Comparison of GFL-GFRalpha complexes: further evidence relating GFL bend angle to RET signalling

Abstract

Glial cell line-derived neurotrophic factor (GDNF) activates the receptor tyrosine kinase RET by binding to the GDNF-family receptor alpha1 (GFRalpha1) and forming the GDNF(2)-GFRalpha1(2)-RET(2) heterohexamer complex. A previous crystal structure of the GDNF(2)-GFRalpha1(2) complex (PDB code 2v5e) suggested that differences in signalling in GDNF-family ligand (GFL) complexes might arise from differences in the bend angle between the two monomers in the GFL homodimer. Here, a 2.35 A resolution structure of the GDNF(2)-GFRalpha1(2) complex crystallized with new cell dimensions is reported. The structure was refined to a final R factor of 22.5% (R(free) = 28%). The structures of both biological tetrameric complexes in the asymmetric unit are very similar to 2v5e and different from the artemin-GFRalpha3 structure, even though there is a small change in the structure of the GDNF. By comparison of all known GDNF and artemin structures, it is concluded that GDNF is more bent and more flexible than artemin and that this may be related to RET signalling. Comparisons also suggest that the differences between artemin and GDNF arise from the increased curvature of the artemin ;fingers', which both increases the buried surface area in the monomer-monomer interface and changes the intermonomer bend angle. From sequence comparison, it is suggested that neuturin (the second GFL) adopts an artemin-like conformation, while persephin has a different conformation to the other three.

Figure 1

The GDNF and ARTN monomer structures. (a) The GDNF monomer from the previous GDNF–GFRα1 complex (PDB code 2v5e). The structure, coloured from blue to red, consists of two two-β-strand fingers (finger 1 and 2) and a helical heel. The cystine knot is shown in magenta. (b) Structural superposition of selected ARTN monomers. ARTN structures are colour-coded: 2gh0, magenta; 2ask, green; 2gyz, yellow; 2gyr (chains A and B), cyan. The two other independent ARTN monomers in 2gyr (not shown here) are essentially identical.

Figure 2

The components of RET signalling: GFLs, GFRαs and RET. GFLs (GDNF, NRTN, ARTN and PSPN) each bind a specific coreceptor GFRα (GFRα1, GFRα2, GFRα3 and GFRα4) and activate the common signalling receptor RET (in light pink). The promiscuity of GFRα1 is shown, as it interacts with noncognate GFLs (dotted arrows) apart from PSPN. PM, plasma membrane; TK, tyrosine kinase domain; GPI, glycosylphosphatidyl inositol.

Figure 3

The crystal structure of the GDNF–GFRα1 complex. (a) Heterodimer AB is shown in blue (GFRα1) and cyan (GDNF), while heterodimer CD is shown in light pink and red. The two heterodimers (GDNF–GFRα1) are superimposed on each other and the differences are boxed. (b) Stereoview of the 2F _o − F _c electron-density map contoured at 1.2σ at the GDNF–GFRα1 interface. The important interface residues surrounding the ion triplet Arg171 ^GFRα1–Glu61^GDNF–Arg224^GFRα1 are shown with sticks colour-coded as follows: carbon (GFRα1), salmon; nitrogen, blue; oxygen, red; carbon (GDNF), cyan.

Figure 4

Comparison with previous ligand–coreceptor structures. (a) Heterodimer superposition of GDNF–GFRα1 structures. 2v5e (GDNF, red; GFRα1, yellow) was superimposed on 3fub (GDNF, cyan; GFRα1, blue). The GFRα1s were superimposed. The differences in the loop (GFRα1) and heel (GDNF) regions are marked with boxes. The same colour coding is used in (b) and (c), which show the heterotetramer superposition of the GDNF₂–GFRα1₂ structure. The left-hand heterodimer was superimposed to show the differences in the right-hand heterodimer. The twofold axis in the two heterotetramers is thus in a slightly different position in each structure; the one shown is for 3fub. The GDNF bend angle is essentially the same in both structures. (c) is rotated 90° from (b) about the horizontal axis. The red arrow represents the direction of motion between the two right-hand GFRα1s.

Figure 5

GFL bend angle and comparison of the GDNF₂–GFRα1₂ and ARTN₂–GFRα3₂ structures. (a) The bend angle for the GDNF complex structure (PDB code 3fub). Using PyMOL (DeLano, 2002 ▶), the bend angle is measured between two finger domains on both monomers (in black spheres) from the intermonomer disulfide bridge (see §2). The monomers in the GDNF homodimer are in cyan and green and the GFRα1s are in blue. (b) The bend angle for the ARTN complex structure. The ARTN homodimer is shown in magenta and yellow and the GFRα3s are in salmon. (c) Heterotetramer superposition of the ARTN₂–GFRα3₂ (PDB code 2gh0) and GDNF₂–GFRα1₂ (PDB code 3fub) structures. The left-hand heterodimers were superimposed as in Fig. 4 ▶(b). The GDNF homodimer is shown in cyan and ARTN in magenta. GFRα1 and GFRα3s are shown in blue and salmon as in (a) and (b).

Figure 6

Superposition of selected GFL homodimers. Structural superposition of ARTN and four GDNF structures. The monomer finger domains were superimposed. The ARTN structure is in magenta (PDB code 2gh0). Unbound GDNF (PDB code 1agq; Eigenbrot & Gerber, 1997 ▶) exists in two conformations: chain AB (in lemon) and chain CD (in orange). GDNF from 2v5e is in red and that from 3fub is in cyan. Finger 1 in the right-hand monomer is not shown for clarity. Only one of the five independent ARTN structures is shown as they are almost identical (see Fig. 1 ▶ b).

Figure 7

Stereoviews of the GDNF and ARTN homodimer interface. (a) Interaction between the finger domains and the heel at the GDNF and ARTN homodimer interface. The heel regions of the GDNF and ARTN structures are superimposed to show the differences. Finger 1 residues 50–61 are not shown for clarity. The cartoon loop structure of GDNF is in green, while that of ARTN is in yellow. The buried residues at the homodimer interface are shown in sticks: carbon, green (GDNF) and yellow (ARTN); oxygen, red; nitrogen, blue. Only GDNF residues are numbered. (b) Interaction of the GDNF heel with the finger domain and the intermonomer ion pair. One monomer is in surface representation (in pale green), while the heel of the other monomer is shown in green. The important interface residues that are not conserved among GFLs are shown as a brown surface for the bottom monomer and as sticks for the heel. The intermonomer ion pair between Asp80 and Arg103 is also shown. (c) The ARTN homodimer as in (b). The finger domain is shown as a pale yellow surface and heel is shown as a yellow loop.

Figure 8

Sequence alignment between GFLs. Based on the GDNF structure, human and mouse GFL sequences are aligned. The secondary structure is shown at the top together with the numbering according to human GDNF. GDNF β-strands 3 and 4 are split because of the insertion of loops L4 and L6. GDNF fingers 1 and 2 are composed of β-strands 1 and 2 and β-strands 3 and 4, respectively, as described previously (Eigenbrot & Gerber, 1997 ▶). The residues in the larger ARTN interface region have a green background. The residues Asn80 and Arg103 forming an ion pair in the GDNF and ARTN structures are shown in bold. The nonconserved buried residues are marked with an asterisk under the sequence alignment. Three critical segments for GFRα1–RET activation identified in previous studies (Baloh et al., 2000 ▶) are boxed.