Journal: Structure / Year: 2021 Title: A two-site flexible clamp mechanism for RET-GDNF-GFRα1 assembly reveals both conformational adaptation and strict geometric spacing. Authors: Sarah E Adams / Andrew G Purkiss / Phillip P Knowles / Andrea Nans / David C Briggs / Annabel Borg / Christopher P Earl / Kerry M Goodman / Agata Nawrotek / Aaron J Borg / Pauline B McIntosh ...Authors: Sarah E Adams / Andrew G Purkiss / Phillip P Knowles / Andrea Nans / David C Briggs / Annabel Borg / Christopher P Earl / Kerry M Goodman / Agata Nawrotek / Aaron J Borg / Pauline B McIntosh / Francesca M Houghton / Svend Kjær / Neil Q McDonald / Abstract: RET receptor tyrosine kinase plays vital developmental and neuroprotective roles in metazoans. GDNF family ligands (GFLs) when bound to cognate GFRα co-receptors recognize and activate RET ...RET receptor tyrosine kinase plays vital developmental and neuroprotective roles in metazoans. GDNF family ligands (GFLs) when bound to cognate GFRα co-receptors recognize and activate RET stimulating its cytoplasmic kinase function. The principles for RET ligand-co-receptor recognition are incompletely understood. Here, we report a crystal structure of the cadherin-like module (CLD1-4) from zebrafish RET revealing interdomain flexibility between CLD2 and CLD3. Comparison with a cryo-electron microscopy structure of a ligand-engaged zebrafish RET-GDNF-GFRα1a complex indicates conformational changes within a clade-specific CLD3 loop adjacent to the co-receptor. Our observations indicate that RET is a molecular clamp with a flexible calcium-dependent arm that adapts to different GFRα co-receptors, while its rigid arm recognizes a GFL dimer to align both membrane-proximal cysteine-rich domains. We also visualize linear arrays of RET-GDNF-GFRα1a suggesting that a conserved contact stabilizes higher-order species. Our study reveals that ligand-co-receptor recognition by RET involves both receptor plasticity and strict spacing of receptor dimers by GFL ligands.
History
Deposition
Oct 9, 2020
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Header (metadata) release
Jan 13, 2021
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Map release
Jan 13, 2021
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Update
Jul 14, 2021
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Current status
Jul 14, 2021
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 12.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: 45 mA
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK II / Details: 90 s wait time, 5 s blot time.
Details
Crosslinked using the GraFIX method with a gradient of 0-0.1% glutaraldehyde, 5-20% sucrose. Further purified by size exclusion chromatography.
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Electron microscopy
Microscope
FEI TITAN KRIOS
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 2 / Number real images: 12475 / Average exposure time: 9.0 sec. / Average electron dose: 48.6 e/Å2 Details: 6105 movies were collected without stage tilt, 6375 movies were collected with a tilt angle of 30 degrees.
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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