RESUMEN
The conformational dynamics of a kinase's activation loop have been challenging to assess due to the activation loop's intrinsic flexibility. To directly probe the conformational equilibrium of the activation loop of mitogen-activated protein kinase p38α, we present an approach based on site-directed spin labeling, electron paramagnetic resonance (EPR) distance restraints, and multilateration. We demonstrate that the activation loop of apo p38α resides in a highly flexible equilibrium state and we reveal that binding of small molecules significantly alters this equilibrium and the populated sub-states.
Asunto(s)
Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Inhibidores de Proteínas Quinasas/metabolismo , Proteína Quinasa 14 Activada por Mitógenos/química , Modelos Moleculares , Unión Proteica , Conformación Proteica , Inhibidores de Proteínas Quinasas/farmacología , Especificidad por SustratoRESUMEN
Conformational transitions in protein kinases are crucial for the biological function of these enzymes. Here, we characterize and assess conformational equilibria of the activation loop and the effect of small molecule inhibitors in the MAP kinase p38α. Our work experimentally revealed the existence of a two-state equilibrium for p38α while the addition of inhibitors shifts the equilibrium between these two states.