Solvent accessibility of the thrombin-thrombomodulin interface.

Area of Science:

• Biochemistry
• Structural Biology
• Protein Interactions

Background:

• Thrombin plays a critical role in hemostasis and thrombosis.
• Thrombomodulin (TM) modulates thrombin's activity, regulating coagulation.
• Understanding the structural basis of thrombin-TM interaction is crucial for therapeutic development.

Purpose of the Study:

• To investigate the kinetics of solvent accessibility at the protein-protein interface between thrombin and thrombomodulin epidermal growth factor-like domain 45 (TMEGF45).
• To characterize the dynamic changes in thrombin's structure upon binding to TMEGF45.
• To correlate structural changes with functional implications in thrombin activity.

Main Methods:

• Amide hydrogen/deuterium (H/2H) exchange monitored by MALDI-TOF mass spectrometry to assess solvent accessibility.
• Surface plasmon resonance (SPR) to measure association and dissociation rate constants.
• H/2H exchange rate measurements at varying pH and TMEGF45 concentrations.

Main Results:

• Thrombin-TMEGF45 interaction is rapid and reversible.
• Binding of TMEGF45 renders two thrombin segments, part of anion-binding exosite I, completely solvent-inaccessible.
• Other thrombin regions show partial solvent-inaccessibility, with pH-dependent exchange rates, suggesting allosteric modulation.

Conclusions:

• TMEGF45 binding induces significant, localized structural changes in thrombin, particularly at anion-binding exosite I.
• These structural alterations are linked to functional modulation and allosteric effects.
• The study provides insights into the dynamic interface of thrombin-thrombomodulin complex formation.