Role of the I16-D194 ionic interaction in the trypsin fold

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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.

Section: Methodsmentioning

confidence: 99%

Zymogen and activated protein C have similar structural architecture

Stojanovski

Pelc

Zuo

et al. 2020

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“…Our understanding of the conformational nature of trypsinlike proteases and their zymogens has been deeply influenced by the celebrated Huber-Bode mechanism of zymogen activation (4). Activity is assumed to result from proteolytic cleavage of a conserved Arg residue in the activation domain followed by an ionic interaction that is established between the new N terminus of Ile 16 and the side chain of the highly conserved Asp 194 . The newly formed H-bond between Ile 16 and Asp 194 organizes the oxyanion hole around Gly 193 and the catalytic Ser 195 and the primary specificity pocket around Asp 189 .…”

Section: Discussionmentioning

confidence: 99%

“…Activity is assumed to result from proteolytic cleavage of a conserved Arg residue in the activation domain followed by an ionic interaction that is established between the new N terminus of Ile 16 and the side chain of the highly conserved Asp 194 . The newly formed H-bond between Ile 16 and Asp 194 organizes the oxyanion hole around Gly 193 and the catalytic Ser 195 and the primary specificity pocket around Asp 189 . This transition, however, is neither necessary nor sufficient to generate a fully active protease.…”

Section: Discussionmentioning

confidence: 99%

“…In the E* form, substrate cannot bind to the active site, and catalysis is impeded. Importantly, the balance between E* and E changes between zymogen and protease, with the E* form pre-dominating in the former (15,16) and presaging little overlap between the free forms of protease and its zymogen precursor.…”

Section: Discussionmentioning

confidence: 99%

“…The E* form prevails in the zymogen and gradually shifts to the E form during transition to the mature protease (15). In fact, the E*-E equilibrium complements the Huber-Bode mechanism and contributes to organization of the active site for efficient binding and catalysis (16).…”

mentioning

confidence: 99%

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19F NMR reveals the conformational properties of free thrombin and its zymogen precursor prethrombin-2

Ruben

Gandhi

Chen

et al. 2020

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The conformational properties of trypsin-like proteases and their zymogen forms remain controversial because of a lack of sufficient information on their free forms. Specifically, it is unclear whether the free protease is zymogen-like and shifts to its mature form upon a ligand-induced fit or exists in multiple conformations in equilibrium from which the ligand selects the optimal fit via conformational selection. Here we report the results of 19F NMR measurements that reveal the conformational properties of a protease and its zymogen precursor in the free form. Using the trypsin-like, clotting protease thrombin as a relevant model system, we show that its conformation is quite different from that of its direct zymogen precursor prethrombin-2 and more similar to that of its fully active Na+-bound form. The results cast doubts on recent hypotheses that free thrombin is zymogen-like and transitions to protease-like forms upon ligand binding. Rather, they validate the scenario emerged from previous findings of X-ray crystallography and rapid kinetics supporting a pre-existing equilibrium between open (E) and closed (E*) forms of the active site. In this scenario, prethrombin-2 is more dynamic and exists predominantly in the E* form, whereas thrombin is more rigid and exists predominantly in the E form. Ligand binding to thrombin takes place exclusively in the E form without significant changes in the overall conformation. In summary, these results disclose the structural architecture of the free forms of thrombin and prethrombin-2, consistent with an E*–E equilibrium and providing no evidence that free thrombin is zymogen-like.

The active site region plays a critical role in Na+ binding to thrombin

Pelc

Koester

Kukla

et al. 2022

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