Heparin Binds Lamprey Angiotensinogen and Promotes Thrombin Inhibition through a Template Mechanism

Area of Science:

• Biochemistry
• Structural Biology
• Evolutionary Biology

Background:

• Lamprey angiotensinogen (l-ANT) functions as a hormone carrier and a heparin-dependent thrombin inhibitor.
• The precise mechanisms of angiotensin transport and heparin-mediated thrombin inhibition by l-ANT are not fully understood.

Purpose of the Study:

• To elucidate the crystal structure of cleaved l-ANT.
• To characterize l-ANT's heparin binding and protease inhibition properties.
• To investigate the role of heparin in mediating thrombin inhibition by l-ANT.

Main Methods:

• X-ray crystallography to determine the 2.7 Å structure of cleaved l-ANT.
• Biochemical assays to measure heparin binding affinity (dissociation constant) and thrombin inhibition kinetics.
• Site-directed mutagenesis to create an l-ANT mutant (P1 Ile to Arg).

Main Results:

• The crystal structure revealed a conserved serpin fold with a labile N-terminal angiotensin peptide.
• Heparin binds tightly to a positively charged surface around helix D (K_d ≈ 10 nm).
• High molecular weight heparin significantly accelerates l-ANT's thrombin inhibition (90-fold), with shorter chains being ineffective.
• A P1 mutant l-ANT showed greatly enhanced thrombin inhibition, further boosted by heparin.

Conclusions:

• Heparin binds to a conserved site on l-ANT, promoting thrombin interaction via a template mechanism.
• This heparin-mediated inhibition mechanism is conserved in vertebrates.
• Structural and functional insights into l-ANT provide a basis for understanding angiotensinogen function and serpin evolution.