Structure of the human galanin receptor 2 bound to galanin and Gq reveals the basis of ligand specificity and how binding affects the G-protein interface

Introduction

Human galanin is a neuropeptide composed of 30 amino acids whose highly conserved N-terminus is linked to biological activities [1]. Its GPCR family receptors (from GALR1 to GALR3) are associated with different disease states and have been used as markers for certain cancer types [2]. Fragments of galanin may have distinct roles from the integral neuropeptide; for example, galanin^2–11 binds to GALR2 roughly 500 times more tightly than GALR1 [3–5]. Nerve damage induces synthesis of galanin, which appears to show neuroprotective effects mediated by GALR2 [6]. Agonists for GALR2 are believed to have a potential as treatments for a variety of nervous disorders by acting as antidepressants or anticonvulsants as well as having analgesic properties, stimulating nerve growth, and reducing neuronal damage [7–10]. Human GALR2 and human GALR3 show roughly 55% sequence conservation with each other, while human GALR2 shows only 37% sequence identity with human GALR1 [11]. All of them signal via Gi/o and inhibit the adenylyl cyclase, but GALR2 can also activate phospholipase C (PLC) through Gq/11, leading to inositol triphosphate accumulation and a subsequent increase of the concentration of intracellular calcium [12]. In order to understand the signaling pathway of GALR2 and interactions between GALR2 and galanin, we determined the cryo-electron microscopy (cryo-EM) structure of human GALR2 with holo-galanin and associated G-proteins.

Results and discussion

To determine the molecular structure of activated GALR2, a complex was purified composed of GALR2, galanin, heterotrimeric mGαq_iN/Gβ1γ2, and scFv16 (S1 Fig). The sample was plunge-frozen, and micrographs were collected using a 300-kV Titan Krios G4 with a Gatan K3 direct detector in the movie mode. The data were processed with cryoSPARC (v.3.3.1) and were refined with PHENIX (v.1.19.2) (Table 1 and S2A and S2B Fig). The density map was determined to a resolution of 3.11 Å (with FSC cutoff of 0.143). Side chains of GALR2 TM helices are visible for most residues (S2B and S2C Fig). The final model of GALR2 includes all residues from Glu24^1.31 to Arg300^8.51, including the intracellular loops (ICLs) and extracellular loops (ECLs). The scFv16 stabilizes the complex by binding to the N-terminal helix of mGαq_iN and a surface loop of Gβ1 (Fig 1A).

Activated GPCRs act as nucleotide exchange factors through direct interactions with heterotrimeric G-proteins. The C-terminal helix of mGαq_iN fits into a hydrophobic pocket formed by TM 5–7 of GALR2 (Fig 1B). Phe228, Ile235, Asn239, and Tyr243 of mGαq_iN form the principal interface with GALR2, and a salt bridge forms between Glu135^ICL2 of GALR2 and Arg32 of mGαq_iN (Fig 1B). The TM helices of GALR2 form a relatively shallow galanin-binding pocket at the extracellular face of the protein (Fig 1C), where the first 16 residues of galanin were modeled into the density map (Fig 2A). The modeled fragment adopts a compact shape with helix-like conformation from Leu4^P to Leu11^P (Fig 2A). Galanin touches each ECL, and it also forms hydrophobic contacts near the N-termini of TM2 and TM7 (Fig 2B). Galanin has a similar sequence to galanin-like peptide (GALP) and spexin (S3A Fig), which can also act as endogenous ligands and activate GALR2 [13]. Trp2^P, Thr3^P, Tyr9^P, Leu10^P, and Gly12^P of galanin are common to these peptides (S3A Fig). Galanin is well conserved among various species, but the last 15 residues are more variable than the N-terminal region, which is known to interact with GALRs (S3B Fig). Replacing Trp2^P of galanin with alanine prevents binding to GALRs [4]. This tryptophan side chain makes a hydrophobic interaction with Leu266^ECL3 of GALR2 (Fig 2C). The side chain of Tyr9^P reaches furthest into the pocket, where it makes interactions with Ile85^2.64×63, His102^3.29 and Tyr164^4.64×65 of the receptor (Fig 2C). Leu10^P interacts with Phe264^ECL3, while Pro13^P packs against His176^ECL2 and Pro177^ECL2 (Fig 2C). His14^P, Ala15^P, and Val16^P are completely exposed on the surface of the complex and do not make any interactions with the receptor (Fig 2C). While the majority of the observed galanin residues interact with the receptor to some extent, Trp2^P, Asn5^P, and Tyr9^P appear to be central to receptor binding, while Leu10^P also makes substantial hydrophobic interactions (Fig 2C).

