Structural insights into the activation of human calcium-sensing receptor

Abstract

Human calcium-sensing receptor (CaSR) is a G-protein-coupled receptor that maintains Ca²⁺ homeostasis in serum. Here, we present the cryo-electron microscopy structures of the CaSR in the inactive and agonist+PAM bound states. Complemented with previously reported structures of CaSR, we show that in addition to the full inactive and active states, there are multiple intermediate states during the activation of CaSR. We used a negative allosteric nanobody to stabilize the CaSR in the fully inactive state and found a new binding site for Ca²⁺ ion that acts as a composite agonist with L-amino acid to stabilize the closure of active Venus flytraps. Our data show that agonist binding leads to compaction of the dimer, proximity of the cysteine-rich domains, large-scale transitions of seven-transmembrane domains, and inter- and intrasubunit conformational changes of seven-transmembrane domains to accommodate downstream transducers. Our results reveal the structural basis for activation mechanisms of CaSR and clarify the mode of action of Ca²⁺ ions and L-amino acid leading to the activation of the receptor.

Keywords: CaSR; G-protein-coupled receptor; GPCR; calcium ions; calcium-sensing receptor; cryo-EM; cryo-electron microscopy; molecular biophysics; nanobody; none; structural biology.

Figure 1.. The function and binding affinity of NB-2D11 and NB88.

(A) Dose-dependent NB-2D11-inhibited intracellular Ca²⁺ mobilization in response to Ca²⁺ ions. N = 3, data represent mean ± SEM (Figure 1—source data 1). (B) Dose-dependent NB-88-inhibited intracellular Ca²⁺ mobilization in response to Ca²⁺ ions. N = 3, data represent mean ± SEM (Figure 1—source data 1). (C) SPR sensorgram showing that NB-2D11 bound to CaSR with 0.24 nM affinity (Figure 1—source data 2). (D) SPR sensorgram showing that NB88 bound to CaSR with 3.9 nM affinity (Figure 1—source data 2).

Figure 2.. Cryo-EM maps and models of full-length CaSR.

(A) Left panel shows the view of CaSR in the active conformation (purple) from front view, and the right panel shows the view after a 90° rotation as indicated. (B) Left panel shows the view of CaSR in the inactive conformation (cyan) bound to NB-2D11 (orange) from front view, and the right panel shows the view after a 90° rotation as indicated. (C) Model (Ribbon representation) of CaSR shows the structure of the active state (purple) bound to TNCA (red) and Ca²⁺ ion (green) viewed from the side. (D) Model (Ribbon representation) of CaSR shows the structure of the inactive state (cyan) bind with NB-2D11 (orange).

Figure 2—figure supplement 1.. Cryo-EM processing workflow of CaSR bound to agonist+PAM.

(A) The flow chart displaying the cryo-EM processing of agonist+PAM bound CaSR in GDN. (B) Cryo-EM class averages of agonist+PAM bound CaSR in GDN micelles. (C) Local resolution map of agonist+PAM bound CaSR. (D) Gold standard Fourier shell correlation (FSC) curve indicates that the map for agonist+PAM bound CaSR reaches nominal resolutions of 2.99 Å at FSC = 0.143. (E) Particle angular distribution of the final cryo-EM reconstruction of agonist+PAM bound CaSR.

Figure 2—figure supplement 2.. Agreement between the cryo-EM map of CaSR bound to agonist+PAM and the model.

(A) The cryo-EM densities and fitted atomic models of B and C helices of CaSR in agonist+PAM bound conformation. (B) The cryo-EM densities and fitted atomic models of TNCA and related amino acids.

Figure 2—figure supplement 3.. Cryo-EM maps and models of CaSR.

(A, B) The full-length CaSR cryo-EM maps and models present view from front view in the agonist+ PAM bound state (purple) (A), and the fully inactive state (cyan) with deletion of NB-2D11 for clarity (B). Positions in the VFT (red, E228), CRD (green, E558), CRD/7TMD interface (blue, T609), and 7TM domain (yellow, P823) show that the active state is characterized by smaller intersubunit distances. The P823 position in the active model (yellow) shows the contact of 7TMDs. (C, D) Comparison of the intersubunit interfaces in active and inactive CaSR are shown for active (C) and inactive (D) CaSR. Contact regions (red) show residues within 4 Å of the opposite subunit. It should be noted that there is only one LB1–LB1 interaction interface for the inactive CaSR, and its total buried surface area is much smaller than that of the active CaSR with four different contact interfaces.

Figure 2—figure supplement 4.. Cryo-EM processing workflow of inactive CaSR bound to NB-2D11 in GDN.

(A) The flow chart displaying the cryo-EM processing of inactive CaSR bound to NB-2D11 in GDN. (B) Cryo-EM class averages of inactive CaSR bound to NB-2D11 in GDN micelles. (C) Local resolution map of inactive CaSR bound to NB-2D11. (D) Gold standard Fourier shell correlation (FSC) curve indicates that the overall map for inactive CaSR bound to NB-2D11 reaches nominal resolutions of 6.0 Å at FSC = 0.143. (E) Particle angular distribution of the final cryo-EM reconstruction of inactive CaSR bound to NB-2D11.

