Structure of the photoreceptor synaptic assembly of the extracellular matrix protein pikachurin with the orphan receptor GPR179

Abstract

Precise synapse formation is essential for normal functioning of the nervous system. Retinal photoreceptors establish selective contacts with bipolar cells, aligning the neurotransmitter release apparatus with postsynaptic signaling cascades. This involves transsynaptic assembly between the dystroglycan-dystrophin complex on the photoreceptor and the orphan receptor GPR179 on the bipolar cell, which is mediated by the extracellular matrix protein pikachurin (also known as EGFLAM). This complex plays a critical role in the synaptic organization of photoreceptors and signal transmission, and mutations affecting its components cause blinding disorders in humans. Here, we investigated the structural organization and molecular mechanisms by which pikachurin orchestrates transsynaptic assembly and solved structures of the human pikachurin domains by x-ray crystallography and of the GPR179-pikachurin complex by single-particle, cryo-electron microscopy. The structures reveal molecular recognition principles of pikachurin by the Cache domains of GPR179 and show how the interaction is involved in the transsynaptic alignment of the signaling machinery. Together, these data provide a structural basis for understanding the synaptic organization of photoreceptors and ocular pathology.

Fig. 1.. Role of pikachurin in photoreceptor synapse organization and crystal structures of the constituent domains of pikachurin.

(A) pikachurin forms a transsynaptic connection by interacting with the pre-synaptic photoreceptor dystroglycan complex (DGC) and the post-synaptic receptor GPR179 at the ON-bipolar cell (ON-BC). (B) pikachurin is composed of an N-terminal FN3 tandem followed by three EGF-LG tandems (middle). Left: A cartoon representation of the crystal structure of the N-terminal FN3 tandem is shown in green. The FN3 connecting loop harboring the 3₁₀ helix and disulfide bridge provides rigidity to the bent FN3 tandem assembly. Glycosylation at Asn⁴⁷ (N47) is shown with cyan-colored carbohydrate moieties. Right: The crystal structure of the C-terminal LG3 domain is represented in orange. Glycosylation and disulfide bridge sites are shown in cyan and blue, respectively. CT, C terminus; NT, N terminus.

Fig. 2.. Cryo-EM structure of pikachurin complexed with GPR179.

(A) Cryo-EM map of a GPR179 homodimer complexed with the EGF3-LG3 of pikachurin. GPR179 protomers are shown in cyan and green, whereas the pikachurin domains are shown in orange and purple. (B) Focused refinement of the GPR179 ectodomains and the pikachurin domains provides well-resolved density for the GPR179 dimeric Cache domain and the LG3 domains. Regions with poor density were excluded from the final model.

Fig. 3.. Structural organization of the GPR179 ectodomain featuring the Cache domain.

(A) The GPR179 ectodomain is composed of an N-terminal domain, a Cache domain, and a stalk region. Its dimeric assembly through the Cache domain is shown as a side view in the cartoon representation. (B) GPR179 contains a central globular domain, a Cache domain that is composed of six antiparallel β-sheets bordered by α-helices from the backside and a β-hairpin in the lever loop. (C) Top view of Cache domain dimerization, which generates a β-barrel–like shape that captures α-helices at the barrel pore. The β-hairpin fills the gap between the two antiparallel β-sheets cores of the Cache domains at the dimeric interface resembling the barrel fold. (D) The β-hairpin is also involved in the dimeric interface, interacting with the α2 and β3 residues of another Cache domain (shown in cyan).

Fig. 4.. Mechanism of interaction between pikachurin and GPR179.

(A) Cartoon representation of overall interaction between the Cache domains of GPR179 and the LG3 domains of pikachurin, in which each Cache domain mainly targets one LG3 domain, forming a tetrameric assembly. The glycosylation site at Asn⁹¹⁷ (N917, blue sphere) is distant from the interaction interface. (B) Binding site I involves the lower leaflet of the β-sandwich core of LG3, which passes perpendicularly over the α-helix α2' and the β-hairpin at the top of the Cache domain. Sites II and III stabilize the binding at opposing ends involving loop regions. (C) Maps of surface electrostatic potentials. The binding of pikachurin generates a complementary electrostatic surface for favorable complex formation with GPR179.

Fig. 5.. Analysis of the effects of CSNB-associated mutations on GPR179 structure.

(A) The mutations D126H and Y220C are found in the Cache domain of GPR179 (represented as sticks). Asp¹²⁶ (D126, blue) is present in the α2' helix, whereas Tyr²²⁰ (Y220) is located in the β3" β-hairpin sheet and can participate in stabilization of GPR179 homodimeric assembly. (B) Tyr²²⁰ (Y220) can form a π-π interaction with Tyr²⁵⁴ (Y254) of the β5 strand and participate in hydrogen bonding with Arg²⁰⁰ (R200) of β3'.0 The Y220C mutation would be expected to hamper these interactions, ultimately destabilizing the dimeric interface involving the β-hairpin.