Structural insights into the activation of human calcium-sensing receptor

Abstract
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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 7-transmembrane domains, and inter- and intrasubunit conformational changes of 7-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.

Data availability

All data is available in the main text or the supplementary materials. Cryo-EM maps of active CaSR in complex with TNCA and inactive CaSR in complex with NB-2D11 have been deposited in the Electron Microscopy Data Bank under accession codes: EMD-30997 (NB-2D11 bound CaSR), EMD-30996 (TNCA bound CaSR). Atomic coordinates for the CaSR in complex with TNCA or NB-2D11 have been deposited in the Protein Data Bank under accession codes: 7E6U (NB-2D11 bound CaSR), 7E6T (TNCA bound CaSR).

The following data sets were generated

1. Xiaochen Chen
2. Lu Wang
3. Zhanyu Ding
4. Qianqian Cui
5. Li Han
6. Yongjun Kou
7. Wenqing Zhang
8. Haonan Wang
9. Xiaomin Jia
10. Mei Dai
11. Zhenzhong Shi
12. Yuying Li
13. Xiyang Li
14. Yong Geng
(2021) Cryo-EM structure of CaSR in complex with NB-2D11
RCSB Protein Data Bank,7E6U.

http://www.rcsb.org/structure/unreleased/7E6U
1. Xiaochen Chen
2. Lu Wang
3. Zhanyu Ding
4. Qianqian Cui
5. Li Han
6. Yongjun Kou
7. Wenqing Zhang
8. Haonan Wang
9. Xiaomin Jia
10. Mei Dai
11. Zhenzhong Shi
12. Yuying Li
13. Xiyang Li
14. Yong Geng
(2021) Cryo-EM structure of CaSR in complex with TNCA
RCSB Protein Data Bank,7E6T.

http://www.rcsb.org/structure/unreleased/7E6T

The following previously published data sets were used

1. Geng
2. Y.
3. Mosyak
4. L.
5. Kurinov
6. I.
7. Zuo
8. H.
9. Sturchler
10. E.
11. Cheng
12. T.C.
13. Subramanyam
14. P.
15. Brown
16. A.P.
17. Brennan
18. S.C.
19. Mun
20. H.-C.
21. Bush
22. M.
23. Chen
24. Y.
25. Nguyen
26. T.
27. Cao
28. B.
29. Chang
30. D.
31. Quick
32. M.
33. Conigrave
34. A.
35. Colecraft
36. H.M.
37. McDonald
38. P.
39. Fan
40. Q.R.
(2016) Crystal structure of the inactive form of human calcium-sensing receptor extracellular domain
Publicly available at the RCSB Protein Data Bank (accession no. 5K5T).

http://www.rcsb.org/ pdb/search/structid- Search.do?structureId= 5K5T
1. Geng
2. Y.
3. Mosyak
4. L.
5. Kurinov
6. I.
7. Zuo
8. H.
9. Sturchler
10. E.
11. Cheng
12. T.C.
13. Subramanyam
14. P.
15. Brown
16. A.P.
17. Brennan
18. S.C.
19. Mun
20. H.-C.
21. Bush
22. M.
23. Chen
24. Y.
25. Nguyen
26. T.
27. Cao
28. B.
29. Chang
30. D.
31. Quick
32. M.
33. Conigrave
34. A.
35. Colecraft
36. H.M.
37. McDonald
38. P.
39. Fan
40. Q.R.
(2016) Crystal structure of the active form of human calcium-sensing receptor extracellular domain
Publicly available at the RCSB Protein Data Bank (accession no. 5K5S).

http://www.rcsb.org/ pdb/search/structid- Search.do?structureId= 5K5S
1. Koehl
2. A.
3. Hu
4. H.
5. Feng
6. D.
7. Sun
8. B.
9. Weis
10. W.I.
11. Skiniotis
12. G.S.
13. Mathiesen
14. J.M.
15. Kobilka
16. B.K.
(2019) Metabotropic Glutamate Receptor 5 bound to L-quisqualate and Nb43
RCSB Protein Data Bank,6N51.

https://www.rcsb.org/structure/6N51
1. Mao
2. C.
3. Shen
4. C.
5. Li
6. C.
7. Shen
8. D.
9. Xu
10. C.
11. Zhang
12. S.
13. Zhou
14. R.
15. Shen
16. Q.
17. Chen
18. L.
19. Jiang
20. Z.
21. Liu
22. J.
23. Zhang
24. Y.
(2020) Cryo-EM structure of the baclofen/BHFF-bound human GABA(B) receptor in active state
RCSB Protein Data Bank,7C7Q.

https://www.rcsb.org/structure/7C7Q

Article and author information

Author details

Xiaochen Chen

The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-0426-9547
Lu Wang

The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

Competing interests
The authors declare that no competing interests exist.
Qianqian Cui

The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

Competing interests
The authors declare that no competing interests exist.
Zhanyu Ding

The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-8136-2243
Li Han

The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

Competing interests
The authors declare that no competing interests exist.
Yongjun Kou

The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

Competing interests
The authors declare that no competing interests exist.
Wenqing Zhang

The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

Competing interests
The authors declare that no competing interests exist.
Haonan Wang

The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

Competing interests
The authors declare that no competing interests exist.
Xiaomin Jia

The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

Competing interests
The authors declare that no competing interests exist.
Mei Dai

The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

Competing interests
The authors declare that no competing interests exist.
Zhenzhong Shi

The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

Competing interests
The authors declare that no competing interests exist.
Yuying Li

The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

Competing interests
The authors declare that no competing interests exist.
Xiyang Li

The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

Competing interests
The authors declare that no competing interests exist.
Yong Geng

The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

For correspondence
gengyong@simm.ac.cn

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0001-7144-3878

Funding

National Natural Science Foundation of China (No. 31670743)

Yong Geng

Shanghai Institute of Materia Medica, Chinese Academy of Sciences (5112345601)

Yong Geng

Shanghai Institute of Materia Medica, Chinese Academy of Sciences (2015123456005)

Yong Geng

National Natural Science Foundation of China (118180359901)

Yong Geng

Science and Technology Commission of Shanghai Municipality (No. 18JC1415400)

Yong Geng

Joint Research Fund for Overseas Chinese Scholars and Scholars in Hong Kong and Macao (No. 81628013)

Yong Geng

Natural Science Foundation of Shanghai (16ZR1442900)

Yong Geng

Shanghai Institute of Materia Medica, Chinese Academy of Sciences (CASIMM0120164013)

Yong Geng

Shanghai Institute of Materia Medica, Chinese Academy of Sciences (SIMM1606YZZ-06)

Yong Geng

Shanghai Institute of Materia Medica, Chinese Academy of Sciences (SIMM1601KF-06)

Yong Geng

Shanghai Institute of Materia Medica, Chinese Academy of Sciences (55201631121116101)

Yong Geng

Shanghai Institute of Materia Medica, Chinese Academy of Sciences (55201631121108000)

Yong Geng

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: The animal work was approved and under the supervision of Shanghai Institute of Materia Medica, Chinese Academy of Sciences (Permit Number: SYXK 2015-0027)

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.