National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R35GM141871
United States
Citation
Journal: Nature / Year: 2024 Title: Promiscuous G-protein activation by the calcium-sensing receptor. Authors: Hao Zuo / Jinseo Park / Aurel Frangaj / Jianxiang Ye / Guanqi Lu / Jamie J Manning / Wesley B Asher / Zhengyuan Lu / Guo-Bin Hu / Liguo Wang / Joshua Mendez / Edward Eng / Zhening Zhang / ...Authors: Hao Zuo / Jinseo Park / Aurel Frangaj / Jianxiang Ye / Guanqi Lu / Jamie J Manning / Wesley B Asher / Zhengyuan Lu / Guo-Bin Hu / Liguo Wang / Joshua Mendez / Edward Eng / Zhening Zhang / Xin Lin / Robert Grassucci / Wayne A Hendrickson / Oliver B Clarke / Jonathan A Javitch / Arthur D Conigrave / Qing R Fan / Abstract: The human calcium-sensing receptor (CaSR) detects fluctuations in the extracellular Ca concentration and maintains Ca homeostasis. It also mediates diverse cellular processes not associated with Ca ...The human calcium-sensing receptor (CaSR) detects fluctuations in the extracellular Ca concentration and maintains Ca homeostasis. It also mediates diverse cellular processes not associated with Ca balance. The functional pleiotropy of CaSR arises in part from its ability to signal through several G-protein subtypes. We determined structures of CaSR in complex with G proteins from three different subfamilies: G, G and G. We found that the homodimeric CaSR of each complex couples to a single G protein through a common mode. This involves the C-terminal helix of each Gα subunit binding to a shallow pocket that is formed in one CaSR subunit by all three intracellular loops (ICL1-ICL3), an extended transmembrane helix 3 and an ordered C-terminal region. G-protein binding expands the transmembrane dimer interface, which is further stabilized by phospholipid. The restraint imposed by the receptor dimer, in combination with ICL2, enables G-protein activation by facilitating conformational transition of Gα. We identified a single Gα residue that determines G and G versus G selectivity. The length and flexibility of ICL2 allows CaSR to bind all three Gα subtypes, thereby conferring capacity for promiscuous G-protein coupling.
Entire : Human CaSR in complex with miniGi1 protein in detergent
Entire
Name: Human CaSR in complex with miniGi1 protein in detergent
Components
Complex: Human CaSR in complex with miniGi1 protein in detergent
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Supramolecule #1: Human CaSR in complex with miniGi1 protein in detergent
Supramolecule
Name: Human CaSR in complex with miniGi1 protein in detergent type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)
Organism: Homo sapiens (human)
Molecular weight
Theoretical: 301 KDa
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Experimental details
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Structure determination
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
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Sample preparation
Concentration
3.5 mg/mL
Buffer
pH: 7.5 Component:
Concentration
Formula
Name
20.0 mM
C8H18N2O4S
HEPES
100.0 mM
NaCl
Sodium chloride
2.0 mM
MgCl2
Magnesium chloride
10.0 mM
CaCl2
Calcium chloride
20.0 uM
C12H12N2O2
TNCA
20.0 uM
C18H22ClNOHCl
R568 HCl
C56H92O25
0.005% glycol-diosgenin (GDN)
C31H50O4.C4H11NO3
0.001% cholesterol hemisuccinate Tris salt (CHS)
Grid
Model: Quantifoil R0.6/1 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 50 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 25 sec. / Pretreatment - Atmosphere: OTHER
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV Details: The sample was blotted for 6s before plunge-frozen..
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Electron microscopy
Microscope
FEI TITAN KRIOS
Temperature
Max: 100.0 K
Specialist optics
Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Software
Name: Leginon (ver. 3.6)
Image recording
Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 15713 / Average exposure time: 2.5 sec. / Average electron dose: 70.14 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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