The gamma-carboxyglutamic acid domain of anticoagulant protein S is involved in activated protein C cofactor activity, independently of phospholipid binding.
We expressed 2 chimeras between human protein S (PS) and human prothrombin (FII) in which the prothrombin gamma-carboxyglutamic acid (Gla) domain replaced the PS Gla domain in native PS (Gla(FII)-PS) or in PS deleted of the thrombin-sensitive region (TSR) (Gla(FII)-DeltaTSR-PS). Neither PS/FII chimera had activated protein C (APC) cofactor activity in plasma clotting assays or purified systems, but both bound efficiently to phospholipids. This pointed to a direct involvement of the PS Gla domain in APC cofactor activity through molecular interaction with APC. Using computational methods, we identified 2 opposite faces of solvent-exposed residues on the PS Gla domain (designated faces 1 and 2) as potentially involved in this interaction. Their importance was supported by functional characterization of a PS mutant in which the face 1 and face 2 PS residues were reintroduced into Gla(FII)-PS, leading to significant APC cofactor activity, likely through restored interaction with APC. Furthermore, by characterizing PS mutants in which PS face 1 and PS face 2 were individually replaced by the corresponding prothrombin faces, we found that face 1 was necessary for efficient phospholipid binding but that face 2 residues were not strictly required for phospholipid binding and were involved in the interaction with APC.
Saller
François
F
Institut Natioanl de la Santé et de la Recherche Médicale U428, Faculté des Sciences Pharmaceutique et Biologiques, IFR 71 Sciences du Médicament, Université Paris V, France.
Villoutreix
Bruno O
BO
Amelot
Aymeric
A
Kaabache
Tahar
T
Le Bonniec
Bernard F
BF
Aiach
Martine
M
Gandrille
Sophie
S
Borgel
Delphine
D
D015055:1-Carboxyglutamic Acid / Q000378:metabolism*; D001665:Binding Sites; D002460:Cell Line; D006801:Humans; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding; D011486:Protein C / Q000378:metabolism*; D017293:Protein S / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
D010743:Phospholipids; D011486:Protein C; D017293:Protein S; D011993:Recombinant Fusion Proteins; D015055:1-Carboxyglutamic Acid