Calcium triggers reversal of calmodulin on nested anti-parallel sites in the IQ motif of the neuronal voltage-dependent sodium channel NaV1.2

Abstract

Several members of the voltage-gated sodium channel family are regulated by calmodulin (CaM) and ionic calcium. The neuronal voltage-gated sodium channel Na_V1.2 contains binding sites for both apo (calcium-depleted) and calcium-saturated CaM. We have determined equilibrium dissociation constants for rat Na_V1.2 IQ motif [IQRAYRRYLLK] binding to apo CaM (~3nM) and (Ca²⁺)₄-CaM (~85nM), showing that apo CaM binding is favored by 30-fold. For both apo and (Ca²⁺)₄-CaM, NMR demonstrated that Na_V1.2 IQ motif peptide (Na_V1.2_IQp) exclusively made contacts with C-domain residues of CaM (CaM_C). To understand how calcium triggers conformational change at the CaM-IQ interface, we determined a solution structure (2M5E.pdb) of (Ca²⁺)₂-CaM_C bound to Na_V1.2_IQp. The polarity of (Ca²⁺)₂-CaM_C relative to the IQ motif was opposite to that seen in apo CaM_C-Na_v1.2_IQp (2KXW), revealing that CaM_C recognizes nested, anti-parallel sites in Na_v1.2_IQp. Reversal of CaM may require transient release from the IQ motif during calcium binding, and facilitate a re-orientation of CaM_N allowing interactions with non-IQ Na_V1.2 residues or auxiliary regulatory proteins interacting in the vicinity of the IQ motif.

Keywords: Allostery; Binding; Biosensor; FRET; Free energy; Linkage; Molecular recognition; NMR; Sodium channels; Thermodynamics; Titration.

Figure 1. Schematics and Structures of CaM Binding to Na_V IQ Motifs

In all structures of CaM, residues 1–75 are blue, 76–80 are black, and 81–148 are red. A: Cartoon of a voltage-gated sodium channel (Na_V) α-subunit (green) with 4 multi-helical transmembrane units DI, DII, DIII, and DIV (cylinders). The C-terminal domain (CTD) contains a 4-helix bundle domain dubbed EF-like (EFL) and an lQ motif bound to apo CaM_C (red) with apo CaM_N (blue) connected by a flexible linker (black). The Na_V inactivation gate (purple) links DIII and DIV, and contains a site for binding (Ca²⁺)₄-CaM. B: Solution structures of the Na_V1.2 EF-like domain (PDB: 2KAV) depicting residues E1777 (blue sphere) to R1881 (red sphere); cylindrical helices are colored in shades of green correspond to the sections labeled H in the linear diagram of the Na_V1.2 CTD residues 1777 to 1937 below. C: Crystallographic structure of apo (calcium-depleted) CaM bound to a CTD fragment of Na_V1.5 (PDB: 4DCK); shades of green match panel B. The Na_V1.2 CTD in 4DCK was aligned (using PyMOL (Schrödinger, LLC)) to the corresponding residues in 2KAV in panel B based on EFL-located helical residues 1791–1866 and 1855–1866. D: Overlay of structures having apo or Mg²⁺-bound CaM associated with Na_V IQ motifs (PDB: 2KXW, 2L53, 4DCK, 4OVN, and 3WFN). For structures determined by NMR (2KXW and 2L53), only the minimized average structure is shown. Structures aligned with PyMOL (Schrödinger, LLC) based on position of CaM residues 102–112 and 117–128 (helices F and G). E: Structures of individual CaM-IQ complexes in overlay in panel D; Mg²⁺ ions are gray spheres. For 2L53, all deposited models consistent with the NMR data are illustrated. Structures were aligned as in panel D. Spheres highlight IQ motif residues at positions 1, 2, 5 and 8. In, Nav1.2IQp these are I₁₉₁₂ (cyan), Q₁₉₁₃ (green), Y₁₉₁₆ and Y₁₉₁₉ (gold). In Nav1.5_IQp and Na_v1.6_IQp, colors for corresponding residues match WebLogos in panel F. F: WebLogo 3.3[26] analyses comparing IQ motif sequences for Na_V1.2 from 53 species, Na_V1.5 from 40 species and Na_V1.6. from 45 species (sequence alignments shown in Supplemental Tables S1A, S1B, S1C). The height of each letter denotes relative conservation of residues. A star indicates the fully conserved Q residue of all three channels which is also labeled in panel E. Within the set of residues binding apo CaM_C, complete conservation is observed for positions corresponding to Q₁₉₁₃, R₁₉₁₄, A₁₉₁₅, R₁₉₁₇ in Na_V1.2.

