Ca2+-saturated calmodulin binds tightly to the N-terminal domain of A-type fibroblast growth factor homologous factors

Figure 1. Architecture of NaV, CaM, and FGFs.A, schematic of human NaV. The α-subunit N-terminal domain (NTD, cyan), transmembrane domains DI-DIII (pale green) and DIV (gray), the linker connecting DIII to DIV (orange), and C-terminal domain (CTD, green) with 4-helix bundle EFL (dark green) that binds FGFs (light gray), and IQ motif that binds CaM (CaMN/blue, CaMC/red) are shown. Auxiliary β-subunit that contains a transmembrane helix and extracellular domain is lavender. B, model of apo CaM and FGF12B bound to NaV. Model is comprised of NaVPAS (5X0M, DI-DIII/pale green surface, DIV voltage-sensing domain/gray surface, DIV pore domain/black surface, DIII-DIV linker/orange cartoon), the NaV1.5CTD (4DCK, forest green), the apo CaM+NaV1.2IQp ensemble (6BUT, CaMN/blue, CaMC/red), and FGF12B (1Q1U, gray). The NaV1.5CTD was aligned to NaVPAS EFL (a.a. 1426–1521), the apo CaM+NaV1.2IQp ensemble was aligned with 4DCK via CaM a.a. 101–112 and 117–128, and FGF12B was aligned with 4DCK using FGF13B a.a. 11–158. For simplicity NaV1.2IQp in 6BUT and CaM FGF13B in 4DCK are not shown. C, schematics of FGF11 (orange), FGF12A (purple), FGF13A (red), and FGF14 (blue) splice variants. The β-trefoil core is…

Figure 2. (Ca2+)4-CaM binds the FGF12ALTPand FGF CaMBD biosensors.A, schematic of FGF12A. Folded β-trefoil core is shown as a rectangle, and LTP (green) and CaMBD (limon) are shown as cylinders. The black box indicates the position of the FGF12A LTP. B, steady-state emission spectra of the FGF12ALTP biosensor alone (solid black), in the presence of apo (dashed green) and (Ca2+)4-CaM (solid green). C, equilibrium titration of FGF12ALTP biosensor with (Ca2+)4-CaM (green). D, schematic of an A-type FGF. Folded β-trefoil core is shown as a rectangle, and LTP (green) and CaMBD (limon green) are shown as cylinders. The black box indicates the position of the A-type FGF CaMBD. E–H, steady-state fluorescence spectra of FGF11ACaMBD (E, orange), FGF12ACaMBD (F, purple), FGF13ACaMBD (G, red), FGF14ACaMBD (H, blue) biosensors alone (all shown in solid black), and in the presence of apo (dashed/colored) and (Ca2+)4-CaM (solid/colored). I–L, equilibrium titrations of FGF11ACaMBD (I), FGF12ACaMBD (J), FGF13ACaMBD (K), and FGF14ACaMBD (L) biosensors with (Ca2+)4-CaM. FGF isoforms are colored as in panels E–H.

Figure 3. Energetic contribution of CaMNand CaMCto FGF12A and FGF13A CaMBD binding by CaM.A, position of E31 (blue), E67 (blue), E104 (red), and E140 (red), shown as ball-and-stick, in apo CaM (1CFD, CaMN/light blue, CaMC/salmon). B, (Ca2+)4-CaM site I (1CLL): Ca2+ (yellow sphere) is surrounded by residues D20 (blue) to E31 (red). The coordinating O atoms of E31 are shown as red spheres. C, schematic of FGF12A. Folded β-trefoil core is shown as a rectangle, and LTP (green) and CaMBD (limon) are shown as cylinders. The black box indicates the position of the FGF12A CaMBD. D and E, equilibrium titrations of the FGF12ACaMBD biosensor with E31Q/E67Q (D, blue) or E104A/E140Q CaM (E, red). A reference titration with WT CaM is shown in black. F, ΔG of WT (black), E31Q/E67Q (blue), and E104Q/E140Q (red) CaM binding the FGF12ACaMBD biosensor. G, schematic of FGF13A. Folded β-trefoil core is shown as a rectangle, and LTP (green) and CaMBD (limon) are shown as cylinders. The black box indicates the position of the FGF13A CaMBD. H and I, equilibrium titrations of the FGF13ACaMBD biosensor with E31Q/E67Q (H, blue) or E104A/E140Q CaM (I, red). A reference titration with WT CaM is shown in black. J, ΔG…

