Backbone resonance assignments of complexes of human voltage-dependent sodium channel NaV1.2 IQ motif peptide bound to apo calmodulin and to the C-domain fragment of apo calmodulin

Abstract

Human voltage-gated sodium channel NaV1.2 has a single pore-forming α-subunit and two transmembrane β-subunits. Expressed primarily in the brain, NaV1.2 is critical for initiation and propagation of action potentials. Milliseconds after the pore opens, sodium influx is terminated by inactivation processes mediated by regulatory proteins including calmodulin (CaM). Both calcium-free (apo) CaM and calcium-saturated CaM bind tightly to an IQ motif in the C-terminal tail of the α-subunit. Our thermodynamic studies and solution structure (2KXW) of a C-domain fragment of apo 13C,15N-CaM (CaMC) bound to an unlabeled peptide with the sequence of rat NaV1.2 IQ motif showed that apo CaMC (a) was necessary and sufficient for binding, and (b) bound more favorably than calcium-saturated CaMC. However, we could not monitor the NaV1.2 residues directly, and no structure of full-length CaM (including the N-domain of CaM (CaMN)) was determined. To distinguish contributions of CaMN and CaMC, we used solution NMR spectroscopy to assign the backbone resonances of a complex containing a 13C,15N-labeled peptide with the sequence of human NaV1.2 IQ motif (NaV1.2IQp) bound to apo 13C,15N-CaM or apo 13C,15N-CaMC. Comparing the assignments of apo CaM in complex with NaV1.2IQp to those of free apo CaM showed that residues within CaMC were significantly perturbed, while residues within CaMN were essentially unchanged. The chemical shifts of residues in NaV1.2IQp and in the C-domain of CaM were nearly identical regardless of whether CaMN was covalently linked to CaMC. This suggests that CaMN does not influence apo CaM binding to NaV1.2IQp.

Backbone resonance assignments of complexes of apo human calmodulin bound to IQ motif peptides of voltage-dependent sodium channels NaV1.1, NaV1.4 and NaV1.7

“…Crosspeaks were not observed for the first two residues (A1 and D2) in all complexes. The majority of the other missing resonances correspond to residues in calcium-binding site IV and flanking regions, consistent with previous findings from solution NMR studies of apo CaM complexes with Na V 1.2 and Na V 1.5 IQ motifs (Feldkamp, Yu, and Shea 2011; Chagot and Chazin 2011; Mahling, Kilpatrick, and Shea 2017). The backbone 1 H N and 15 N assignments of non-proline residues in the IQ motif peptides are 97% complete in the apo CaM+Na V 1.4 IQp , and apo CaM+Na V 1.1 IQp complexes, missing only the first residue [G(−4)] in the GPGS tag, and 94% complete in the apo CaM+Na V 1.1 IQp complex, missing G(−4) and G(−2) in the GPGS tag.…”

“…4A, 4B, 4C) using 1 H N , 15 N, 13 Cα, 13 Cβ, and 13 C’ chemical shifts (Shen et al 2009). The values predicted for apo CaM were similar to those observed in the apo CaM+Na V 1.2 IQp complex (Mahling, Kilpatrick, and Shea 2017), and agree well with previously determined high-resolution structures of free apo CaM such as 1CFD (Kuboniwa et al 1995) and apo CaM in complex with the Na V 1.5 IQ motif (Chagot and Chazin 2011). A colored bar above Fig.…”

“…4G, 4H, and 4I, show that, in all three complexes, resonances for CaM C residues underwent large chemical shift perturbations upon peptide binding, with the largest perturbations observed in the loop connecting the two EF-hands of CaM C . The large and numerous CSPs in CaM C , and the very small CSPs in CaM N (judged to be at the level of noise), demonstrate that apo CaM binds to the Na V 1.1, Na V 1.4, and Na V 1.7 IQ motifs exclusively through CaM C , as was observed previously in NMR studies of apo CaM bound to an IQ motif of Na V 1.2 IQ motif (Mahling, Kilpatrick, and Shea 2017), 2KXW (Feldkamp, Yu, and Shea 2011)) and Na V 1.5 (Chagot and Chazin 2011). …”

“…Our previous thermodynamic and solution NMR studies showed that both calcium-depleted (apo) CaM and calcium-saturated CaM bind the IQ motif of Na V 1.2 with high affinity, and that CaM C was necessary and sufficient for binding in the absence or the presence of calcium (Theoharis et al 2008; Feldkamp, Yu, and Shea 2011; Hovey et al 2017; Mahling, Kilpatrick, and Shea 2017). CaM N did not influence CaM binding to the Na V 1.2 IQ motif, and it retained structural and energetic properties of the free domain.…”

Effect of Ca2+on the promiscuous target-protein binding mechanism of calmodulin

“…Key hydrophobic residues found in this paper's analysis are highlighted in different colors. The residues are shown in S3 Fig and S1 Fig. favorable than holo-CaM binding [8,9].…”