Crystal structure of thrombin-ecotin reveals conformational changes and extended interactions

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biology

Background:

• Ecotin is a serine protease inhibitor with broad specificity.
• Wild-type ecotin exhibits weak inhibition of thrombin.
• Thrombin is a key serine protease involved in blood coagulation.

Purpose of the Study:

• To investigate the structural basis for the enhanced inhibition of thrombin by a mutated ecotin.
• To determine the binding interactions between thrombin and the ecotin M84R mutant.
• To understand how structural changes in thrombin accommodate the inhibitor.

Main Methods:

• Site-directed mutagenesis to create the ecotin M84R mutant.
• Crystallography to determine the complex structure of bovine thrombin with ecotin M84R at 2.5 A resolution.
• Analysis of structural changes in thrombin's active site and surface loops.

Main Results:

• The ecotin M84R mutant displays a 10(4)-fold stronger affinity for thrombin (1.5 nM) compared to wild-type ecotin.
• The crystal structure reveals significant conformational changes in thrombin's surface loops surrounding the active site cleft.
• Specific interactions include polar and nonpolar contacts between thrombin's C-terminus/residue 99 loop and ecotin, and a defined preference for Val at the P4 position of the inhibitor.

Conclusions:

• The M84R mutation dramatically enhances ecotin's affinity for thrombin.
• Structural rearrangements in thrombin, particularly at insertion loops 60 and 148, are crucial for accommodating the M84R inhibitor.
• The findings provide insights into thrombin's substrate specificity and the design of potent protease inhibitors.