Figure 4. Interaction of (Ca2+)4-CaM with the FGF12ACaMBDpand FGF12ANTD.A, schematics depicting models of (Ca2+)4-CaM (CaMN/blue, CaMC/red) binding to the FGF12ACaMBDp (limon cylinder) in a 1: 1 M ratio. B and C, overlay of the 15N-HSQC spectra of 15N-(Ca2+)2-CaMN (blue, B) or 15N-(Ca2+)2-CaMC (red, C) and 15N-(Ca2+)4-CaM+14N-FGF12ACaMBDp (black). Peaks labeled in the spectrum of 15N-(Ca2+)2-CaMN (B) or 15N-(Ca2+)2-CaMC (C) are shifted in the 15N-(Ca2+)4-CaM+14N-FGF12ACaMBD spectrum. Insets show the Cα of labeled CaMN (blue spheres, B) or CaMC (red spheres, C) residues on a structure of (Ca2+)4-CaM (1CLL, gray helices). D, schematics depicting models of (Ca2+)4-CaM (CaMN/blue, CaMC/red) binding to the FGF12ANTD in a 1:1 M ratio. FGF12A LTP (green) and CaMBD (limon) are shown as cylinders. E, overlay of the 15N-HSQC spectra of 15N-(Ca2+)4-CaM bound to the 14N-FGF12ACaMBDp (black) or 14N-FGF12ANTD (purple). F, schematic showing that the interface is essentially identical between (Ca2+)4-CaM (CaMN/blue, CaMC/red) and the FGF12ACaMBDp (limon cylinder) or FGF12ANTD (LTP/green cylinder, CaMBD/limon cylinder). G and H, 15N-HSQC spectrum of 14N-(Ca2+)4-CaM bound to 15N-FGF12ACaMBDp (G, black) or…

Figure 5. Equilibrium Ca2+titrations of CaM+FGF12ACaMBDpand CaM+FGF12ANTD. A, equilibrium Ca2+ titrations of sites I and II of CaM alone (black) and in the presence of the FGF12ANTD (teal). B, equilibrium Ca2+ titrations of sites III and IV of CaM alone (black) and in the presence of the FGF12ANTD (pink). C, equilibrium Ca2+ titrations of sites I and II of CaM alone (black) and in the presence of the FGF12ACaMBDp (blue). D, equilibrium Ca2+ titrations of sites III and IV of CaM alone (black) and in the presence of the FGF12ACaMBDp (red). E and F, ΔG of Ca2+ binding to sites I and II (E) in CaM alone (black), +FGF12ANTD (teal) or +FGF12ACaMBDp (blue), and sites III and IV (F) in CaM alone (black), +FGF12ANTD (pink) or +FGF12ACaMBDp (red). G, schematic depicting that the interface is essentially identical between (Ca2+)4-CaM (CaMN/blue, CaMC/red) and the FGF12ACaMBDp (lime green cylinder) or FGF12ANTD (LTP/green cylinder, CaMBD/limon cylinder).

Figure 6. Equilibrium Ca2+titrations of CaM+FGF12B+NaV1.2CTDand CaM+FGF12A+NaV1.2CTD.A and B, equilibrium Ca2+ titrations of sites I and II (A), and sites III and IV (B) of CaM in CaM+NaV1.2CTD. Solid gray lines show reference Ca2+ titrations sites I and II (A) and sites III and IV (B) of CaM alone. C, E, equilibrium Ca2+ titrations of sites I and II of CaM in CaM+NaV1.2CTD (C, E, black), CaM+FGF12B+NaV1.2CTD (C, blue) and CaM+FGF12A+NaV1.2CTD (E, teal). D, F, equilibrium Ca2+ titrations of sites III and IV of CaM in CaM+NaV1.2CTD (D, F, black), CaM+FGF12B+NaV1.2CTD (D, red) and CaM+FGF12A+NaV1.2CTD (F, pink).

