Structure and biophysical characterization of the human full-length neurturin–GFRa2 complex: A role for heparan sulfate in signaling

Abstract

Neurturin (NRTN) provides trophic support to neurons and is considered a therapeutic agent for neurodegenerative diseases, such as Parkinson's disease. It binds to its co-receptor GFRa2 and the resulting NRTN:GFRa2 complex activates the transmembrane receptors RET or NCAM. We report the crystal structure of NRTN, alone and in complex with GFRa2. This is the first crystal structure of a GFRa with all three domains and shows that domain 1 does not interact directly with NRTN, but may support an interaction with RET and/or NCAM, via a highly conserved surface. In addition, biophysical results show that the relative concentration of GFRa2 on cell surfaces can affect the functional affinity of NRTN through avidity effects. We have identified a heparan sulfate binding site on NRTN and a putative binding site in GFRa2, suggesting that heparan sulfate has a role in assembly of the signalling complex. We further show that mutant NRTN with reduced affinity for heparan sulfate may provide a route forward for delivery of NRTN with increased exposure in preclinical in vivo models and ultimately to Parkinson's patients.

Revisiting the Role of Neurotrophic Factors in Inflammation

“…However, GFRα may also signal independently of Ret via the neural cell adhesion molecule (NCAM) pathway [2]. In addition, heparan sulfate proteo-glycosaminoglycans (HSPG) can serve as co-receptors of Ret to transduce GFLs signals, leading to downstream activation via Src kinases [3]. This family is well described for its role in the growth and the maintenance of the peripheral and central nervous systems and acts on dopaminergic, sensory, motor, hippocampal, midbrain, enteric, sympathetic and parasympathetic neurons [4].…”

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

“…N-acetylglucosamine (GlcNAcβ1-Asn) glycan rings linked to asparagine sites were also evident in the map. Density was also evident for zGFRα1a D1 , sandwiched between zGFRα1a D3 and zRET CLD1 , at a similar position to GFRα2 D1 (Bigalke et al, 2019;Li et al, 2019;Sandmark et al, 2018) Figure 11).…”

“…We then fitted crystal structures for zRET CLD(1-4) and zGDNF mat -zGFRα1a ΔD1 into the symmetry-expanded map ( Figure 2C) together with homology models for the zRET CRD and zGFRα1a D1 . An initial model for zRET CRD was generated from the hRET ECD -hGFRα2-NRTN structure (Li et al, 2019) and for zGFRα1a D1 from the hGFRα2-NRTN (Sandmark et al, 2018) structure by substituting zebrafish sequences followed by model optimisation using Swiss-Model (Schwede et al, 2003) and Modeller (Webb and Sali, 2016), respectively. The initial structure was refined against the symmetry-expanded map and rebuilt, before placing it into the C2 averaged map for further refinement in PHENIX (Adams et al, 2010) (Supplementary Table 2, Supplementary Figure 10).…”

“…To build a full ligand-co-receptor complex, the zGDNF mat. -zGFRα1 ΔD1 crystal structure described here was used together with a homology model of domain D1 (zGFRα1 generated by MODELLER from the GFRα2-neurturin crystal structure (PDB 5MR4) (Sandmark et al, 2018;Webb and Sali, 2016). For zRET, chain A of the CLD(1-4) module described here was used together with a CRD model generated with SwissPROT (Schwede et al, 2003) using the structure of hRET ECD in complex with GFRα2-neurturin (PDB 6Q2O) (Li et al, 2019;Webb and Sali, 2016).…”

Improving therapeutic potential of GDNF family ligands

“…Thus, in the presence of soluble co-receptors, GDNF family ligands can active RET also in cells which lack endogenous co-receptor expression. The crystal structures of GDNF/GFRa1 (Parkash et al 2008 ), NRTN/GFRa2 (Sandmark et al 2018 ), and ARTN/GFRa3 (Wang et al 2006 ) complexes have been solved. Even the structures of the full GDNF/GFRa1/RET and NRTN/GFRa2/RET complexes have been resolved using a combination of electron microscopy and low-angle X-ray scattering (Goodman et al 2014 ), or cryo-EM (Bigalke et al 2019 ).…”