 nM unmodified APC-PTGla. The initial net emission intensity (F o ) was obtained by the subtraction of the signal of B from the signals of DA, A, and D. Samples D and B were then titrated with phospholipid vesicles lacking the OR acceptor, whereas samples DA and A were titrated with an equivalent amount of phospholipid vesicles containing OR.…”

“…Samples containing OR were prepared in the same way except that the desired amount of OR (in ethyl acetate) was added to the phospholipid before lyophilization and sonication (13). The concentrations of phospholipid and OR in a purified vesicle sample were determined as before (12), as was , the OR acceptor density at the vesicle surface.…”

“…Spectral Measurements-All spectral measurements, including determinations of Q, J DA , and R 0 were performed as before (12).…”

“…Fl-FPR-labeled APC-PTGla and human APC were then generated essentially as described for bovine APC (12).…”

“…Light scattering experiments indicated that two elongated vitamin K-dependent proteins, prothrombin (PT) and factor X, project radially from the surface when bound to the membrane (11), and our fluorescence resonance energy transfer (FRET) experiments showed that the active site of each of the vitamin K-dependent enzymes is located far (Ͼ70 Å) above the membrane surface (12)(13)(14)(15)(16), thereby indicating that they project approximately perpendicularly from the membrane surface. In the case of membrane-bound APC, its active site is located an average of 94 Å above the surface (assuming 2 ϭ 2/3; Ref.…”