Probing prothrombin structure by limited proteolysis

Laura Acquasaliente,Leslie A Pelc,Enrico Di Cera

doi:10.1038/s41598-019-42524-z

Laura Acquasaliente, Leslie A Pelc + Show 1 more

Open Access

https://doi.org/10.1038/s41598-019-42524-z

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Journal: Scientific Reports	Publication Date: Apr 16, 2019
Citations: 19	License type: open-access

Affiliation: Saint Louis University

Abstract

Prothrombin, or coagulation factor II, is a multidomain zymogen precursor of thrombin that undergoes an allosteric equilibrium between two alternative conformations, open and closed, that react differently with the physiological activator prothrombinase. Specifically, the dominant closed form promotes cleavage at R320 and initiates activation along the meizothrombin pathway, whilst the open form promotes cleavage at R271 and initiates activation along the alternative prethrombin-2 pathway. Here we report how key structural features of prothrombin can be monitored by limited proteolysis with chymotrypsin that attacks W468 in the flexible autolysis loop of the protease domain in the open but not the closed form. Perturbation of prothrombin by selective removal of its constituent Gla domain, kringles and linkers reveals their long-range communication and supports a scenario where stabilization of the open form switches the pathway of activation from meizothrombin to prethrombin-2. We also identify R296 in the A chain of the protease domain as a critical link between the allosteric open-closed equilibrium and exposure of the sites of cleavage at R271 and R320. These findings reveal important new details on the molecular basis of prothrombin function.

Highlights

The response of the body to vascular injury entails activation of a cascade of proteolytic events where zymogens are converted into active proteases[1]
Www.nature.com/scientificreports the flexible autolysis loop of the protease domain, as observed for thrombin[17], but the closed form is resistant to proteolysis because of the intramolecular collapse of residue Y93 onto residue W547 shaping the west wall of the active site in the protease domain (Fig. 1)
Limited proteolysis by chymotrypsin reveals that the closed form is resistant to proteolysis, as seen for wild-type, but the open form is readily accessible to cleavage at W468 (Fig. 2)

Summary

Introduction

The response of the body to vascular injury entails activation of a cascade of proteolytic events where zymogens are converted into active proteases[1]. The closed form predominates in solution and features a collapse of kringle-1 into the active site of the protease domain (Fig. 1), with a concomitant preferential exposure of R320 for cleavage by prothrombinase. The open and closed forms initiate activation along the prethrombin-2 or meizothrombin pathways, respectively[16]. The flexible autolysis loop of the protease domain, as observed for thrombin[17], but the closed form is resistant to proteolysis because of the intramolecular collapse of residue Y93 onto residue W547 shaping the west wall of the active site in the protease domain (Fig. 1). The open-closed equilibrium is directly linked to accessibility of the autolysis loop in the protease domain and limited proteolysis by chymotrypsin becomes useful to interrogate individual domains of prothrombin in their contribution to the allosteric equilibrium and mechanism of activation

Methods

Results

Conclusion