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 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).
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Cryo-EM structure of CaSR in complex with NB-2D11RCSB Protein Data Bank,7E6U.
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Cryo-EM structure of CaSR in complex with TNCARCSB Protein Data Bank,7E6T.
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Crystal structure of the inactive form of human calcium-sensing receptor extracellular domainPublicly available at the RCSB Protein Data Bank (accession no. 5K5T).
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Crystal structure of the active form of human calcium-sensing receptor extracellular domainPublicly available at the RCSB Protein Data Bank (accession no. 5K5S).
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Metabotropic Glutamate Receptor 5 bound to L-quisqualate and Nb43RCSB Protein Data Bank,6N51.
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Cryo-EM structure of the baclofen/BHFF-bound human GABA(B) receptor in active stateRCSB Protein Data Bank,7C7Q.
Article and author information
Author details
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
© 2021, Chen et al.
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.
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Further reading
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We present near-atomic-resolution cryoEM structures of the mammalian voltage-gated potassium channel Kv1.2 in open, C-type inactivated, toxin-blocked and sodium-bound states at 3.2 Å, 2.5 Å, 3.2 Å, and 2.9 Å. These structures, all obtained at nominally zero membrane potential in detergent micelles, reveal distinct ion-occupancy patterns in the selectivity filter. The first two structures are very similar to those reported in the related Shaker channel and the much-studied Kv1.2–2.1 chimeric channel. On the other hand, two new structures show unexpected patterns of ion occupancy. First, the toxin α-Dendrotoxin, like Charybdotoxin, is seen to attach to the negatively-charged channel outer mouth, and a lysine residue penetrates into the selectivity filter, with the terminal amine coordinated by carbonyls, partially disrupting the outermost ion-binding site. In the remainder of the filter two densities of bound ions are observed, rather than three as observed with other toxin-blocked Kv channels. Second, a structure of Kv1.2 in Na+ solution does not show collapse or destabilization of the selectivity filter, but instead shows an intact selectivity filter with ion density in each binding site. We also attempted to image the C-type inactivated Kv1.2 W366F channel in Na+ solution, but the protein conformation was seen to be highly variable and only a low-resolution structure could be obtained. These findings present new insights into the stability of the selectivity filter and the mechanism of toxin block of this intensively studied, voltage-gated potassium channel.
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xCas9 is an evolved variant of the CRISPR-Cas9 genome editing system, engineered to improve specificity and reduce undesired off-target effects. How xCas9 expands the DNA targeting capability of Cas9 by recognising a series of alternative protospacer adjacent motif (PAM) sequences while ignoring others is unknown. Here, we elucidate the molecular mechanism underlying xCas9’s expanded PAM recognition and provide critical insights for expanding DNA targeting. We demonstrate that while wild-type Cas9 enforces stringent guanine selection through the rigidity of its interacting arginine dyad, xCas9 introduces flexibility in R1335, enabling selective recognition of specific PAM sequences. This increased flexibility confers a pronounced entropic preference, which also improves recognition of the canonical TGG PAM. Furthermore, xCas9 enhances DNA binding to alternative PAM sequences during the early evolution cycles, while favouring binding to the canonical PAM in the final evolution cycle. This dual functionality highlights how xCas9 broadens PAM recognition and underscores the importance of fine-tuning the flexibility of the PAM-interacting cleft as a key strategy for expanding the DNA targeting potential of CRISPR-Cas systems. These findings deepen our understanding of DNA recognition in xCas9 and may apply to other CRISPR-Cas systems with similar PAM recognition requirements.