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.
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.
François
F
Saller
Bruno O
BO
Villoutreix
Aymeric
A
Amelot
Tahar
T
Kaabache
Bernard F
BF
Le Bonniec
Martine
M
Aiach
Sophie
S
Gandrille
Delphine
D
Borgel
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