Biophysical analysis of the dynamics of calmodulin interactions with neurogranin and Ca2+/calmodulin-dependent kinase II

Abstract

Calmodulin (CaM) functions depend on interactions with CaM-binding proteins, regulated by Ca2+. 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 Ca2+/calmodulin-dependent kinase II (CaMKII290−309) have been studied using biophysical methods. These proteins have opposite Ca2+ dependencies for CaM binding. Surface plasmon resonance biosensor analysis confirmed that Ca2+ and CaM interact very rapidly, and with moderate affinity (KSPRD = 3μM). Calmodulin-CaMKII290−309 interactions were only detected in the presence of Ca2+, exhibiting fast kinetics and nanomolar affinity (KSPRD = 7.1nM). The CaM–Ng interaction had higher affinity under Ca2+ -depleted (KSPRD = 480 nM, k1=3.4×105M−1S−1 and k−1 = 1.6 × 10−1s−1) than Ca2+ -saturated conditions (KSPRD = 19 μM). The IQ motif of Ng (Ng27−50) had similar affinity for CaM as Ng under Ca2+ -saturated conditions (KSPRD = 14μM), but no interaction was seen under Ca2+ -depleted conditions. Microscale thermophoresis using fluorescently labeled CaM confirmed the surface plasmon resonance results qualitatively, but estimated lower affinities for the Ng (KMSTD = 890 nM) and CaMKII290−309(KMSTD = 190 nM) interactions. Although CaMKII290−309 showed expected interaction characteristics, they may be different for full-length CaMKII. The data for full-length Ng, but not Ng27−50, agree with the current model on Ng regulation of Ca2+ /CaM signaling.