Structural mechanism of cooperative activation of the human calcium-sensing receptor by Ca2+ ions and L-tryptophan

Abstract

The human calcium-sensing receptor (CaSR) is a class C G protein-coupled receptor (GPCR) responsible for maintaining Ca²⁺ homeostasis in the blood. The general consensus is that extracellular Ca²⁺ is the principal agonist of CaSR. Aliphatic and aromatic L-amino acids, such as L-Phe and L-Trp, increase the sensitivity of CaSR towards Ca²⁺ and are considered allosteric activators. Crystal structures of the extracellular domain (ECD) of CaSR dimer have demonstrated Ca²⁺ and L-Trp binding sites and conformational changes of the ECD upon Ca²⁺/L-Trp binding. However, it remains to be understood at the structural level how Ca²⁺/L-Trp binding to the ECD leads to conformational changes in transmembrane domains (TMDs) and consequent CaSR activation. Here, we determined the structures of full-length human CaSR in the inactive state, Ca²⁺- or L-Trp-bound states, and Ca²⁺/L-Trp-bound active state using single-particle cryo-electron microscopy. Structural studies demonstrate that L-Trp binding induces the closure of the Venus flytrap (VFT) domain of CaSR, bringing the receptor into an intermediate active state. Ca²⁺ binding relays the conformational changes from the VFT domains to the TMDs, consequently inducing close contact between the two TMDs of dimeric CaSR, activating the receptor. Importantly, our structural and functional studies reveal that Ca²⁺ ions and L-Trp activate CaSR cooperatively. Amino acids are not able to activate CaSR alone, but can promote the receptor activation in the presence of Ca²⁺. Our data provide complementary insights into the activation of class C GPCRs and may aid in the development of novel drugs targeting CaSR.

Fig. 1. Cryo-EM structures of CaSR in active and inactive states.

a Map and cartoon representations of the dimeric CaSR structure in the Ca²⁺/L-Trp-bound state, representing the structure of active CaSR. The two subunits are colored in green and cyan, respectively. Ca²⁺ ions are shown as magenta spheres. b Detailed view of the L-Trp and Ca²⁺ binding sites of CaSR in the active CaSR structure. L-Trp is shown as yellow stick, and Ca²⁺ is shown as magenta sphere. Residues in CaSR that participate in the interaction with Ca²⁺ and L-Trp are shown as sticks. c Representative 2D class average images (upper panel) and three distinct classes of 3D reconstruction density maps are obtained with resolutions of 4.5 Å, 5.7 Å, and 6.8 Å, respectively (lower panel). d Cartoon representation of the dimeric inactive CaSR structure in the Icc conformation (CaSR^Icc). Extra density located at the cleft between LB1 and LB2 in the VFT domain is shown in the inset. e Corresponding coordinates fit into the reconstruction maps of the LB1/LB2 of each VFT domain in the three models shown in c. The dimeric VFT domains with Icc conformation are shown in orange, and Ioc and Ioo VFT domains are shown in wheat and gray, respectively. f Superimposition of the closed VFT domains of CaSR in the Acc and Icc conformations, showing the structural similarity of the two closed VFT domains. g Superimposition of a single VFT domain in Acc and Ioo conformations, showing the opening of the VFT in the Ioo conformation (gray) and the closure of the VFT in the Acc conformation (green).

Fig. 2. Cryo-EM structure of CaSR in the L-Trp-bound state.

a Map and cartoon representations of the dimeric CaSR structure in the L-Trp-bound state (CaSR^Trp). The two subunits of CaSR are colored in cyan. L-Trp molecules are shown as yellow sticks. b Cartoon representation of a single VFT domain structure of CaSR^Trp. The density that was assigned to L-Trp is shown as mesh. c Superposition of the overall structures of CaSR^Trp and CaSR in the Icc conformation, indicating high structural similarity. d Structural superposition of single VFT domains derived from CaSR^Trp (blue) and CaSR^Icc (orange). The VFT domains from CaSR in both CaSR^Trp and CaSR^Icc reveal “closed” conformations. e Structural superimposition of single VFT domains in CaSR^Trp and CaSR^Ioo, showing the opening of the VFT in the Ioo conformation (gray) and the closure of the VFT in the CaSR^Trp (blue).