Figure 2—figure supplement 5.. Comparisons of the structures of CaSR in different conformations.

(A) The superimposition of CaSR^agonist+PAM (purple) and CaSR^Acc (lavender, PDB:7DTV) displays the similar structures with the r.m.s.d of 1.299 Å. (B–D) The superimpositions of CaSR^agonist+PAM (purple) and CaSR^Acc (lavender, PDB:7DTV) in intersubunit (B), the VFT (C), and 7TMD (D) regions. (E) The superimposition of CaSR^{fully inactive} (cyan) and CaSR^Icc (brown, PDB:7DTW) presents a significant difference with the r.m.s.d of 8.007 Å. (F–H) The alignment of CaSR^{fully inactive} (cyan) and CaSR^Icc (brown, PDB:7DTW) in intersubunit (F), the VFT (G), and 7TMD (H) regions. (H) The superimpositions of CaSR^agonist+PAM (purple), CaSR^Acc (lavender, PDB:7DTV), and CaSR^Icc (brown, PDB:7DTW) for VFT domain.

Figure 3.. Ca²⁺ and TNCA as a composite agonist activate the full-length CaSR dimer directly.

(A) Ribbon representation of the active CaSR (gray), showing the location of the Ca²⁺-binding sites (green sphere) and TNCA (red). (B) Specific contacts between CaSR (gray) and TNCA (red space-filling model), mesh represents the final density map contoured at 17σ surrounding. (C) Specific interactions between CaSR and newly identified Ca²⁺ ion (green sphere), the mesh represents the cryo-EM density map contoured at 6.0σ surrounding Ca²⁺. (D) Highlighting the newly identified Ca²⁺ and TNCA sharing two common binding residues S170 and E297 (cyan space-filling model). (E) Dose-dependent intracellular Ca²⁺ mobilization expressing WT (black dots), mutant S170K (red dots), E297K (cyan dots), and D190K (brown dots) of CaSR. The single mutations were designed based on Ca²⁺ binding sites. N = 4, data represent mean ± SEM (Figure 3—source data 1). (F) Dose-dependent intracellular Ca²⁺ mobilization expressing WT (black dots), mutant Y489K (red dots) of CaSR. The single mutation was designed based on Ca²⁺ binding sites. N = 4, data represent mean ± SEM (Figure 3—source data 1). (G) Dose-dependent TNCA-activated intracellular Ca²⁺ mobilization in response to 0.5 mM Ca²⁺ ions. N = 3, data represent mean ± SEM (Figure 3—source data 2).

Figure 3—figure supplement 1.. Cell surface expression.

Cell surface expression of indicated CaSR mutants. N = 3, data represent mean ± SEM.

Figure 4.. Comparisons of intersubunit LB1 domains interfaces in the inactive and active states of CaSR.

(A) Left panel: The Cα trace of VFT module of CaSR^{fully inactive} cryo-EM structure (cyan). The B-C Helix angle is 117°. Right panel: The Cα trace of VFT module of CaSR^agonist+PAM cryo-EM structure (purple). The B-Helix angle is 89°. (B) Left panel: The Cα trace of VFT module of crystal structure of CaSR-ECD^Ioo (yellow) (PDB:5K5T). The B-Helix angle is 89°. Right panel: The Cα trace of VFT module of CaSR-ECD^active crystal structure (red) (PDB:5K5S). The B-Helix angle is 89°. (C) Left panel: The Cα trace of VFT module of CaSR^Icc cryo-EM structure (brown) (PDB:7DTW). The B-Helix angle is 79°. Right panel: The Cα trace of VFT module of CaSR^Acc cryo-EM structure (lavender) (PDB:7DTV). The B-Helix angle is 80°.

Figure 4—figure supplement 1.. Comparisons of intersubunit LB1 domains interfaces in the inactive and active states of CaSR.

(A, B) The interface is a hydrophobic patch between residues on the B and C helices of each protomer. In the inactive conformation, it is an interface involving V115, V149, and L156 residues (A, cyan), whereas LB1 interface of the agonist+PAM bound state (B, purple) is packed with residues L112, L156, L159, and F160. (C) Left panel: The Cα trace of VFT module of inactive mGluR cryo-EM structure (raspberry) (PDB: 6N52). Right panel: The B-Helix angle is 125°. (D) Left panel: The Cα trace of VFT module of active mGluR cryo-EM structure (green) (PDB: 6N51). Right panel: The B-Helix angle is 66°. (E, F) Alignment of LB1 domain of CaSR in three conformations: fully inactive (cyan), intermediate (yellow, PDB:5K5T), and agonist+PAM bound (purple) states, ligand-binding residues (red). (E) Fully inactive and intermediate LB1 domain, the conformation of the ligand-binding region in LB1 domain is significantly different in two states. (F) intermediate and agonist+PAM LB1 domain, showing a well superposition.

Figure 5.. The conformational changes of LB2 domains in three states.

