Structural mechanism of ligand activation in human calcium-sensing receptor
- Columbia University, United States
- Chinese Academy of Sciences, China
- Cornell University, United States
- The Scripps Translational Science Institute, United States
- University of Sydney, Australia
Figures
Figure 1 with 2 supplements
Crystal structures of human CaSR ECD.
(A) Inactive-state structure of CaSR ECD homodimer in the presence of 2 mM Ca2+. (B) Active-state structure of CaSR ECD homodimer in the presence of 10 mM Ca2+ and 10 mM L-Trp. Each structure is …
Figure 1—figure supplement 1
Purification of the CaSR ECD homodimer.
(A) Superdex-200 size exclusion chromatography of secreted wild-type (wt) CaSR ECD. (B) SDS gel of wt-CaSR ECD before and after Endo H digestion under reducing condition. (C) Superdex-200 size …
Figure 1—figure supplement 2
Different conformational states of CaSR ECD.
(A) Cartoon representation of the inactive (form I) CaSR ECD crystal structure. (B) Cartoon representation of the active (form II) CaSR ECD crystal structure. Each homodimer structure is shown in …
Figure 2
Agonist-induced conformational changes.
(A) Superposition of the inactive (orange) and active (blue) CaSR ECD structures based on the LB1 domain of one protomer (front view, left; side view, right). Green line is the axis of rotation that …
Figure 3 with 2 supplements
Homodimer interface.
(A) Cα trace of inactive structure with elements involved in homodimer formation highlighted by cartoons. The interface is divided into three regions I, II, and IV. Site II is further separated into …
Figure 3—figure supplement 1
Homodimer interface.
(A, B) Cα traces of inactive (A) and active (B) CaSR ECD structures. Each structure is presented in three views: top (left), front (center) and side (right). Structural elements involved in …
Figure 3—figure supplement 2
Homodimer interface.
Specific contacts at the interface areas I, II_a, and II_b of the active CaSR ECD structure.
Figure 4 with 2 supplements
L-Trp recognition by CaSR ECD.
(A) Molecular surface of a L-Trp-bound CaSR ECD protomer in the active structure. L-Trp is displayed as a space-filling model. (B) Specific contacts between CaSR ECD (gray) and L-Trp (yellow). Mesh …
Figure 4—figure supplement 1
L-Trp recognition by CaSR ECD.
(A) Molecular surface of L-Trp-bound CaSR ECD homodimer. L-Trp is displayed as a space filling model. (B, C) Specific contacts between CaSR ECD (gray) and L-Trp (yellow) within each protomer of the …
Figure 4—figure supplement 2
Endogenous ligand of CaSR.
(A) Active-state structure of CaSR ECD homodimer showing unexplained electron density at the interdomain crevice of each protomer when crystals of CaSR ECD were grown in the presence of 10 mM Ca2+ …
Figure 5 with 1 supplement
Ca2+-binding sites.
(A) Active-state structure showing peaks in anomalous difference Fourier map (magenta mesh; 3σ contour level) that correspond to bound Ca2+ ions. Sites are labeled 1–4 or 1'-4' for each protomer. (B)…
Figure 5—figure supplement 1
Ca2+-binding sites in the active homodimer.
(A) Active-state structure of CaSR ECD showing peaks in anomalous difference Fourier map (magenta mesh; 3σ contour level) that correspond to bound Ca2+ ions. Sites are labeled 1–4 or 1'-4' for each …
Figure 6
Mutational analysis of Ca2+-binding sites.
(A, B) Dose-dependent Ca2+-stimulated IP accumulation (A) and intracellular Ca2+ mobilization (B) in cells transiently expressing wt or mutant CaSR. Naturally occurring inactivating mutations I81M …
Figure 7 with 1 supplement
Anion-binding sites.
(A) Inactive-state structure showing peaks in anomalous difference Fourier map (green mesh; 3σ) that correspond to bound SO42- ions. Sites are labeled 1–3 or 1'-3' for each protomer. (B) Specific …
Figure 7—figure supplement 1
Anion-binding sites in the active homodimer.
