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Deciphering Conformational Changes Associated with the Maturation of Thrombin Anion Binding Exosite I

Author:
Billur, Ramya, Ban, David, Sabo, T. Michael, Maurer, Muriel C.
Source:
Biochemistry 2017 v.56 no.48 pp. 6343-6354
ISSN:
1520-4995
Subject:
crystal structure, crystallography, hydrophobicity, ligands, nuclear magnetic resonance spectroscopy, peptides, platelet activation, prothrombin, stable isotopes, thrombin
Abstract:
Thrombin participates in procoagulation, anticoagulation, and platelet activation. This enzyme contains anion binding exosites, ABE I and ABE II, which attract regulatory biomolecules. As prothrombin is activated to thrombin, pro-ABE I is converted into mature ABE I. Unexpectedly, certain ligands can bind to pro-ABE I specifically. Moreover, knowledge of changes in conformation and affinity that occur at the individual residue level as pro-ABE I is converted to ABE I is lacking. Such changes are transient and were not captured by crystallography. Therefore, we employed nuclear magnetic resonance (NMR) titrations to monitor development of ABE I using peptides based on protease-activated receptor 3 (PAR3). Proton line broadening NMR revealed that PAR3 (44–56) and more weakly binding PAR3G (44–56) could already interact with pro-ABE I on prothrombin. ¹H–¹⁵N heteronuclear single-quantum coherence NMR titrations were then used to probe binding of individual ¹⁵N-labeled PAR3G residues (F47, E48, L52, and D54). PAR3G E48 and D54 could interact electrostatically with prothrombin and tightened upon thrombin maturation. The higher affinity for PAR3G D54 suggests the region surrounding thrombin R77a is better oriented to bind D54 than the interaction between PAR3G E48 and thrombin R75. Aromatic PAR3G F47 and aliphatic L52 both reported on significant changes in the chemical environment upon conversion of prothrombin to thrombin. The ABE I region surrounding the 30s loop was more affected than the hydrophobic pocket (F34, L65, and I82). Our NMR titrations demonstrate that PAR3 residues document structural rearrangements occurring during exosite maturation that are missed by reported X-ray crystal structures.
Agid:
5864873