Mutagenesis studies toward understanding allostery in thrombin.
Research article published in Biochemistry (2009)
Abstract
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.
Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.
Резюме
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...
Почему это важно для гирудотерапии
This study used rationally designed thrombin mutants with engineered disulfide bonds to probe whether exosite-1 (thrombomodulin binding), the Na+-binding 225-loop, and the active site are energetically linked, finding that Na+-binding and exosite-1 are conformationally coupled while exosite-2 (heparin binding) functions independently. This work is indirectly relevant to ASH's domain because thrombin is the primary target of hirudin from the leech secretome, and the allosteric communication between exosite-1 and the active site directly informs how hirudin engages thrombin's fibrinogen recognition site. The caveat is that the study involves no leech-derived molecules, hirudin, or secretome components whatsoever — it is a protein engineering/kinetics study using mammalian cell–expressed mutants — and the connection to hirudotherapy is purely conceptual through the shared target thrombin.
Цитирование
Mutagenesis studies toward understanding allostery in thrombin.
Qureshi et al. · Biochemistry, 2009
Связанный клинический контекст
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