Biophysical analysis of the dynamics of calmodulin interactions with neurogranin and Ca 2+ 2+ /calmodulin‐dependent kinase II

摘要

Abstract Calmodulin (CaM) functions depend on interactions with CaM‐binding proteins, regulated by Ca 2 + . Induced structural changes influence the affinity, kinetics, and specificities of the interactions. The dynamics of CaM interactions with neurogranin (Ng) and the CaM‐binding region of Ca 2 + /calmodulin‐dependent kinase II (CaMKII 290?309 ) have been studied using biophysical methods. These proteins have opposite Ca 2 + dependencies for CaM binding. Surface plasmon resonance biosensor analysis confirmed that Ca 2 + and CaM interact very rapidly, and with moderate affinity ( K D S P R = 3 μ M ). Calmodulin‐CaMKII 290?309 interactions were only detected in the presence of Ca 2 + , exhibiting fast kinetics and nanomolar affinity ( K D S P R = 7 . 1 n M ). The CaM–Ng interaction had higher affinity under Ca 2 + ‐depleted ( K D S P R = 4 8 0 n M , k 1 = 3 . 4 × 1 0 5 M ? 1 s ? 1 and k ?1 = 1.6 × 10 ?1 s ?1 ) than Ca 2 + ‐saturated conditions ( K D S P R = 1 9 μ M ). The IQ motif of Ng (Ng 27?50 ) had similar affinity for CaM as Ng under Ca 2 + ‐saturated conditions ( K D S P R = 14 μ M ), but no interaction was seen under Ca 2 + ‐depleted conditions. Microscale thermophoresis using fluorescently labeled CaM confirmed the surface plasmon resonance results qualitatively, but estimated lower affinities for the Ng ( K D M S T = 8 9 0 n M ) and CaMKII 290?309 ( K D M S T = 1 9 0 n M ) interactions. Although CaMKII 290?309 showed expected interaction characteristics, they may be different for full‐length CaMKII. The data for full‐length Ng, but not Ng 27?50 , agree with the current model on Ng regulation of Ca 2 + /CaM signaling.