NaV1.2 EFL domain allosterically enhances Ca2+ binding to sites I and II of WT and pathogenic calmodulin mutants bound to the channel CTD

Abstract

Neuronal voltage-gated sodium channel Na_V1.2 C-terminal domain (CTD) binds calmodulin (CaM) constitutively at its IQ motif. A solution structure (6BUT) and other NMR evidence showed that the CaM N domain (CaM_N) is structurally independent of the C-domain (CaM_C) whether CaM is bound to the Na_V1.2_IQp (1,901-1,927) or Na_V1.2_CTD (1,777-1,937) with or without calcium. However, in the CaM + Na_V1.2_CTD complex, the Ca²⁺ affinity of CaM_N was more favorable than in free CaM, while Ca²⁺ affinity for CaM_C was weaker than in the CaM + Na_V1.2_IQp complex. The CTD EF-like (EFL) domain allosterically widened the energetic gap between CaM domains. Cardiomyopathy-associated CaM mutants (N53I(N54I), D95V(D96V), A102V(A103V), E104A(E105A), D129G(D130G), and F141L(F142L)) all bound the Na_V1.2 IQ motif favorably under resting (apo) conditions and bound calcium normally at CaM_N sites. However, only N53I and A102V bound calcium at CaM_C sites at [Ca²⁺] < 100 μM. Thus, they are expected to respond like wild-type CaM to Ca²⁺ spikes in excitable cells.

Keywords: FRET; NMR; affinity; allostery; binding; biosensor; free energy; linkage; molecular recognition; titration; voltage-gated sodium channel.

Figure 1.. Architecture of Na _V1.2 and CaM

(A) Transmembrane structure of human Na_V1.2 α-subunit (6J8E, pale green). DIII–DIV linker (orange) includes inactivation gate connecting helix 6 (pink) of DIII pore-forming domain to voltage-sensing domain of DIV (gray). Schematics show unresolved Na_V1.2 N-(NTD, cyan) and C-terminal domains (CTD, green) containing EFL and IQ motif, and CaM (CaM_N, blue, CaM_C, red) bound at IQ motif. Pore-forming DIV (black) connects to the CTD. (B) Vacuum electrostatics surface (positive, blue; negative, red; neutral, white) of human Na_V1.2_CTD (1,777–1,937, 4JPZ/cylinders). (C) Human Na_V1.2_IQp (1,901–1,927) aligned with canonical IQ motif sequence over α-helix showing positions of I, cyan; Q, green; B (R,K/blue); and fJ, orange (ball and stick) and vacuum electrostatics surface (colors match B). (D) Apo CaM (25 models, 1CFC, aligned by aa 82–142). (E and F) Apo (2KXW) and (Ca²⁺)₂-PCaM_C (2M5E) bound to rat Na_V1.2_IQp, aligned by Na_V1.2 1,904 (blue) to 1,924 (red); I1912, cyan, and Q1913, green, are spheres. “N” (blue sphere) at residue 76 would connect CaM_N; helices F (red) and G (orange) show rotation of (Ca²⁺)₂-CaM_C on Na_V1.2_IQp. (G) Putative CaMBD in human Na_V1.2 NTD (NT_BAA). F, orange, and I, cyan (ball and stick) on ideal α helix (cyan). WebLogo (Crooks et al., 2004) of NT_BAA sequence in nine human Na_V isoforms. (H) Vacuum electrostatics surface of Na_V1.2 NT_BAA (colors match B). (I) Steady-state emission spectra of Na_V1.2 NT_BAA biosensor (5 nM) alone (solid black), with 10 μM apo (dashed red) or (Ca²⁺)₄-CaM (solid blue). Inset: Na_V1.2 NT_BAA schematic.