Figure 7. Equilibrium Ca2+titrations of a 2:1:1 complex of CaM+FGF12A+NaV1.2CTD.A, schematic depicting the possible binding of two CaM (CaMN/blue, CaMC/red) molecules the FGF12A (purple)+NaV1.2CTD (green) complex. The FGF12A LTP (green) and CaMBD (limon) are shown as cylinders. B, equilibrium Ca2+ titrations of CaM sites I and II in CaM+FGF12A+NaV1.2CTD (light blue, CaM:FGF12A:NaV1.2CTD = 2:1:1). The solid black and gray lines show reference titrations of CaM sites I and II in CaM+FGF12ANTD and CaM+FGF12B+NaV1.2CTD, respectively. C, Equilibrium Ca2+ titrations of sites III and IV of CaM in CaM+FGF12A+NaV1.2CTD (hot pink, CaM:FGF12A:NaV1.2CTD = 2:1:1). The solid black and gray lines show reference titrations of CaM sites III and IV in CaM+FGF12ANTD and CaM+FGF12B+NaV1.2CTD, respectively. D, maximum decrease in Phe fluorescence intensity in equilibrium Ca2+ titrations of CaM+FGF12B+NaV1.2CTD (blue), CaM+FGF12A+NaV1.2CTD (teal, CaM:FGF12A:NaV1.2 = 1:1:1) and CaM+FGF12A+NaV1.2CTD (light blue, CaM:FGF12A:NaV1.2CTD = 2:1:1). E, maximum increase in Tyr fluorescence intensity in equilibrium Ca2+ titrations of CaM+FGF12B+NaV1.2CTD (red), CaM+FGF12A+NaV1.2CTD (pink, CaM:FGF12A:NaV1.2 = 1:1:1), and…

Figure 8. Models of (Ca2+)4-CaM bound to the FGF12A and FGF13A CaMBD. In all structures CaM residues 1–75 are blue, 76–80 are black and 81–148/red, and Ca2+ are yellow spheres. A, top, model 1 in the Robetta (54) generated ensemble of each full-length A-type FGF. Bottom, orientation of the LTP and CaMBD regions in the modeled ensemble of FGF11, FGF12A, FGF13A, and FGF14. The FGF isoforms are colored as follows: FGF11A is orange, FGF12A is purple, FGF13A is red, and FGF14A is blue. The LTP and CaMBD regions of each FGF are shown in green and limon, respectively. Models were aligned with FGF12A a.a. 71–204. B, alignment of the NMDA receptor CaMBD, FGF12A CaMBD, and FGF13A CaMBD sequences. The positions that correspond to F880 and T886in the NMDA receptor are shaded (hydrophobic/tan, polar/green). C: Structure of (Ca2+)4-CaM bound to the NMDA receptor CaMBD (2HQW, gray), F880 (orange), and T886 (green) of the NMDA receptor are shown as spheres (top) and positions of F19, L32, M51, and M71 in CaMN (blue) and F92, L105, M124, and M144 in CaMC (red) are shown as sticks relative to F880 (orange spheres) and T886 (green spheres) in the NMDA receptor CaMBD (lower). D: Model of (Ca2+)4-CaM bound to…

Figure 9. States of CaM bound to FGF12B+NaV1.2CTDor FGF12A+NaV1.2CTD.A and B, abundance of ligated CaM species in the CaM+FGF12B+NaV1.2CTD (A, gray) and CaM+FGF12A+NaV1.2CTD (B, purple) complexes. Simulations are based on the Ca2+-binding affinities reported in Table 4. States of CaM with an abundance >0.25 are shown in the inset schematics that depict the possible orientations of the CaM domains (CaMN/blue, CaMC/red) relative to FGF12B (gray) and NaV1.2CTD (green) (A) or FGF12A (purple) and NaV1.2CTD (green) (B), Ca2+ are shown as yellow circles. The yellow box is centered at physiologically relevant Ca2+ concentration of 10 μM. Representing the occupancy of the four Ca2+-binding sites in CaM (in order I, II, III, IV) with binary, where 0 is emtpy and 1 if filled, the discernible (abundances >0.05) species of CaM in the CaM+FGF12B+NaV1.2CTD complex (A) are 0000 (dotted line), 1100 (dashed and dotted line), 1101 or 1110 (dashed line), and 1111 (solid line). The discernible (abundance >0.5) species of CaM in the CaM+FGF12A+NaV1.2CTD complex (B) are 0000 (dotted line), 1000 or 0100 (dashed line), 1100 (dashed and dotted line), 1111 (solid line). C, model of CaM (CaMN/blue, CaMC/red) bound to…