Figure 2. Ca²⁺-Induced Changes in Calmodulin Structure and Linkage to Target Binding

A. Schematic showing structural changes of CaM_C upon binding Ca²⁺ (horizontal) and a peptide target (vertical). Structures were aligned by CaM residues 117–128 (helix G). Interhelical angles for helices E and F (http://calcium.sci.yokohama-cu.ac.jp/efhand.html) shown in black are as follows: apo CaM_C (1CFC, 54.8°), apo CaM_C bound to an IQ motif in myosin V (2IX7, 79.5°), (Ca²⁺)₂-CaM_C (1CLL, 89.7°), and (Ca²⁺)₂-CaM_C bound to CaMKII (1CDM, 88.5°). For 2IX7 and 1CDM, the termini of the green target peptide are blue (amino) and red (carboxyl). B. (Ca²⁺)₄-CaM in 1CLL. CaM residues 1–75 are blue, 76–80 are black, and 81–148 are red; Ca²⁺ ions are yellow. C. (Ca²⁺)₄-CaM bound to CaMKII peptide in 1CDM. Color scheme the same as panel B, with peptide in green. D. Alignment of CaM_C residues in B and C showing E104 and E140 in 1CLL (orange) and 1CDM (red) as sticks in Ca²⁺-binding sites. Label gives the average distance between carboxyl oxygens of E140 and the Ca²⁺ in site IV. CaM_C in each structure is shown in gray; structures were aligned using CaM residues 102–112 and 117–128 (helices F and G). E. Overlay of apo CaM bound to a myosin V IQ motif in 2IX7, and half-saturated CaM (CaM_N Ca²⁺-saturated but CaM_C apo) bound to an SK Channel peptide (1G4Y) aligned by CaM_C helices F and G. F. Apo (Mg²⁺-bound) CaM and (Ca²⁺)₄-CaM bound to Na_V1.5 IQ motif (4DCK and 4JQ0). In sites III and IV, Mg²⁺ is gray, and Ca²⁺ ions listed in 4JQ0 are hot pink. Structures were aligned as in panel A. G. Alignment of apo CaM_C bound to SK Channel (1G4Y) shown in panel E, and apo (Mg²⁺ bound CaM_C) bound to Na_V1.5 (4DCK) and (Ca²⁺)₄-CaM bound to Na_V1.5 IQ motif (4JQ0) shown in panel F. Side chains of E104 and E140 in 1G4Y (cyan) and 4DCK (blue) are shown as sticks; only backbone atoms for E104 and E140 were assigned in 4JQ0 (hot pink). Label gives the average distance between carboxyl oxygens of E140 and the Mg²⁺ ion in site IV in 4DCK. Mg²⁺ is gray, and Ca²⁺ assigned in 4JQ0 is hot pink. H. Dimer of heterotrimers (Na_V-FGF13U-CaM) in the 4JPZ crystal structure of (Ca²⁺)₄-CaM bound to Na_V1.2 CTD (residues 1777–1937, green) and FGF13U (purple). Circled regions correspond to regions circled in panels I and J. I. Blue circles highlight multiple contacts (≤ 6Å) between CaM in one heterotrimer of 4JPZ with FGF13U in the other heterotrimer. Heterotrimers are labeled with subscripts “a” and “b” for tracking interactions. They are identical in sequence, and nearly identical structurally. J. Orange circle highlights highlight contacts (≤ 6Å) between CaM_C in one heterotrimer of 4JPZ with CaM_C in the other.

