Dissertation

Thrombomodulin Binding to Thrombin Reveals the Backbone Dynamics Required for the Serine Protease Catalytic Mechanism

Peacock, Riley Bryant

2020

Overview

The serine protease thrombin plays an essential regulatory role in blood clotting. Prothrombin is converted to the active α-thrombin in response to tissue damage, allowing the enzyme to cleave procoagulative substrates such as fibrinogen and PAR, enabling the formation of the clot. On the other hand, when thrombin binds its protein cofactor thrombomodulin (TM), the enzyme loses substrate specificity for procoagulative substrates, and engages the anticoagulative pathway by selectively targeting the substrate protein C for proteolytic activation. Despite decades of study, the mechanism by which TM enables thrombin to cleave protein C has yet to be fully characterized, indicating a need for further investigation. Crystallographic evidence shows no notable difference between the thrombin and thrombin-TM structures, suggesting the influence of TM is dynamic in nature. The work presented in this thesis uses two biophysical techniques, hydrogen-deuterium exchange mass spectrometry (HDXMS) and nuclear magnetic resonance spectrometry (NMR) to report the effects of TM on thrombin dynamics.In Chapter II, HDXMS and NMR were used to evaluate the regions of thrombin effected by the presence of TM. The results show two paths of communication between the active site and TM binding site of thrombin, as well as large scale conformational changes throughout all of thrombin. From these data we generated a model of the dynamic influence of TM on thrombin that describes the global protein dynamics necessary for the execution of the serine protease catalytic mechanism. Chapter III presents a study of the dynamics of the W215A thrombin mutant using HDXMS. Previous studies have shown that mutations at Trp 215 impart dramatic changes to the substrate specificity of thrombin, and the W215A mutation appeared to favor protein C activation over procoagulative substrate activation. We found that alterations to the hydrophobic environment at and around Trp 215 increased the dynamics of the surrounding thrombin loops, as well as the N-terminus of the thrombin heavy chain over 10 Å away from the sites of mutation.In Chapter IV, the dynamics of the W215A/E217A mutant was investigated in the presence and absence of TM through HDXMS. The W215A/E217A mutant heavily prefers protein C over procoagulative thrombin substrates, causing us to ask whether TM binding recovers the destabilizing effects of these mutations. We found that TM was capable of stabilizing parts of the W21A/E217A thrombin mutant, but could not recover the increased dynamics of the primary substrate recognition pocket. These results suggest that the primary cause of the decreased activity of the W215A/E217A mutant towards procoagulative substrates results from the mutation of Trp 215, which is not important recognition element for protein C binding but is so for procoagulative substrates.

• Publisher: ProQuest Dissertations & Theses
• Subject: Biochemistry / Biophysics / Molecular biology
• ISBN: 9798557069595