Figure 2.. Solution structure of apo CaM bound to Na _V1.2_IQp

(A and B) ¹⁵N-HSQC overlay of free apo CaM_N (1–75), blue, (A) or apo CaM_C (76–148/red), (B) on apo CaM + Na_V1.2_IQp (black). (C) Apo CaM + Na_V1.2_IQp ensemble (20 models, 6BUT) aligned by CaM 82–142. (D) Apo CaM (putty diameter proportional to CSP) bound to Na_V1.2_IQp (cylinder). (E) Backbone alignment of 6BUT subsets: CaM 1–75 aligned by 6–75; CaM 76–148 and Na_V1.2_IQp, aligned by CaM 82–142. (F and G) Ensemble of interfacial (F) Na_V1.2_IQp residues I1912, Q1913, Y1916, Y1919 (sticks), and (G) aliphatic CaM_C residues (sticks) contacting them. (H) CaM residues ≤6 Å from Na_V1.2_IQp 1,904–1,923 (≤4.5 Å red). Na_V1.2 I1912, cyan; Q1913, green; Y1916/Y1919, orange. (I) CaM atoms (small red spheres) %4.5 Å of Na_V1.2 I1912, cyan; Q1913, green; Y1916, orange; and Y1919, orange (spheres at van der Waals radius), Cα (green). (J) I1912, cyan; Q1913, green; Y1916, orange; and Y1919, orange of Na_V1.2_IQp (ball and stick) relative to CaM_C CSP. See Figures S1–S5.

Figure 3.. Mutational analysis of CaM-Na _V1.2 IQ motif interface

(A) Equilibrium titrations of WT Na_V1.2 IQ biosensor with apo (◇/dashed) or (Ca²⁺)₄-CaM (◆/solid). (B) Cleft of apo CaM_C + Na_V1.2_IQp (6BUT), I1912, cyan; Q1913, green; R1914, marine, Y1916, salmon; R1917, firebrick; and Y1919, wheat (ball and stick). CaM_C surface: orange atoms ≤4.5 Å of Na_V1.2_IQp. (C) Ensemble of R1914, blue, and R1917, red (sticks), positions in 6BUT. WebLogo compares Na_V1.2 1,906–1,922 in 53 eukaryotes. (D–L) CaM binding to mutant Na_V1.2 IQ biosensors. Reference titrations (gray) of apo (dashed) and (Ca²⁺)₄-CaM (solid) from (A). Apo (◇/dashed) or (Ca²⁺)₄-CaM (◆/solid) binding to (D and G) I1912A, cyan; Q1913A, green; and IQ/AA, magenta; (E and H) Y1916A, pink; Y1919A, gold; or YY/AA, orange; (F and I) R1914A, blue; R1917A, orange; or RR/AA, violet; (J) R1902C, gold; (K) I1911V, pink; and (L) Q1913R, blue (blue arrow shows difference in apo versus (Ca²⁺)₄-CaM titration curve midpoints). (D–I) Square brackets show difference in titration curve midpoints. (M) CaM_C (6BUT, gray). CaM_N connects at aa 76 (blue sphere). Na_V1.2_IQp 1,902–1,924 (yellow to orange, thin helix). Sidechains R1902, gold; I1911, magenta; I1912, cyan; Q1913, green; R1914, marine; Y1916, salmon; R1917, firebrick; Y1919, wheat; and Cα atoms, gray, are spheres. (N) ΔG_Ca2+ versus ΔG_Apo of WT CaM-binding Na_V1.2 IQ biosensors. See Figure S6.