Figure 3. CaM Binding to Na_V1.2IQ Biosensor and Na_V1.2 IQp

Na_v1.2_IQ biosensor samples excited at 430 nm; peaks in emission intensity correspond to CFP (475 nm) and YFP (525 nm). A. Steady-state emission spectra of Na_v1.2_IQ biosensor alone (solid green) and saturated with apo CaM (black dashed) or calcium-saturated CaM (black solid) (>10³-fold excess). B. Emission spectra of Na_v1.2_IQ biosensor alone (solid green) and with >10³-fold excess apo CaMN (blue solid) and calcium-saturated CaMN (blue dashed). C. Equilibrium titrations of biosensor binding by apo full-length CaM (black dashed), CaMC (red dashed), and CaM_N (blue dashed). D. Equilibrium titrations of biosensor binding by calcium-saturated full-length CaM (black solid), CaMC (red solid), and CaMN (blue solid). Simulations in panels C and D are based on fits to the experimental data set shown in each panel. For CaMN, a horizontal line is provided to guide the eye. E. ¹⁵N-HSQC spectral overlay of (Ca²⁺)₂-CaM_C (red) and (Ca²⁺)₄-CaM (black) bound to Na_v1.2_IQp (black). Backbone resonance assignments are shown for residues of peptide-bound CaM_C. Green ellipses encircle resonances for the same residue in each complex. F. Expansion of crowded region of spectral overlay in panel E.

Figure 4. NMR Models of Ca²⁺-Saturated CaM_C Bound to Na_V1.2_IQp

A. Wireframe backbone representations of the 20 lowest energy structures and the ensemble minimized average model of (Ca²⁺)₂-CaM_C bound to Na_v1.2_IQp (2M5E). For clarity, only well ordered helical residues 1904–1923 of Na_v1.2_IQp are shown. B. Cartoon representation of minimized average model in 2M5E. Na_v1.2_IQp helix shown as a ribbon with gradient from blue (N-terminus) to magenta (C-terminus). Cylinders represent CaM helices E (gray), F (red), G (orange) and H (gray) with yellow spheres for calcium ions. Residues of the IQ motif making ≥ 3 short-range contacts (< 4.5 Å) with the hydrophobic cleft of CaM are shown in ball-and-stick representation along with Q₁₉₁₃ for reference. C. Analysis by CSU (Contacts of Structural Units)[73] of the minimized average model of (Ca²⁺)₂-CaM_C-Na_v1.2_IQp. The primary sequence of residues 1904 to 1923 of the IQ motif is shown on the x-axis (I₁₉₁₂ in cyan, Q₁₉₁₃ in green). Boxes listing residues of CaM located within 4.5 Å of each IQ motif residue are shaded in orange; longer range contacts (4.5 to 6 Å) are in white boxes. D. NMR strip plots corresponding to three CaM residues: A88α, V91γ2 and M124ε. For each residue, the top panel represents a ¹³C-edited, ¹²C,¹⁴N-filtered NOESY experiment, the middle panel represents an HCCH-TOCSY experiment, and the bottom panel is a ¹³C-edited NOESY with carbon chemical shift of the planes denoted in the label. Labels for atoms in Na_v1.2_IQp are green and in CaM are red. Residues surrounding A₈₃, V₉₁ and M₁₂₄ of CaM are shown in images made with PyMOL (Schrödinger, LLC) next to the strip plots.

Figure 5. Chemical Shift Differences and Contacts in (Ca²⁺)₂-CaM_C ± Na_v1.2|_Qp

A. HSQC spectral overlay of (Ca²+)₂-CaM_C alone (red) and bound to Na_v1.2_IQp (black). Backbone resonance assignments are shown for residues of peptide-bound CaM_C. B. Effects of Na_v1.2_IQp on chemical shifts of (Ca²+)₂-CaM_C. Changes in chemical shifts are calculated as described in Methods. Location of CaM helices and calcium-binding loops are shown above the plot. Residues in calcium-binding sites III (93–104) and IV (128–140) are indicated by yellow shading. C. Chemical shift perturbations mapped onto the structure of (Ca²⁺)₂-CaMC in 2M5E. The greatest effect of Na_v1.2_IQp binding is indicated by the red/wide cartoon, and the weakest effect is shown by the blue/narrow cartoon.