(A) Active-state structure of CaSR ECD showing the peaks in anomalous difference Fourier map (3σ) that correspond to bound PO43- ions. The sites are labeled 2 and 4 or 2' and 4' for each protomer. (B…
Figure 8
Mutational analysis of anion-binding sites.
(A, B) Dose-dependent Ca2+-stimulated IP accumulation (A) and intracellular Ca2+ mobilization (B) in cells transiently expressing wt or mutant CaSR. Naturally-occurring inactivating mutations R66H, …
Figure 9
Activation mechanism of CaSR.
The schematic diagram shows the equilibrium between the resting and active states of CaSR and the effects of L-amino acid and Ca2+ binding.
Author response image 1
Effect of SO42- on Ca2+-stimulated IP accumulation in cells transiently expressing wild-type (WT) or mutant CaSR.
The mutations R62M and R66H affect arginine residues at anion-binding sites 1 and 3 that form hydrogen bonds formed across the interdomain cleft in the absence of any bound anion.
Author response image 2
Effect of various concentrations of L-Trp (1, 2, 0, 10 mM) on intracellular Ca2+ mobilization in the presence of 2 mM extracellular Ca2+.
(A) Wild-type (WT) CaSR. (B) S147A mutant. (C) Y218A mutant.
Author response image 3
Superposition of the CaSR and mGluR1 structures in the region of the Gd3+-binding site in mGluR1 structure.
https://doi.org/10.7554/eLife.13662.025
Author response image 4
Dose-dependent Ca2+-stimulated IP accumulation in cells transiently expressing the D234A mutant CaSR.
https://doi.org/10.7554/eLife.13662.026Tables
Table 1
Data collection and refinement statistics.
| Functional state | Inactive (2 mM Ca2+) | Active (10 mM Ca2+, 10 mM L-Trp) |
|---|---|---|
| Crystal | Form I | Form II |
| Data collection | ||
| Space group | F222 | C2 |
| Wavelength (Å) | 0.9792 | 1.7712 |
| Cell dimensions | ||
| a, b, c (Å) | 126.3, 150.2, 214.6 | 107.7, 127.5, 146.8 |
| α, β, γ (°) | 90.0, 90.0, 90.0 | 90.0, 108.7, 90.0 |
| Resolution (Å) | 88.1 - 3.1 (3.6 - 3.1) | 139.0 - 2.6 (2.9 - 2.6) |
| Rsym or Rmerge | 0.051 (0.702) | 0.043 (0.575) |
| I / σI | 21.3 (2.1) | 22.1 (2.3) |
| Completeness (%) | 99.9 (100.0) | 98.0 (97.1) |
| Redundancy | 6.6 (6.8) | 6.9 (6.8) |
| CC1/2 (%) | 100.0 (93.1) | 99.9 (96.9) |
| Refinement | ||
| Resolution (Å) | 107.2 - 3.1 | 37.5 - 2.6 |
| No. of reflections | 16,747 | 48,839 |
| Rwork / Rfree (%) | 22.2 / 23.9 | 21.1 / 22.2 |
| No. of atoms | ||
| Protein | 4564 | 8454 |
| Ligand (Trp) | - | 30 |
| Cation (Ca2+) | 1 | 8 |
| Anion | 15 (SO42-) | 20 (PO43-) |
| Sugar | 98 | 70 |
| Water | 43 | 331 |
| B-factors (Å2) | ||
| Protein | 110.4 | 68.3 |
| Ligand | - | 39.9 |
| Cation (Ca2+) | 105.4 | 96.3 |
| Anion | 102.1 (SO42-) | 61.0 (PO43-) |
| Sugar | 152.9 | 80.4 |
| Water | 76.9 | 53.8 |
| R.m.s. deviations | ||
| Bond lengths (Å) | 0.008 | 0.009 |
| Bond angles (°) | 1.15 | 1.14 |
-
Values in parentheses are for highest-resolution shell.
-
CC1/2 is defined in reference (Karplus and Diederichs, 2012).
-
Table 1—source data 1
Statistics for anomalous data collection.
- https://doi.org/10.7554/eLife.13662.004
-
Table 1—source data 2
Data collection and refinement statistics for endogenous ligand-bound CaSR ECD.
- https://doi.org/10.7554/eLife.13662.005