Figure 4.. Ca²⁺ binding by CaM + Na_V1.2_IQp

(A) ¹⁵N-HSQC overlay of apo (black) or (Ca²⁺)₄-¹⁵N-CaM (red) bound to ¹⁴N-Na_V1.2_IQp. Arrows show CSP of some CaM residues. (B) ¹⁵N-HSQC overlay of ¹⁵N-Na_V1.2_IQp bound to apo (black) or (Ca²⁺)₄-¹⁴N-CaM (red). Inset shows Na_V1.2_IQp 1,901–1,911 (pre-IQ, light blue), 1,912–1,919 (IQ motif/green), and 1,920–1,927 (post-IQ/red). Arrows show CSP of some Na_V1.2_IQp residues bound to apo versus (Ca²⁺)₄-CaM. (C) Ca²⁺ titration of CaM_N (sites I and II) in ¹⁵N-CaM+¹⁴N-Na_V1.2_IQp. Normalized chemical shift changes shown for T5 ○, E6 ☒, A10 ▻, E11 ☐, F16 ⧅, S17 △, F19 , D24 ◪, K30 ▽, G33 , R37 , S38 , L39 ◧, G40 , E45 , E54 G61 ◨, T62 ⊟, D64 ▷, F68 , T70 ⧄, R74 ⊡. Inset: monitored CaM_N residues (blue spheres) on 6BUT (gray). (D and E) Ca²⁺ titration of CaM_C (sites III and IV) in ¹⁵N-CaM+¹⁴N-Na_V1.2_IQp. (D) Normalized peak intensities of apo CaM residues E82 ○, E83 ☒, F89 ⊡, V91 ☐, F92 ⧅, D93 △, K94 , D95 ◪, Y99 ▽, S101 , A102 , E104 , L105 ◧, H107 , V108 ⧄, G113 ◨, E114 ⊟, K115 , D122 , M124 ◩, I130 ⊞, Y138 , E139 ◩, E140 ⊲, T146 ⊳, A147 ✚, K148 . (E) Normalized peak intensities of (Ca²⁺)₄-CaM residues E82 ○, E83 ☒, K94 , D95 ☐, Y99 ⧅, S101 △, A 102 , A103 ⧄, E104 ◪, G113 ⊞, E114 ▽, K115 ⊟, T117 ◩, R126 , D129 , I130 , N137 ⊟, E139 ⊙, T146 , A147 ◫, K148 ◨. Inset: monitored CaM_C residues (red spheres) on 6BUT (gray). (F–H) Ca²⁺ titration of ¹⁴N-CaM+¹⁵N-Na_V1.2_IQp. Normalized peak intensities of Na_V1.2_IQp residues. (F) K1901 ○, R1902 ◇, K1903 △, Q1904 ▽, E1905 ☐, E1906 ⧅, V1907 , S1908 ☒, A1909 , I1910 ⊟, I1911 ⧄; (G) I1912 ○, Q1913 , R1914 △, A1915 ☐, Y1916 , R1917 , R1918 ⊞, Y1919 , L1920 ⧅, L1921 ☒; and (H) Q1923 ◇, K1924 △, K1927 . Insets: Na_V1.2_IQp residues monitored: pre-IQ/blue, IQ/green, post-IQ/red (spheres) on 6BUT (gray). Solid gray lines are for reference. (I and J) E1906/purple, A1909/black, I1912/cyan, Q1913/green, Y1916/orange, Y1919/orange (spheres) of Na_V1.2_IQp relative to apo (I) and (Ca²⁺)₂-CaM_C (J) surface (gray). 6BUT and 2M5E aligned by CaM 117–128 (blue spheres at 76). Na_V1.2_IQp 1,904–1,923 (gray helix), Cα of Q1904/blue and Q1923/red are small spheres. CaM_C residues ≤4.5 Å of Na_V1.2_IQp with Na_V1.2_IQp-induced CSPs > (average plus 1 SD) are orchid (apo, I) or red ((Ca²⁺)₄-CaM, J). See Figure S7.

Figure 5.. Interaction of CaM with Na _V1.2 CTD

(A and B) Models of (A) CaM_C (red) bound to Na_V1.2_CTD (green) with alternate positions of CaM_N, blue, and (B) Ca²⁺-induced rotation of CaM on Na_V1.2_CTD. (C) ¹⁵N-HSQC overlay of apo ¹⁵N-CaM+¹⁴N-Na_V1.2_IQp (black) or + ¹⁴N-Na_V1.2_CTD (green). (D) Histogram of Δδ of labeled CaM residues in CaM + Na_V1.2_CTD versus CaM + Na_V1.2_IQp; Δδ scaled as in Figure S1A. Inset: Cα of CaM residues (CaM_N, blue; CaM_C, red, spheres) labeled in (C) on a model of apo CaM (6BUT, gray helices) bound to Na_V1.2_CTD (4JPZ, cylinders). (E) ¹⁵N-HSQC overlay of (Ca²⁺)₄-¹⁵N-CaM+¹⁴N-Na_V1.2_IQp (black) or + N-Na_V1.2_CTD (green). (F) Histogram of Δδ of labeled CaM residues in CaM + Na_V1.2_CTD versus CaM + Na_V1.2_IQp; Δδ scaled per Figure S1A. Inset: Cα of CaM residues (CaM_N, blue; CaM_C, red, spheres) labeled in (E) on a model of (Ca²⁺)₄-CaM (2K0E, gray helices) bound to Na_V1.2_CTD (4JPZ, cylinders, see transparent methods).

