Zymogen and activated protein C have similar structural architecture

Abstract

Activated protein C is a trypsin-like protease with anticoagulant and cytoprotective properties that is generated by thrombin from the zymogen precursor protein C in a reaction greatly accelerated by the cofactor thrombomodulin. The molecular details of this activation remain elusive due to the lack of structural information. We now fill this gap by providing information on the overall structural organization of these proteins using single molecule Förster resonance energy transfer and small angle X-ray scattering. Under physiological conditions, both zymogen and protease adopt a conformation with all domains vertically aligned along an axis 76 Å long and maximal particle size of 120 Å. This conformation is stabilized by binding of Ca2+ to the Gla domain and is affected minimally by interaction with thrombin. Hence, the zymogen protein C likely interacts with the thrombin-thrombomodulin complex through a rigid body association that produces a protease with essentially the same structural architecture. This scenario stands in contrast to an analogous reaction in the coagulation cascade where conversion of the zymogen prothrombin to the protease meizothrombin by the prothrombinase complex is linked to a large conformational transition of the entire protein. The presence of rigid EGF domains in protein C as opposed to kringles in prothrombin likely accounts for the different conformational plasticity of the two zymogens. The new structural features reported here for protein C have general relevance to vitamin K-dependent clotting factors containing EGF domains, such as factors VII, IX, and X.

Role of sequence and position of the cleavage sites in prothrombin activation

“…A quick change lightning site-directed mutagenesis kit (Agilent Technologies) was used to introduce substitutions into a plasmid expressing the human prothrombin gene carrying a C-terminal HPC-4 tag. Transfection of baby hamster kidney cells and selection of stably expressing clones were done as described for protein C ( 39 ). Prothrombin secreted in media (6–8 L) collected from CellSTACK culture factories with ten chambers (Corning) was initially purified by immunoaffinity chromatography ( 40 ).…”

Comparative sequence analysis of vitamin K‐dependent coagulation factors

“…The VK‐dependent coagulation proteins of the EGF type are synthesized as inactive zymogens that are activated through proteolytic cleavage at position 15 (the scissile site occupies the P1 position; Table S1 presents chymotrypsin and Human Genome Variation Society [HGVS] 34 numbering). 8 , 9 , 10 , 11 , 12 The position of the activation site is highly conserved among >1300 primary sequences of PC, FVII, FIX, and FX, with the scissile residue being always spaced by exactly 12 amino acids from the N‐terminus of the perfectly conserved P28 in the protease domain (Figures S2 – S5 ). G19 can be used as another marker in locating the position of the scissile site, although this residue is less conserved than P28.…”

“…Protein C, FVII, FIX, and FX are vitamin K (VK)‐dependent coagulation factors whose multidomain architecture comprises an N‐terminal ‐carboxyglutamic acid (Gla) domain that promotes membrane binding, two epidermal growth factor (EGF1 and EGF2) domains that serve as spacers and a serine protease domain that hosts the active site (Figure S1 ). 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 Prothrombin also belongs to the family of VK‐dependent coagulation proteins, but its structure comprises a Gla, kringle 1, kringle 2, and serine protease domains (Figure S1 ). 13 , 14 All VK‐dependent coagulation proteins circulate in plasma as inactive zymogens.…”

Neutrophil, neutrophil extracellular traps and endothelial cell dysfunction in sepsis

“…123 During coagulation, thrombin and PC bind to their receptors, TM and EPCR, respectively, which form a thrombin-TM-PC-EPCR complex on the endothelium. This further contributes to the formation of APC, a proteinase generated by thrombin from the zymogen precursor protein C. 124 This proteinase has many anti-coagulation properties, such as inactivating the procoagulant factors, FVa and FVIIIa and promoting the release of plasminogen activator. 125 As noted above, NETs can activate NF-κB signalling in endothelial cells.…”