A two-site flexible clamp mechanism for RET-GDNF-GFRα1 assembly reveals both conformational adaptation and strict geometric spacing

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 RETECD-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 RETECD-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. [Display omitted] •X-ray structure of zebrafish RETCLD1-4 module reveals conformational flexibility•Conformational differences between RETCLD1-4 and a liganded RETECD cryo-EM structure•Spatial separation of RETECD CRD dimer C termini imposed by each ligand dimer•Differences in GDNF and GDF15 co-receptor engagement of RET and multimerization evidence Adams et al. use X-ray crystallography and cryo-electron microscopy to probe conformational changes in RET due to binding of GDNF ligand-GFRα1 co-receptor. The study shows a two-site clamping mechanism with flexible adaptations near the RET calcium sites but near identical spacings of the RET cysteine-rich domains established by GDNF/GDF15 ligand dimers.