Comparing the sequences of the human galanin receptors (GALR1–GALR3), the majority of the neuropeptide binding site is found to be preserved, but not perfectly. Cys98^3.25 and Cys175^ECL2 of GALR2 form a disulfide bridge, and an equivalent bond is presumably also found in the other 2 receptors (S3C Fig). This bond, connecting the N-terminus of TM3 and ECL2 will create a more rigid platform for galanin to pack against. Next to the disulfide bond sits His176^ECL2, which contacts Asn5^P, Gly8^P, and Pro13^P. This histidine is unique to GALR2 (S3C Fig) and makes a modest contribution to galanin binding; the H176^ECL2A mutant shows a 5-fold higher EC₅₀ value (Table 2). This residue is replaced by tryptophan (Trp188^ECL2) in GALR1 and valine in GALR3 (S3C Fig), suggesting that the 3 receptors make interactions of different strengths at this position.

The GALR2 model was used to design a number of mutants that were assayed for galanin binding, considering the expression level of the mutants (Figs 3, S4A, and S4B). The Y164^4.64×65F mutant shows only slightly weakened galanin binding, while the Y164^4.64×65A mutant shows none at all, due to the loss of hydrophobic contact with galanin Tyr9^P (Figs 2C and 3). His102^3.29 is common to all 3 receptors, and H102^3.29A of GALR2 showed strongly decreased binding affinity to galanin, again emphasizing the importance of Tyr9^P for binding (Fig 3). Phe264 and Leu266 in ECL3 contact Leu10^P and Trp2^P, respectively, and replacing either residue with alanine gave roughly 200-fold drops in binding affinity, presumably because ECL3 becomes highly flexible in these mutants, and the same interactions with galanin are lost (Fig 2C and Table 2). Leu255^6.54 and Arg274^7.35×34 lie close together and contact Leu10^P, so that the mutants L255^6.54A and R274^7.35×34A both showed 12- to 15-fold drops in affinity for galanin (Fig 2C and Table 2). Mutant R184^5.35×36V also showed a significant drop in galanin binding, possibly due to its role in stabilizing ECL2 (Fig 3). Although Arg184^5.35×36 forms a contact with Pro13^P, the galanin residue is not required for interaction with GALR2. The truncated peptide galanin^2–11 has been reported to show specificity for GALR2 and GALR3 over GALR1 [3,14]. Duan and colleagues [15] reported that the N-terminal amino group of galanin forms a hydrogen bond with Glu32^1.31 of GALR1, but no such bond forms with GALR2.