Figure 6.. Effects of pathogenic CaM mutations on Ca²⁺ and Na_V1.2 IQ motif binding

(A) Apo CaM + Na_V1.2_IQp (6BUT): N53, blue; D95, brown; A102, purple; E104, dark green; D129, orange; and F141L, red (ball and stick) (B) Ca²⁺-saturated site III (1CLL): Ca²⁺ (yellow spheres) and coordinating O atoms (red) of D93, blue, to E104, red. (C–E) Equilibrium Ca²⁺ titrations of full-length or domain fragment of WT/black or mutant (N53I, blue; D95V, brown; A102V, purple; E104A, dark green; D129G, orange; F141L, red) CaM. (C) Sites I and II in N53I CaM_N (○/dashed) and full-length CaM (●/solid), sites III and IV of (D) CaM_C and (E) CaM. (F) Equilibrium titrations of WT Na_V1.2 IQ biosensor with WT or mutant apo CaM. Colors match (C). (G–I) Equilibrium Ca²⁺ titrations of CaM or CaM_C fragment bound to Na_V1.2_IQp. (G) Sites I and II of CaM, sites III and IV of (H) CaM_C, and (I) CaM. Colors match (C). (J) ¹⁵N-HSQC overlay of (Ca²⁺)₄-¹⁵N-CaM+¹⁴ N-Na_V1.2_IQp (red) and free (Ca²⁺)₂-¹⁵N-CaM_N (1–75, ,blue). Inset: Cα (blue spheres) of labeled CaM_N residues on (Ca²⁺)₄-CaM (1CLL, gray). See Figures S8 and S9 and Table S1.

Figure 7.. Equilibrium Ca²⁺ binding titrations of CaM bound to Na_V1.2 CTD

(A and B) WT CaM (A) sites I and II (alone, black; +Na_V1.2_IQp, blue; +Na_V1.2_CTD, light blue) and (B) sites III and IV (alone, black; +Na_V1.2_IQp, red; +Na_V1.2_CTD, pink). (C and D) Sites I and II (C), or III and IV (D) of WT/black or mutant (N53I, blue; D95V, brown; E104A, dark green; D129G, orange; F141L, red) CaM + Na_V1.2_CTD. (E) Models of apo (6BUT) and (Ca²⁺)₄-CaM (2K0E) (1–75 pale blue, 76–80 black, 81–148 pale red cylinders) bound to Na_V1.2_CTD (green). CaM mutations (spheres) match (C). See Figure S9, Table S1.

Figure 8.. Modes of CaM interaction with NaV

(A) High-resolution structures of Na_V fragments bound to CaM (aligned by 101–112 and 117–128) overlaid with models from the apo CaM + Na_V1.2_IQp ensemble (20 models, 6BUT/NMR). 6BUT_model9 aligned with apo CaM fused to Na_V1.6 IQ motif (3WFN/XRD). 6BUT_model19 aligned with apo (Mg²⁺) CaM + Na_V1.5 CTD (4OVN/XRD). 6BUT_model9 aligned with apo (Mg²⁺) CaM + Na_V1.5 CTD with auxiliary FGF (4DCK/XRD). 6BUT_model13 aligned with intermediate (Ca²⁺)₂-CaM + Na_V1.2 CTD with auxiliary FGF (4JPZ/XRD). Mg²⁺, Ca²⁺ are spheres. (B and C) Cylinder models of apo (6BUT, (B) and (Ca²⁺)₄-CaM (2K0E), (C) bound to the IQ motif of Na_V CTD (4DCK/5X0M) relative to Na_V IG (5XOM, orange) and auxiliary FGF (4DCK) shown as surfaces. Na_V residues not in CTD or IG are light green. Ca²⁺ are spheres. (D) Structures of (i) (Ca²⁺)₂-CaM bound to SK channel (1G4Y/green and pink) and (ii) (Ca²⁺)₄-CaM bound to two distinct regions of STRA6 retinol receptor (5SY1/green and magenta), SK channel (6CNO, green and orange), and MUNC13 (2KDU, orange), aligned by CaM 43–56. Schematics show CaM bridging Na_V1.2 IQ motif and CaMBD X (light pink or purple) or Na_V IG, or two sites in Na_V IG.