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.

Figure 1

Illustration of structures of CaM and sequences of the target peptides. (a) Solution structure of Ca²⁺-free CaM (apoCaM) (PDB: 1CFD) and crystal structure of Ca²⁺/CaM (holoCaM) (PDB: 1CLL). CaM is colored as follows, red → nCaM (residue 1–76), gray → central linker (residue 77–82), blue → cCaM (residue 83–148), and the Ca²⁺ ions are colored in green; Ca²⁺ binding sites are labeled from I to IV. (b) Structure of Ca²⁺/CaM-dependent kinase II (CaMKII) peptide from the holoCaM-CaMKII crystal structure (PDB: 1CDM) and the sequence of the peptide. (c) X-ray structure of holoCaM-CaMKII (PDB: 1CDM). CaM and the Ca²⁺ ions are colored as in (a) and the CaMKII peptide is colored in green. (d) Sequence of the neurogranin peptide (Ng_13–49). The two minimum compositions of the Ng_13–49 peptide are marked as “acidic region” and “IQ motif.” To see this figure in color, go online.

Figure 2

Reweighted PMF of apoCaM-Ng_13–49 binding and changes in chemical shifts for apoCaM upon Ng_13–49 binding. (a and b) The residues that experienced significant changes in chemical shifts are projected on the apoCaM structure (PDB: 1CFD) as a ball-and-stick representation from experimental measurement in (a) (30) and from our calculations according to the coarse-grained molecular simulations in (b). (c) The PMF from the coarse-grained molecular simulations is plotted against the center of mass distance between apoCaM and Ng_13–49 (d_COM) and the number of intermolecular contacts (Z). The coarse-grained molecular simulations were performed at pH = 6.3 and ionic strength = 0.1 M according to the conditions of the nuclear magnetic resonance experiments (30). We used a reduced unit of length σ = 3.8Å. The color is scaled in k_BT, T = 1.1 ε/k_B, where ε is reduced unit of energy and k^B is Boltzmann constant. For visual guidance, we provided bars above the schematic representation of secondary structures in red and blue segments of apoCaM on the half circles in (a and b). To see this figure in color, go online.

Figure 3

Illustration of the structural changes in unbound, encounter, and bound ensemble of CaM-Ng_13–49 complexes. Structures of CaM in (a) was taken from the unbound state when CaM and Ng_13–49 are well separated at d_COM = 20.0 σ. σ = 3.8 Å. (b) Structures from the encounter of binding when apoCaM and Ng is separated at d_COM = 10.0 σ. (c) Structures from the bound state at d_COM = 2.8 σ. For visual guidance, we superposed 20 sets of structures in each panel. The CaM is colored in black; the residues (residues 99∼101 in Ca²⁺ binding loop III and residues 135∼137 in Ca²⁺ binding loop IV), which form EF-hand β-scaffold in cCaM are colored in blue; the acidic region and IQ motif of Ng_13–49 are colored in pink and yellow, respectively. To see this figure in color, go online.

Figure 4

Illustration of the definitions of the binding free energy ΔG and ΔΔG. ΔG = G_B − G_U. ΔΔG = ΔG^{holoCaM-CaMBT} − ΔG^holoCaM. B and U stand for bound and unbound states of the Ca²⁺, respectively. ΔΔG >0 means that the CaMBT destabilizes the bound state and thus decreases the Ca²⁺ affinity.

Figure 5

Differential intermolecular interactions in holoCaM-Ng_13–49 and holoCaM-CaMKII complex structure tune Ca²⁺ binding affinity for cCaM. (a) The atomistic structure of holoCaM-Ng_13–49 complex was reconstructed from the coarse-grained simulations that led to the largest decrease in Ca²⁺ affinity for site III and site IV. (b) The structure of holoCaM-CaMKII complex, showing a wrap-around binding pattern, is from the X-ray crystallography (PDB: 1CDM). The complete structures of the complexes are on the left, CaM is in black, Ca²⁺ ions are represented by yellow beads, and the CaMBTs are in white. The sequences of the CaMBTs are provided above the figures. For visual guidance, the cCaM and the CaMBTs are enlarged and recolored on the right. The cCaM is in ribbon representation and colored in orange. The residues forming EF-hand β-scaffold in the crystal structure, i.e., residues Y99, I100 from site III, and residues Q135, V136 from site IV, are colored in blue. The invariant Glu residue in the 12th position of each Ca²⁺-binding loop contributes two oxygen atoms to the coordination of the Ca²⁺ ion and is shown in ball-and-stick representation. The dotted lines show the distance between Ca²⁺ and the two oxygen atoms from Glu12 and the distance between the two β-strands in cCaM. The four residues that stick out from Ng_13–49 are D22 (red) D23 (red) P24 (green) G25 (magenta) in (a).The residues from CaM, which form contacts with CaMKII peptides, are colored in light green in (b). To see this figure in color, go online.