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Deciphering Conformational Changes Associated with the Maturation of Thrombin Anion Binding Exosite I
Ramya Billur1, David Ban2, T Michael Sabo2
1Department of Chemistry, University of Louisville , Louisville, Kentucky 40292, United States.
Biochemistry
|November 8, 2017
Summary
Nuclear magnetic resonance (NMR) revealed that protease-activated receptor 3 (PAR3) peptides bind to prothrombin
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Thrombin, a key enzyme in hemostasis, possesses anion binding exosites (ABE I and ABE II) crucial for regulating biomolecule interactions.
- The conversion of prothrombin to thrombin involves the maturation of pro-ABE I into ABE I, a process with incompletely understood conformational and affinity changes.
Purpose of the Study:
- To investigate the transient structural rearrangements and binding dynamics during the maturation of pro-thrombin's ABE I exosite.
- To characterize the interaction of protease-activated receptor 3 (PAR3) peptides with both pro-thrombin and thrombin using nuclear magnetic resonance (NMR).
Main Methods:
- Proton line broadening NMR to assess the interaction of PAR3 (44-56) and PAR3G (44-56) peptides with pro-thrombin.
- 1H-15N heteronuclear single-quantum coherence NMR titrations to probe the binding of individual 15N-labeled PAR3G residues (F47, E48, L52, D54) to prothrombin and thrombin.
Main Results:
- PAR3 (44-56) and PAR3G (44-56) peptides were found to bind to pro-ABE I on prothrombin.
- PAR3G residues E48 and D54 exhibited electrostatic interactions with prothrombin, which strengthened upon thrombin maturation.
- NMR data indicated significant chemical environment changes for PAR3G residues F47 and L52 during prothrombin to thrombin conversion, with the 30s loop region being more affected than the hydrophobic pocket.
Conclusions:
- NMR titrations provide insights into the dynamic structural changes during ABE I exosite maturation, capturing transient events missed by crystallography.
- PAR3 residues serve as valuable reporters of the structural rearrangements occurring during thrombin exosite development.
- The findings elucidate the detailed molecular interactions and conformational shifts involved in thrombin's regulatory functions.
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