Crystal structure and flexibility of the zRET CLD(1-4) module

(A) Schematic of zebrafish RET receptor tyrosine kinase. CLD, cadherin-like domains; CRD, cysteine-rich domain; TM, transmembrane helix; JM, juxtamembrane domain; KD, kinase domain.

(B) Orthogonal views of zRET^CLD1-4 colored as in (A). The calcium-binding site between CLD(2-3) has three calcium ions as green spheres with coordinating ligands as sticks and waters as red spheres.

(C) Close-up view of the coordination shell for the three calcium atoms between CLD2 and CLD3.

(D) Close-up of the interface between CLD3 and CLD4 centered on R272, selected side chains shown as sticks and dashed lines for hydrogen bonds.

(E) Superposition of chains A and B within the crystallographic asymmetric unit.

Cryo-EM structure of the zRET^ECD-zGFRα1a^D1-3-zGDNF^mat. (zRGα1a) complex

(A) Schematic of zRET^ECD, zGFRα1a^D1-3, and zGDNF^mat., color coded according to Figure 1A.

(B) Orthogonal views of the reconstituted zRGα1a complex cryo-EM map, projecting down the approximate molecular dyad or perpendicular to it. The cryo-EM map is segmented and colored by protein, with zRET^ECD cyan, zGFRα1a^D1-3 green, and zGDNF^mat. orange.

(C) Symmetry-expanded map of zRGα1a half-complex, with the map segmented and colored by protein as in (B).

(D) The final model of the zRGα1a complex built into the C2 symmetry map, colored light gray. The domains are colored as in Figure 1 with zRET^ECD: for GFRα1a domains D1-3 are pale green, green, and dark green, respectively; the two molecules of zGDNF^mat. are orange and pale orange. Two sites of interaction within the zRGα1a complex are highlighted in red dashed boxes, labeled as site 1 (zGFRα1a-zRET) and site 2 (zGDNF-zRET). Interaction residues are highlighted as sticks and the backbone represented as a cartoon.

Ligand-co-receptor-induced conformational changes in zRET^ECD

(A) The CLD3-β2-β3 loop is shown in yellow as sticks (i) projects “downward” in the view shown for zRET CLD(1-4) (see the orientation of Y292 side chain) and (ii) projects “upward” to engage the GFRα1^D2 α1 helix (green sticks) in the zRGα1a structure.

(B) The shorter CLD3-β2-β3 loop and extra helix from the human RET^ECD-NRTN-GFRα2 structure (PDB: 6Q2O) shown as olive colored sticks, domains colored as in Figure 1.

(C) Sequence alignment of RET CLD3-β2-β3 loop sequences by Espript (http://espript.ibcp.fr) (Robert and Gouet, 2014).

(D) Binding curves and calculated K_D values for zRET^ECD_wt and mutant (zRET^ECD_{P291-Q296:AAG}) binding to zGFRα1a₂-zGDNF₂ measured by MST, with a minimum of n = 3 repeats for the WT and the mutations with the SE represented.

(E) (i) Electron density map calculated using m2Fo-DFc coefficients over the CLD3-β2-β3 loop, yellow sticks and contoured at 1.0σ. (ii) Coulombic potential cryo-EM map for CLD3-β2-β3 loop from the zRGα1a complex (black mesh). Calcium ions are represented as pale green spheres.

Different GFL ligands establish a conserved spacing between RET CRD-CRD pairs in their respective ternary complexes

(A) Separation between the Cα of E613 (equivalent to E620 of hRET) from both molecules of zRET^ECD within the zRGα1a structure.

(B) Equivalent distance between the Cα E620 from both molecules of hRET^ECD from the hRET^ECD-NRTN-GFRα2 (PDB: 6Q2O) structure.

(C) Equivalent separation between the Cα E620 from the two molecules of hRET^ECD from the hRET^ECD-GDF15-GFRAL (PDB: 6Q2J) structure. The overall structure is represented as a cartoon and the Ca²⁺ ions are represented as spheres. RET is colored cyan, teal, and pale cyan in zRGα1a, hRET^ECD-NRTN-GFRα2, and hRET^ECD-GDF15-GFRAL structures, respectively. GFRα1a, GFRα2, and GFRAL are colored green, dark green, and pale green, respectively. GDNF, NRTN, and GDF15 are colored orange, red, and light pink, respectively.

Mutational analysis of zGDNF and zGFRα1 site 1 and 2 interactions with zRET^ECD

(A) Heatmap of the sequence conservation between hGFRα paralogs, and zGFRα1a mapped onto the structure of zGFRα1a D2-D3 domains reported here. Residues are colored by similarity (red highly similar to yellow through to white, least similar). Two orthogonal views are shown. Right panel, close-up of site 1 and conserved zGFRα1a residues.

(B) Binding curves and K_D values obtained using MST for zGFRα1a^D1-3 and mutations assessed in complex with zGDNF^mat., with a minimum of n = 3 repeats for the WT and the mutations with the SE represented.

(C) Heatmap of the sequence similarity between GDNF paralogs depicted as a surface representation, mapped onto zGDNF^138-235. Right panel, close-up of site 2 contact between RET^CRD and zGDNF dimer.

(D) MST binding curves and K_D values for zGDNF and mutations L156A and Y158A probed in complex with WT zGFRα1a binding to zRET^ECD.

Divergent GFRα1/GFRAL co-receptor D1 domain positions within the RET^ECD ternary complex

(A) The D1-D2 domain linker motif (SPYE), highlighted in cyan is conserved between zGFRα1a, GFRα1, GFRα2, and GFRα3. It is missing from the shorter GFRα4 that lacks a D1 domain altogether and from the divergent GFRAL.

(B) Schematic diagram of human RET^ECD, GFRAL, and GDF15 construct boundaries used and individual domains annotated as in Figure 1.

(C) (i) Negative stain EM envelope of a reconstituted hRET^ECD_2-hGDF15₂-hGFRAL₂ (hR15AL) complex docked with hR15AL (PDB: 6Q2J) revealing additional map potential indicated by a green Gaussian volume (generated from a D1 domain homology model). (ii) Cryo-EM map of zRGα1a (light gray) superposed with the final model (colored as in Figure 2) with GFRα1a^D1 shown (light green Gaussian volume at 5 Å²).

(D) Comparison of co-receptor D1 domain position and interfaces (i) GFRAL^D1 makes different contacts to domains D2-D3 (green), GFRAL^D1 shown as a 30 Å² Gaussian volume (light green), and GDF15 (salmon). (ii) zGFRα1a^D1 contacts and colored as in Figure 2. zGFRα1a^D1 represented as a 5 Å² Gaussian volume (light green).

Evidence for linear arrays of zRGα1a particles on cryo-EM grids

(A) Close-up of a representative micrograph for zRGα1a showing the particle orientation bias by fitting the dominant 2D class average view into picked particles and a recurring linear array of particles highlighted within pale cyan boxes.

(B) Statistical distribution of the difference between the angle psi (Δψ) between two particles and their separation distance. Here the angle ψ is defined for each particle as the angle of rotation of each particle required to align it onto the 2D class average.

(C) 2D class average from automated particle picking containing two adjacent zRGα1a particles.

(D) The zRGα1a-zRGα1a interface highlighted with a black box. The angle and separation between each complex is based on the peak maxima coordinates from (B) assuming both particles are at the same Z height.

(E) An electrostatic potential surface with selected side chains for the homotypic zRET^CLD2 interface.

(F) Close-up of the CLD2 contact, highlighting interface residues.

(G) Conservation of representative RET sequences at the CLD2-CLD2 interface shown with an asterix.