Estudios de mutagénesis para comprender la alostería de la trombina

The binding of thrombomodulin (TM) to exosite-1 and the binding of Na(+) to 225-loop allosterically modulate the catalytic activity and substrate specificity of thrombin. To determine whether the conformation of these two cofactor-binding loops are energetically linked to each other and to the active site, we rationally designed two thrombin mutants in which either the 70-80 loop of exosite-1 or the 225-loop of the Na(+)-binding site was stabilized by an engineered disulfide bond. This was possible by replacing two residues, Arg-67 and Ile-82, in the first mutant and two residues, Glu-217 and Lys-224, in the second mutant with Cys residues. These mutants were expressed in mammalian cells as monomeric molecules, purified to homogeneity and characterized with respect to their ability to bind TM and Na(+) by kinetic and direct binding approaches. The Cys-67/Cys-82 mutant did not bind TM and exhibited a normal amidolytic activity, however, the activity of Cys-217/Cys-224 was dramatically impaired, though TM interacted with this mutant with >20-fold elevated K(D) to partially restore its activity. Both mutants exhibited approximately 2-3-fold higher K(D) for interaction with Na(+), and neither mutant clotted fibrinogen or activated protein C in the presence of TM. Both mutants interacted with heparin with a normal affinity. These results suggest that, while exosite-2 of thrombin is an independent cofactor binding-site, both Na(+)-binding and exosite-1 are energetically linked. Further studies with the fluorescein labeled Cys-195 mutant of thrombin revealed that the catalytic residue of thrombin is modulated by Na(+), but TM has no effect on the conformation of this residue.

Resumen tomado de PubMed (NCBI) para el registro citado, en el idioma original. La versión de referencia es la de la publicación original.

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Se diseñaron dos mutantes de trombina con un disulfuro artificial que estabiliza el asa 70–80 del exosito 1 o el asa 225 de unión al Na+. Cys-67/Cys-82 no unió trombomodulina y conservó actividad amidolítica normal; la de Cys-217/Cys-224 estaba muy afectada y la trombomodulina, con una K(D) más de 20 veces mayor, la restauró parcialmente. Ambos presentaron una K(D) para Na+ aproximadamente 2–3 veces mayor. Los resultados sugieren un acoplamiento energético entre la unión al Na+ y el exosito 1.

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