Identification of the antithrombin III heparin binding site

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Antithrombin III (ATIII) is a crucial anticoagulant protein.
• Previous studies implicated several basic residues in ATIII's interaction with heparin.
• Understanding the precise heparin binding site is essential for elucidating ATIII's mechanism of action.

Purpose of the Study:

• To precisely define the heparin binding site of antithrombin III (ATIII) using alanine scanning mutagenesis.
• To investigate the role of specific basic residues in heparin binding and ATIII activation.
• To provide a structural basis for heparin-induced conformational changes in ATIII.

Main Methods:

• Alanine scanning mutagenesis of 17 basic residues in ATIII.
• Baculovirus expression system for producing recombinant ATIII.
• NaCl gradient elution from heparin columns to determine relative heparin affinities.
• Structural comparisons with related proteins and models.

Main Results:

• A subset of basic residues, not all previously implicated, are critical for heparin binding.
• Key residues (Lys-11, Arg-13, Arg-24, Arg-47, Lys-125, Arg-129, Arg-145) form a channel on the ATIII surface.
• Heparin binding likely disrupts salt bridges, inducing conformational changes essential for ATIII activation.
• Differences in heparin affinity across ATIII conformational states are explained by the identified binding site.

Conclusions:

• The study precisely maps the heparin binding site on antithrombin III.
• Heparin activates ATIII by inducing a conformational change through disruption of stabilizing salt bridges.
• The findings explain the catalytic function of heparin and its release upon complex formation.