Thrombomodulin Binding Selects the Catalytically Active Form of Thrombin

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Chemistry

Background:

• Human alpha-thrombin is a serine protease with dual procoagulant and anticoagulant functions.
• Thrombomodulin (TM) binding switches thrombin's function to anticoagulant by enabling protein C cleavage.
• A soluble, active TM fragment (TM456m) has been developed for studying thrombin-TM interactions.

Purpose of the Study:

• To investigate conformational changes in human alpha-thrombin upon binding to TM456m or PPACK.
• To compare the structural states of apo-thrombin, PPACK-bound thrombin, and TM456m-bound thrombin.

Main Methods:

• Utilized hydrogen-deuterium exchange mass spectrometry (HDX-MS) to monitor backbone amide dynamics.
• Analyzed deuterium uptake in human alpha-thrombin across three states: unbound (apo), bound to PPACK, and bound to TM456m.

Main Results:

• Both PPACK and TM456m binding reduced amide exchange at their respective binding sites.
• A significant decrease in amide exchange was observed in the gamma-loop and heavy chain N-terminus upon binding.
• HDX-MS data suggest the heavy chain N-terminus is not fully inserted in apo-thrombin, unlike in crystal structures.

Conclusions:

• Thrombin's catalytically active conformation may not be present in the unbound state.
• TM binding allosterically induces the catalytically active conformation of thrombin.
• HDX-MS provides insights into dynamic structural changes not evident in static crystal structures.