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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 [1]

['Yunseok Heo', 'Structural Biochemistry', 'Molecular Biophysics Laboratory', 'Department Of Biochemistry', 'College Of Life Science', 'Biotechnology', 'Yonsei University', 'Seoul', 'Naito Ishimoto', 'Drug Design Laboratory']

Date: 2022-08 

Galanin is a neuropeptide expressed in the central and peripheral nervous systems, where it regulates various processes including neuroendocrine release, cognition, and nerve regeneration. Three G-protein coupled receptors (GPCRs) for galanin have been discovered, which is the focus of efforts to treat diseases including Alzheimer’s disease, anxiety, and addiction. To understand the basis of the ligand preferences of the receptors and to assist structure-based drug design, we used cryo-electron microscopy (cryo-EM) to solve the molecular structure of GALR2 bound to galanin and a cognate heterotrimeric G-protein, providing a molecular view of the neuropeptide binding site. Mutant proteins were assayed to help reveal the basis of ligand specificity, and structural comparison between the activated GALR2 and inactive hβ 2 AR was used to relate galanin binding to the movements of transmembrane (TM) helices and the G-protein interface.

Funding: 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 and JP20am0101093 (K.M.) from the Japan Agency for Medical Research and Development (AMED). K.K. 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.

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.

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 (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 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 Glu241.31 to Arg3008.51, including the intracellular loops (ICLs) and extracellular loops (ECLs). scFv16 stabilizes the complex by binding to the N-terminal helix of mGαq iN and a surface loop of Gβ1 (Fig 1A).

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TIFF original image Download: Fig 1. Cryo-EM structure of GALR2 complex with galanin. (A) The molecular structure of GALR2 complex is shown both as cryo-EM map and ribbon diagram. GALR2, mGαq iN , Gβ1, Gγ2, and scFv16 are colored in blue, red, yellow-orange, marine, and lemon, respectively. The bound galanin peptide is colored in light orange. (B) The C-terminal helix of mGαq iN is inserted in the pocket formed by TM5–7 of GALR2 forming hydrophobic and hydrophilic interactions. Arg32 of mGαq iN forms a salt bridge with Glu135ICL2 of GALR2. (C) The galanin is bound to the pocket formed among the 7 TMs of GALR2 at the extracellular side (left panel). GALR2 and galanin are shown as an electrostatic surface representation (right panel). cryo-EM, cryo-electron microscopy; TM, transmembrane. https://doi.org/10.1371/journal.pbio.3001714.g001

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 Glu135ICL2 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 Leu4P to Leu11P (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]. Trp2P, Thr3P, Tyr9P, Leu10P, and Gly12P 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 Trp2P of galanin with alanine prevents binding to GALRs [4]. This tryptophan side chain makes a hydrophobic interaction with Leu266ECL3 of GALR2 (Fig 2C). The side chain of Tyr9P reaches furthest into the pocket, where it makes interactions with Ile852.64×63, His1023.29 and Tyr1644.64×65 of the receptor (Fig 2C). Leu10P interacts with Phe264ECL3, while Pro13P packs against His176ECL2 and Pro177ECL2 (Fig 2C). His14P, Ala15P, and Val16P 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, Trp2P, Asn5P, and Tyr9P appear to be central to receptor binding, while Leu10P also makes substantial hydrophobic interactions (Fig 2C).

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TIFF original image Download: Fig 2. Galanin recognition of GALR2. (A) The cryo-EM map near the galanin ligand in 2 different orientations. The sequence of galanin is written below, and the C-terminal 14 amino acids, which are not visible in the map, are colored in gray. (B) The ligand binding pocket of GALR2 with galanin is shown in 2 different orientations. GALR2 is shown as a ribbon diagram, and galanin as a ribbon diagram and stick model. (C) Detailed interactions between galanin and GALR2. The hydrophobic interactions between Leu10P and Phe264ECL3 and Trp2P and Leu266ECL3 are indicated with yellow dotted lines. cryo-EM, cryo-electron microscopy. https://doi.org/10.1371/journal.pbio.3001714.g002

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. Cys983.25 and Cys175ECL2 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 His176ECL2, which contacts Asn5P, Gly8P, and Pro13P. This histidine is unique to GALR2 (S3C Fig) and makes a modest contribution to galanin binding; the H176ECL2A mutant shows a 5-fold higher EC 50 value (Table 2). This residue is replaced by tryptophan (Trp188ECL2) 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 Y1644.64×65F mutant shows only slightly weakened galanin binding, while the Y1644.64×65A mutant shows none at all, due to the loss of hydrophobic contact with galanin Tyr9P (Figs 2C and 3). His1023.29 is common to all 3 receptors, and H1023.29A of GALR2 showed strongly decreased binding affinity to galanin, again emphasizing the importance of Tyr9P for binding (Fig 3). Phe264 and Leu266 in ECL3 contact Leu10P and Trp2P, 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). Leu2556.54 and Arg2747.35×34 lie close together and contact Leu10P, so that the mutants L2556.54A and R2747.35×34A both showed 12- to 15-fold drops in affinity for galanin (Fig 2C and Table 2). Mutant R1845.35×36V also showed a significant drop in galanin binding, possibly due to its role in stabilizing ECL2 (Fig 3). Although Arg1845.35×36 forms a contact with Pro13P, the galanin residue is not required for interaction with GALR2. The truncated peptide galanin2–11 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 Glu321.31 of GALR1, but no such bond forms with GALR2.

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TIFF original image Download: Fig 3. Gq-signaling activity of GALR2 mutants. Galanin-induced Gq-signaling activity of WT GALR2 (titrated plasmid volume) and mutant GALR2 was assessed by the NanoBiT Gq-PLCβ assay. Symbols and error bars indicate mean and SEM, respectively, of 3 independent experiments with each performed in duplicate. The dashed lines, the dotted lines, and long dashed dotted lines represent response curves of WT, mock transfection, and surface expression–matched WT, respectively. Surface expression levels of WT and the mutants both of which contained the N-terminal FLAG-epitope tag were assessed by the flow cytometry using a FLAG-epitope tag antibody (S4A Fig). Note that, in many data points, error bars are smaller than the size of symbols and thus are not visible. The data underlying this figure can be found in S1 Data. WT, wild type. https://doi.org/10.1371/journal.pbio.3001714.g003

The experimental model of activated GALR2 shows notable differences from the inactive model of hβ 2 AR (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 (Trp6.48) 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, Ser1133.40 of GALR2, shows no significant movement, while Phe2456.44 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 Lys2316.30 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 Trp2496.48.

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TIFF original image Download: Fig 4. Comparison of the conserved motifs between active GALR2 and inactive hβ 2 AR. (A) The overall structures of activated GALR2 and inactive hβ 2 AR in 2 different orientations. GALR2, hβ 2 AR, and galanin are shown as a ribbon diagram. Active GALR2, inactive hβ 2 AR, and galanin are colored in blue, orange, and light orange, respectively. The movements of TM1, TM6, and TM7 are indicated by red arrows. PDB code of the inactive structure of hβ 2 AR is 2RH1. (B) The conserved motifs are shown as a stick model. The movements of the residues are indicated by black arrows, and the movements of TM are indicated by red arrows. TM, transmembrane. https://doi.org/10.1371/journal.pbio.3001714.g004

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 (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 Cα 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; Val2215.72 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 Glu135ICL2 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 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.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001714

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