The experimental model of activated GALR2 shows notable differences from the inactive model of hβ₂AR (PDB code 2RH1) [16]. The conformational changes associated with agonist-induced activation of class A GPCRs are well known [17,18], mainly involving a highly conserved “toggle” tryptophan residue (Trp^6.48) within the CWxP motif of TM6, close to the NPxxY motif of TM7; agonists trigger pronounced movements of TM6 relative to the inactive state, and GALR2 follows the same pattern (Fig 4A and 4B). The DRY and PIF motifs are other motifs common to class A GPCRs, and their conformational changes on activation are shown (Fig 4B). The isoleucine residue of the PIF motif is replaced by serine in GALR2 and GALR3 (S3C Fig). This residue, Ser113^3.40 of GALR2, shows no significant movement, while Phe245^6.44 of PI(S)F motif slides against it as TM6 extends toward the cytoplasmic side and rotates, as in the case of other reported GPCR models [17] (Fig 4B). The movement of TM6 is illustrated by the predicted shift of 8 Å in the position of Lys231^6.30 of the DRY motif (Fig 4B). In common with other class A GPCRs, the shift in TM6 of GALR2 creates a binding site for the cognate G-proteins [17], but unlike the dopamine receptors, the agonist itself (galanin) is considerably distant (>12 Å) from Trp249^6.48.

Recently, Duan and colleagues published cryo-EM models of GALR1 coupled to G_i and GALR2 coupled to G_q, showing the role of ICL2 in partner selectivity [15]. This model of GALR2 (PDB code 7WQ4) includes the antibody Nb35 used to stabilize the complex, instead of scFv16, and 2 cholesterol molecules are modeled lying against TM6. Therefore, our structure provides an independent view of the complex, showing that the antibodies have little effect on the GPCR itself. Several residues (from 219 to 222) of ICL3 that lie close to G_q are not modeled in the structure (PDB code 7WQ4). The RMS deviations between the 273 C^α atoms shared by the GALR2 models is 1.16 Å; the largest difference is found around ICL3, which is slightly shifted by indirect effects of Nb35. No notable bonds are formed between ICL3 of the GPCR and G_q in our model; Val221^5.72 comes within 4 Å of Tyr212 of mGαq_iN. As concluded by the Duan group, the selectivity of GALR2 for G-protein partners is mainly controlled by ICL2 [15]. The protein–protein interface is essentially the same between the model (PDB code 7WQ4) and our model, except that the latter places Glu135^ICL2 of GALR2 close enough to Arg32 of mGαq_iN to form a salt bridge. Although our model includes residues from 1 to 16 of galanin rather than residues from 1 to 13, the ligand overlays closely and makes the same interactions with the protein when compared to each other. Minor changes of rotamer indicate the flexibility of the binding pocket, but there are no significant differences between the ligand interfaces in the 2 GALR2 models.

In conclusion, GALR2 shares the activation mechanism common to class A GPCRs. Our model reveals details of the interactions between GALR2 and galanin, whose N-terminal half adopts a compact form, and this model will assist structure-based drug design selectively targeting one or more galanin receptors to address various human diseases.

Materials and methods

Supporting information

Data Availability

Atomic coordinate of GALR2-galanin-mGαqiN/Gβ1γ2-scFv16 has been deposited in the Protein Data Bank (PDB) under accession code 7XBD. The cryo-EM map has been deposited in the Electron Microscopy Data Bank (EMDB) under accession code EMD-33103. All other relevant data are within the paper and its Supporting information files.

Funding Statement

This work was supported by Japan Agency for Medical Research and Development, AMED under grant numbers JP21fk0310103 (S.-Y.P.), JP20ae0101047 (K.M.), and by JSPS/MEXT KAKENHI grant (JP19H05779, 21H02449 to S.-Y.P.), (JP20H05873 (K.M.)). This work was also supported by a grant (NRF-2020M3A9G7103934 to W.L.) from National Research Foundation (NRF) of Korea. A.I. was funded by KAKENHI 21H04791 and 21H05113 from the Japan Society for the Promotion of Science (JSPS); the LEAP JP20gm0010004 and the BINDS JP20am0101095 from the Japan Agency for Medical Research and Development (AMED). K.M. was funded by the BINDS JP20am0101093 from AMED. A.I. was funded by FOREST Program JPMJFR215T and JST Moonshot Research and Development Program JPMJMS2023 from the Japan Science and Technology Agency (JST); Daiichi Sankyo Foundation of Life Science; Takeda Science Foundation; The Uehara Memorial Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.