Opposing Intermolecular Tuning of Ca2+ Affinity for Calmodulin by Neurogranin and CaMKII Peptides

Abstract

We investigated the impact of bound calmodulin (CaM)-target compound structure on the affinity of calcium (Ca²⁺) by integrating coarse-grained models and all-atomistic simulations with nonequilibrium physics. We focused on binding between CaM and two specific targets, Ca²⁺/CaM-dependent protein kinase II (CaMKII) and neurogranin (Ng), as they both regulate CaM-dependent Ca²⁺ signaling pathways in neurons. It was shown experimentally that Ca²⁺/CaM (holoCaM) binds to the CaMKII peptide with overwhelmingly higher affinity than Ca²⁺-free CaM (apoCaM); the binding of CaMKII peptide to CaM in return increases the Ca²⁺ affinity for CaM. However, this reciprocal relation was not observed in the Ng peptide (Ng_13-49), which binds to apoCaM or holoCaM with binding affinities of the same order of magnitude. Unlike the holoCaM-CaMKII peptide, whose structure can be determined by crystallography, the structural description of the apoCaM-Ng_13-49 is unknown due to low binding affinity, therefore we computationally generated an ensemble of apoCaM-Ng_13-49 structures by matching the changes in the chemical shifts of CaM upon Ng_13-49 binding from nuclear magnetic resonance experiments. Next, we computed the changes in Ca²⁺ affinity for CaM with and without binding targets in atomistic models using Jarzynski's equality. We discovered the molecular underpinnings of lowered affinity of Ca²⁺ for CaM in the presence of Ng_13-49 by showing that the N-terminal acidic region of Ng peptide pries open the β-sheet structure between the Ca²⁺ binding loops particularly at C-domain of CaM, enabling Ca²⁺ release. In contrast, CaMKII peptide increases Ca²⁺ affinity for the C-domain of CaM by stabilizing the two Ca²⁺ binding loops. We speculate that the distinctive structural difference in the bound complexes of apoCaM-Ng_13-49 and holoCaM-CaMKII delineates the importance of CaM's progressive mechanism of target binding on its Ca²⁺ binding affinities.