Molecular interactions of the NaV1.5 C-terminal domain: CaM sequestered the IQ motif from the CTD

Figures

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  Figure 1. All atom models of NaV1.5 alpha subunit provided insight into the C-terminal domain (CTD) intramolecular interactions (Knotts et al. 2024, Lile et al. 2025). Three distinct configurations have been identified with energetic minima in these MD stimulations. CTD bound to inactivation gate (IGATE) (left), CTD dissociated from IGATE (middle), and CTD bound to IQ motif (right) are shown as ribbon models with components color coded: CTD in blue, IQ motif in green and IGATE in pink. Each model contains 2016 amino acids in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine lipid bilayer with explicit salt and water. Models were generated using 4 × 1 micro-second Amber 23 MD simulations using ff19sb and lipid21 forcefields with the optimal point charge water. Lipid bilayer was comprised of Palmitoyl and Oleoyl and Phosphatidylcholine components. CTD, C-terminal domain; IGATE, inactivation gate.

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  Figure 2. Cartoon diagram of NaV components that highlight cytosolic NaV-NaV and NaV-CaM interactions in the absence or presence of Ca2+. Cartoon of: IGATE (black), CTD (blue bundle of four helixes), IQ motif (green), CaM (red dumbbell) and Ca2+ (yellow spheres). Biophysical details of these interactions are provided in Table S1. CaM, calmodulin; CTD, C-terminal domain; IGATE, inactivation gate.

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  Figure 3. NMR spectra provide a signature for complex formation. Overlays of 1H-15N HSQC spectra of isotopically enriched 15N CaM (A–C) or 15N CTD (D–I) in the presence of IQ motif peptide, or unlabeled (14N) CaM. Components of each NMR sample are shown on each left vertical axis, and absence or presence of Ca2+ is displayed on the top of each spectrum. Sample names and Ca2+ conditions are color coded to match the spectra colors. CaM, calmodulin; NMR, nuclear magnetic resonance spectroscopy.

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  Figure 4. Overlays of 1H-15N HSQC spectra of isotopically enriched 15N CTD collected in the: absence (black) and presence of IQ motif peptide (blue) or IQ motif peptide + CaM (red). Data collected in the absence (upper spectra) and presence of Ca2+ (lower spectra). Assignments and resonance frequencies obtained during the course of 15N CTD: 14N CaM (1:1 stoichiometry) titrations with IQ motif peptide are provided in Fig. S7. Chemical shift perturbation color coded unto structure of CTD provided in Figure 5. CaM, calmodulin; CTD, C-terminal domain.

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  Figure 5. Overlay ofHSQC NMR spectra of 15N CTD: 14N CaM with additions of IQ motif peptide in the absence (top) and presence of Ca2+ (bottom). Insets for several amino acids that displayed prominent chemical shift perturbations (CSPs) are shown on the left. Prominent CSPs are color-coded on the surface of a ribbon rendering of the CTD structure (PDB ID: 2KBI). Plots of CSPs obtained during IQ motif titration in the absence (open rectangles) and presence of Ca2+ (closed rectangles) are shown on right. Red dashes indicate loss of cross-peak during titration. CaM, calmodulin; CSP, chemical shift perturbation; CTD, C-terminal domain; NMR, nuclear magnetic resonance spectroscopy.

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  Figure 6. Quantification of CaM domain kinetic rates. Changes in Quin-2 fluorescence monitored over time to report on Ca2+ dissociation rates from (A) isolated CaM (gray), CaM in the presence of (B) IQ motif (green) or (C) IGATE peptide (red). Data were best fit to biexponential equations to obtain fast and slow Ca2+ release rates. D and E, assignment of slow Ca2+ release rates to CaM-C domain were derived from replicate experiments that monitored Tyr fluorescence (from CaM C-domain). F, summary of Ca2+ dissociation rates with individual replicates. Statistical difference between the CaM-IQ motif and CaM-IGATE rates were calculated using an unpaired student's t test with Welch's correction (p < 0.0001). CaM, calmodulin; IGATE, inactivation gate.

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  Figure 7. Summary of empirical structural data of NaV1.5 components in the absence and presence of accessory proteins (CaM and fibroblast growth factors). Ribbon renderings of: CTD (blue), IQ motif (green), IGATE (black), fibroblast growth factors (beige surface rendering), CaM-N domain (pink) and CaM-C domain (red). Data are grouped by protein or interaction: (A and B) CaM bound to CTD-IQ construct, (C) Isolated CTD, (D) CaM bound to IQ motif peptide, (E, F) Ca2+ CaM bound to CTD-IQ construct, (G, H) Ca2+ CaM bound to IGATE fragments, (I) Isolated IGATE. CaM, calmodulin; CTD, C-terminal domain; IGATE, inactivation gate.

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  Figure 8. Model illustrating IQ motif can interact with one binding partner at a time (CaM or CTD) irrespective of Ca2+. IQ motif shown in green, CaM shown as red dumbbell, and CTD shown as blue bundle of four helixes. Arrow size indicates that CaMs ability to sequester the IQ motif was enhanced in the presence of Ca2+. CaM, calmodulin; CTD, C-terminal domain.

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  Figure 9. 1H 15N HSQC spectra of 15N CaM in presence of IQ motif (E1901-S1925) or (E1901-A1932). Data was acquired in both the absence (top) and presence (bottom) of Ca2+ at the same temperature and pH as Figure 3, Figure 4, Figure 5. CaM, calmodulin.