Fig. 3. Cryo-EM structure of CaSR in the Ca²⁺-bound state.

a Representative 2D class average images (upper panel) and three distinct classes of 3D reconstruction density maps are obtained with resolutions of 3.8 Å, 5.6 Å, and 7.3 Å, respectively (lower panel). b Cartoon representation of the dimeric Ca²⁺-bound CaSR structure in the closed-closed conformation (CaSR^Ca). Extra density located in the cleft between LB1 and LB2 in the VFT domain is shown in mesh. The Ca²⁺ ion is shown as a magenta sphere. c Superposition of the overall structures of CaSR^Acc and CaSR in the Ca²⁺-bound closed-closed conformation. d L-Trp concentration-dependent activation of CaSR in the presence of Ca²⁺ ions. e Ca²⁺ concentration-dependent activation of CaSR mutants indicates that mutations of residues in L-Trp binding sites reduce receptor activation by Ca²⁺. The IP₁ accumulation data in d and e represent the means ± SD of three independent experiments.

Fig. 4. Conformational changes of CaSR upon Ca²⁺ binding.

a Structural comparison of CaSR dimers in inactive (orange) and active (green) states. The dimeric CaSR structures are viewed in slices parallel to the membrane plane. b Gradual increase in the rotational angles of different subdomains down to the TMD along the symmetric axis of the inactive (orange) and active (green) CaSR dimers. The rotational angles of each subdomain are indicated. c Conformational changes of the interface between the LB1 loop and helix N. In Ca²⁺/L-Trp-bound active CaSR, the LB1 loop (green) reaches across the dimerization interface between two LB1 regions to contact helix N in the adjacent subunit, leading to domain rotation along the dimer axis. The LB1 loop and helix N in inactive CaSR are shown in orange. d Mutations of residues in hydrophobic sites in the LB1 loop (L51A) and helix N (F444A, W458A) reduced receptor activation by Ca²⁺. The IP₁ accumulation data represent the means ± SD of three independent experiments. e Ca²⁺ ions were coordinated by D234, E231 in the LB2 region of one subunit, and G557 in the CRD of the other subunit, increasing the proximity of G557 residues of the two subunits in the active CaSR compared with that in the inactive CaSR. f The G557E mutation decreased the Ca²⁺-induced receptor response. The IP₁ accumulation data represent the means ± SD of three independent experiments. g Structure model and cryo-EM map showing interactions between ECL2 of the TMD and the CRD. Critical residues at the CRD–TMD interface are shown as spheres at their Cα positions (I761, F762, I763, I603, and F605), indicating hydrophobic interactions between ECL2 and the linker region connecting the CRD and TMD. h Site mutations disrupting hydrophobic interactions between ECL2 and the CRD–TMD linker decreased the sensitivity of CaSR to Ca²⁺, as shown by Ca²⁺-stimulated IP₁ accumulation assay. Data represent the means ± SD of three independent experiments.

Fig. 5. Rotation of the TMDs of CaSR during activation.

a, b Compact dimerized TMDs from inactive CaSR (a) and active CaSR (b) are shown as cartoons in side view (upper panel) and top view (lower panel). A824 and S827, which are located in TM6, are indicated by red spheres. c Representative cryo-EM map and fitted atomic model of TM6 of CaSR^Acc, indicating the agreement between the map and the model. d Mutations in residues contributing to TM6-mediated contact points (A824K and S827K) decreased the Ca²⁺-induced receptor response. The data represent the means ± SD of three independent experiments performed in triplicate. e A hypothetical model for cooperative activation of CaSR by L-Trp and Ca²⁺. The binding of the L-Trp molecule in the cleft of the VFT domain closes and stabilizes the VFT domain. Conformational changes in the LB1 loop region upon Ca²⁺ binding initiate twisting of the VFT domains. Ca²⁺ binds to the VFT domain and the junction between the LB2 region and the CRD, bringing the LB2 region and CRD closer through rigid-body domain twisting. The increased proximity of the LB2 regions is propagated to the TMDs through the interaction between the CRD and TMD-ECL2 region. A series of domain twisting motions between domains of CaSR dimer increase the proximity of the TMDs and produce a new TM6-mediated TMD interface for downstream signal transduction.