NMR reveals a dynamic allosteric pathway in thrombin.

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Serine proteases, the largest peptidase family, have ubiquitous roles but their dynamic motions are not fully understood.
• Thrombin, a key coagulation serine protease, plays critical roles in hemostasis and thrombosis.
• Understanding thrombin's dynamics is crucial for comprehending its allosteric regulation and developing targeted therapies.

Purpose of the Study:

• To investigate and compare the backbone dynamics of apo-thrombin (substrate-free) and substrate-bound thrombin (PPACK-thrombin).
• To elucidate the role of protein dynamics in the allosteric regulation of thrombin's active site and effector binding sites.
• To identify specific regions and pathways involved in allosteric communication within thrombin.

Main Methods:

• Nuclear Magnetic Resonance (NMR) spectroscopy, including R1, R2, 15N-{1H}NOEs, and relaxation dispersion experiments.
• Measurement of protein dynamics across the picosecond (ps) to millisecond (ms) timescale.
• Comparison of dynamic properties between the apo- and substrate-bound forms of thrombin.

Main Results:

• Picosecond-nanosecond (ps-ns) motions were largely unaffected by substrate binding.
• Apo-thrombin exhibited significant microsecond-millisecond (μs-ms) motions throughout the molecule.
• Substrate binding reduced dynamics in active site loops, but specific regions (N-terminal heavy chain, γ-loop, anion-binding exosite 1) retained μs-ms motions, forming a dynamic allosteric pathway.

Conclusions:

• Thrombin's dynamics are significantly altered by substrate binding, particularly in regions involved in allosteric regulation.
• A dynamic allosteric pathway connecting the active site to anion-binding exosite 1 persists even in the substrate-bound form.
• These findings provide insights into the mechanisms of thrombin allostery and its functional regulation.