Figure 6. Comparison of Nested Anti-Parallel Sites in Na_v1.2_IQp

A. HSQC overlay of CaM_C-Na_v1.2_IQp with (black) and without (green) saturating calcium. Backbone resonance assignments are shown for residues of (Ca²⁺)₂-CaM-Na_v1.2_IQp. B. Chemical shift differences between CaM_C-Na_v1.2_IQp ± Ca²⁺ indicated changes in chemical environment of individual CaM residues. Location of CaM helices and calcium-binding loops are shown above the plot. Residues in calcium-binding sites III (93–104) and IV (128–140) are indicated by yellow shading. C. Chemical shift perturbations mapped onto the structure of (Ca²⁺)₂-CaM_C in 2M5E. The greatest effect of Ca²⁺ binding is shown in the cartoon as red/wide, while the weakest effect is blue/narrow. D. Comparison of (Ca²⁺)₂-CaM_C (2M5E) and apo CaM_C (2KXW) bound to Na_v1.2_IQp aligned by superposition of the helical backbone of Na_v1.2_IQp. Residues I₁₉₁₂ (cyan), Q₁₉₁₃ (green), and A₁₉₁₅ (black) are in ball-and-stick. For clarity, the peptide backbone is omitted. The surface of solvent-exposed CaM residues is red and cleft residues are orange in semi-open (2KXW) and open (2M5E) CaM. CaM_N would connect to CaM_C via residue M76 (blue) highlighting the reversed orientations of CaM_C on the IQ motif. E. Comparison of clefts of Ca²⁺-CaM_C (2M5E) and apo CaM_C (2KXW) binding Na_v1.2_IQp. Structures were aligned based on helical residues of the peptide. IQ motif residues I₁₉₁₂ (cyan) Q₁₉₁₃ (green), A₁₉₁₅ (black), Y₁₉₁₆ (magenta), and Y₁₉₁₉ (dark green) are shown in ball-and-stick representation. For clarity, the backbone of the peptide is not shown.

Figure 7. Models of CaM Retention, Release, or Reversal on Na_V1.2

A. Simulations of saturation of the Na_V1.2 IQ motif by apo (dashed) and calcium-saturated (solid) CaM based on ΔG values in Table 1. Molecular models show calcium-induced reversal of CaM_C relative to the IQ motif peptide. B. Simulation of calcium binding to CaM bound to the Na_V1.2 IQ motif (see Methods). Resting calcium levels indicated by blue bar; green bar centered on 10 03BCM indicates range of neuronal intracellular Ca²⁺ levels sufficient to achieve 50–75% calcium-saturated CaM_C bound to Na_V1.2-IQ. Schematics indicate calcium-induced reversal of CaM_C on the IQ motif. C. Schematic for proposed 3-state mechanism of CaM-mediated regulation based on calcium titration of full-length CaM bound to Na_V1.2_IQp monitored by NMR. Apo CaM binds to the IQ motif via CaM_C with no preferred contacts between CaM_N and Na_v1.2_IQp. Calcium binding to sites I and II affects only CaM_N; calcium binding to sites III and IV triggers reversal of CaM_C on the IQ motif. D. CaM-target structures representative of the 3 states shown schematically in part C. Target is green with spheres for the I (cyan) and Q (green) residues of the IQ motif. CaM is gray except for CaM_C helices F (red) and G (orange). Apo CaM bound to the Na_V1.5 IQ motif (4OVN) or an IQ motif of myosin V (2IX7) shows I oriented into the semi-open cleft, and Q oriented towards the FG-linker in semi-open CaM_C. The intermediate state of CaM bound to the Na_V1.2 IQ motif (4JPZ) retains the same orientation of CaM_C as the apo state. Ca²⁺-saturated CaM adopts an open conformation bound to the IQ motif of Na_V1.2 (2M5E) and myosin V (4ZLK) with Q oriented towards the junction between CaM_N and CaM_C. All 5 structures were aligned by residues corresponding to 1909–1915 of Na_V1.2 in 2M5E, where Iis 1912 and Q is at 1913. E. Alternative models of calcium-mediated response include (Ca²⁺)₄-CaM dissociation from Na_V1.2, which might promote (Ca²⁺)₄-CaM binding at the DIII–DIV linker (“inactivation gate”, purple) as seen in 4DJC, or binding at a pair of CaMBD sequences (orange, cyan) that are not in a continuous helix, such as seen in 5SY1, the STRA6 receptor for retinol uptake.