(A) The CaSR^{fully inactive} (cyan) conformation of VFT module. The distance between C termini of the two LB2 domains is 56.26 Å. (B) CaSR-ECD^Ioo (yellow) (PDB:5K5T) conformation of VFT module. The distance between C termini of the two LB2 domains is 45.8 Å. (C) CaSR^Icc (brown) conformation of VFT module (PDB:7DTW). The distance between C termini of the two LB2 domains is 41.8 Å. (D) CaSR^agonist+PAM (purple) conformation of VFT module. The distance between C termini of the two LB2 domains is 29 Å.

Figure 5—figure supplement 1.. Superposition of LB1 and VFT domains of CaSR.

(A) Inactive and intermediate LB2 domains, (B) intermediate (PDB:5K5T) and active LB2 domains, which indicate the three conformations of LB2 domain have no distinguishing differences, suggesting the ligand-binding residues in LB2 domain do not change during approach of LB2 domain. (C–E) Superpositions of VFT module based on LB1 domains in the inactive and intermediate (C), intermediate and active (D), and inactive, intermediate, and active states (E) of CaSR.

Figure 6.. The NB-2D11 blocks the interaction of LB2 domains.

(A) Structure of the inactive CaSR protomer (surface representation, cyan) with NB-2D11 (ribbon diagram, orange) from front view. (B) The NB-2D11 (orange) binds the left lateral of the LB2 (cyan) from the front view of the protomer. (C) NB-2D11 binds the LB2 domain through a series of polar interactions through CDR1 and CDR3 of the nanobody and the Helix F and Strand I of the CaSR. (D) Superposition of NB-2D11 (orange) binding inactive conformations (cyan) and active (purple) conformations based on the LB2 domain of VFT module, showing the whole NB-2D11 in the inactive state crashes with the LB2 domain of another VFT module in the active state.

Figure 7.. The closure of VFT leading to the rearrangement of inter-7TMDs.

(A) Front view of CaSR^{fully inactive} CRDs and 7TMDs (cyan). (B) Front view of CaSR^agonist+PAM CRDs and 7TMDs (purple). (C) The alignment of the part of CRD and 7TMDs in both fully inactive and agonist+PAM bound CaSR. (D) The alignment of inactive and agonist+PAM bound 7TMDs from top view. (E–G) The 7TMDs interface in the fully inactive state of CaSR is mediated by TM5 and TM6 (cyan) from top view and that of the agonist+PAM state is driven by TM6 from top view. Superposition of 7TMD of the inactive (cyan) and agonist+PAM bound CaSR (purple) show the rotation of 7TMDs. (H–J) Dose-dependent intracellular Ca²⁺ mobilization expressing WT (black dots) and mutant (red dots) CaSR (Figure 7—source data 1). The single mutations of F789A (H), F792A (I), and P823R (J) were designed based on the inactive density map. For (H–J), N = 4, data represent mean ± SEM.

Figure 7—figure supplement 1.. The homodimer interface of the CRDs in the active state of CaSR.

(A) The homodimer interface of the CRDs (surface representation, purple). Contact regions (red) show residues within 4 Å of the opposite. It covers approximately 1079.09 Ų of solvent accessible surface area. (B) There is a bound Ca²⁺ coordinated by carboxylate group of D234 and carbonyl oxygen of E231 and G557, holding the interface that is required to activate the receptor. (C) The CR–CR contacts were maintained through the cross-subunit hydrogen bonds between T560 and E558, and hydrophobic interaction of I554 and P569.

Figure 7—figure supplement 2.. Conformational change of the 7TMDs interface during activation.

(A) The 7TMD configuration in the inactive state from front view. There are pairwise symmetrical undefined maps that link the extracellular and intracellular part of TM5 and TM6 in the 7TM dimer interface, blocking the association of 7TMDs could regulate the function of CaSR. (B, C) The 7TMDs interface of the active state of mGluR5 (C, PDB:6N51) and GABA_B (D, PDB:7C7Q) have the interface contact with TM6.

Figure 8.. Upward movement of LB2 is converted into the intra-7TM conformational rearrangement through ECL2.

(A) Model in CaSR^agonist+PAM state (purple) and cryo-EM map (gray) showing the contact between the CRD and the ECL2 of the 7TMDs. Critical residues at this interface are shown as spheres at their Cα positions. (B) Superposition of the interface between the CRD and the ECL2 of the 7TMD between both fully inactive and agonist+PAM bound conformations. (C) Specific contacts between the loop of CR domain and the loop of ECL2 to shift the ECL2 up. (D–F) Deletion of residues D758 and E759 (D), the single mutation of K601E (E) and W590E (F) significantly reduced Ca²⁺-induced receptor activity. (WT in black dots and mutant in red dots). For (D–F), N = 4, data represent mean ± SEM (Figure 8—source data 1).

Author response image 1.. Does-response curves of TNCA-induced intracellular Ca²⁺ mobilization in presence of 0.

5mM extracellular Ca²⁺ ion.

Author response image 2.. Normalized cAMP accumulation upon stimulation with 10 mM ca²⁺ in the absence or presence of NB2D11.