Zymogen and activated protein C have similar structural architecture

Leslie A Pelc,Xiaobing Zuo,Bosko M Stojanovski,Enrico Di Cera

doi:10.1074/jbc.ra120.014789

Leslie A Pelc, Xiaobing Zuo + Show 2 more

Open Access

https://doi.org/10.1074/jbc.ra120.014789

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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 FRET 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 epidermal growth factor 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 epidermal growth factor domains, such as factors VII, IX, and X.

Highlights

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
Results single molecule FRET (smFRET) and small angle X-ray scattering (SAXS) studies smFRET measurements with protein C and Activated protein C (APC) labeled with the AF555/AF647 FRET pair at positions C12/C312 across the Gla and protease domains (Fig. 1A) were carried out to evaluate the overall conformational properties of the two proteins
No perturbation was detected for APC as established from the catalytic activity of the labeled protein compared with the unlabeled protein (Fig. 1C). smFRET measurements reveal an interprobe distance between the FRET pair that does not change between the zymogen protein C and protease APC

Summary

Introduction

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. We fill this gap by providing information on the overall structural organization of these proteins using single molecule FRET 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. We use single molecule FRET (smFRET) and small angle X-ray scattering (SAXS) to probe the structural architecture of the zymogen protein C and the protease APC We find that both proteins assume a nearly identical conformation in solution, with the constitutive domains vertically stacked in a linear arrangement that does not change significantly upon binding of thrombin

Methods

Results

Conclusion