Structural insight into ligand binding and activation of the orphan GPCR Mas1

Figures

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  Figure 1. Mas1 responds to NPFF, AR234958 and shows low-potency response to Ang-(1–7) in HEK293T cells. (A) Primary amino acid sequences of NPFF and Ang 1–7 peptides. Conserved and functionally analogous residues are aligned to highlight structural similarities that may underlie Mas1 binding. (B) Dose–response curves of cAMP inhibition in HEK293T cells transiently expressing Mas1. Cells were stimulated with forskolin and treated with increasing concentrations of NPFF (blue), AR234958 (red), or Ang 1–7 (green). cAMP accumulation was measured and normalized to NPFF-induced maximal response. Data are presented as mean ± SEM from three independent experiments (n = 3). Curves were fitted using nonlinear regression. Source data are available online for this figure.

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  Figure 2. Cryo-EM structures of Mas1 reveal ligand recognition and a predominantly hydrophobic binding pocket. (A) Cryo-EM structure of the Mas1–Gi–scFv16 complex bound to NPFF at 2.54 Å resolution. Left: cryo-EM density map of the full signaling complex with individual components shown in distinct colors (Mas1: orange, Gα: gray, Gβ: dark blue, Gγ: purple, scFv16: brown). Middle: ribbon diagram of the resolved complex. Right: modeled conformation of NPFF (green), occupying the orthosteric site of Mas1. (B) Cryo-EM structure of the Mas1–Gi–scFv16 complex bound to AR234958 at 3.07 Å resolution. Left and middle: density map and structural model of the complex, with Mas1 shown in purple. Right: atomic structure of the synthetic ligand AR234958 (pink), indicating its accommodated pose within the orthosteric binding pocket. (C) Top-down views of Mas1 from the extracellular side, illustrating the ligand-binding cavity formed by transmembrane helices (TM1–TM7). Left and middle: surface representation and ribbon models at two perpendicular angles. Right: cavity-lining residues are shown in stick representation, emphasizing the hydrophobic environment of the orthosteric pocket, which is…

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  Figure 3. Structural and functional dissection of the NPFF binding pocket in Mas1. (A) Cross-sectional view of the Mas1 transmembrane (TM) domain showing NPFF (green) bound within a predominantly hydrophobic orthosteric pocket. Right: close-up of NPFF within the binding site, with key residues labeled, including I172ECL2, Y1684.61, S1093.29, Y2486.55, H2627.35, L2667.39, I842.57, L872.60, I391.39, H361.36 and Y912.64. Ligand–receptor interactions are mediated largely through hydrophobic contacts, complemented by polar anchoring residues. (b–d) Structural snapshots highlighting specific ligand-interacting residues: (B) Contacts at the extracellular entrance (e.g., the hydrogen bond between S1093.29 and the backbone nitrogen of NPFF-1F) (e.g., S1093.29, I172ECL2). (C) Mid-pocket interactions along TM5–TM6 (e.g., Y2486.55, H2627.35). (D) Basal pocket residues contributing to ligand stabilization (e.g., L2667.39, Y912.64, L872.60, I842.57, I391.39, H361.36). (E–G) Functional characterization of individual residues via cAMP assays in HEK293T cells expressing wild-type (WT) or Mas1 mutants. (E) Mutation of S109A, Y168A, or I172A abolishes ligand response. Data are presented as mean ± SEM from…

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  Figure 4. Structural basis of AR234958 recognition by Mas1 and functional impact of key binding pocket residues. (A) Cross-sectional view of the Mas1 transmembrane domain (purple) with AR234958 (pink) occupying the orthosteric ligand-binding pocket. Right: schematic diagram illustrating the three binding subareas (SA1, FA1, FA2) of AR234958, which engages key residues via hydrophobic and aromatic interactions. Critical residues include I172ECL2, Y1684.61, Y2486.55, H2627.35, L2667.39, L872.60, Y912.64, and Y95ECL1. (B–D) Structural snapshots of specific residue interactions with AR234958. (B) The extracellular entrance. The FA2 moiety is oriented toward I172ECL2 and Y1684.61, which define the structural boundary of the binding pocket. (C) Central pocket coordination involving H2627.35, Y2486.55 and L2667.39. (D) Lower cavity residues I842.57, Y95ECL1, Y912.64, and L872.60 contribute to stabilizing the aromatic FA1 moiety and SA1 moiety of AR234958. (E–G) Concentration-dependent inhibition of cAMP accumulation in HEK293T cells expressing wild-type (WT) or mutant Mas1 variants. (E) I172A and Y168A mutations abrogate AR234958 activity. Data are presented as mean ± SEM from three independent…

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  Figure 5. Comparison of NPFF- and AR234958-bound mas1 structures reveals a conserved hydrophobic binding core and ligand-triggered conformational pressure switch. (A, B) Structural overlays of Mas1 bound to NPFF (green) or AR234958 (pink), viewed from the side (A) and the extracellular surface (B). Ligands occupy similar spatial positions within the transmembrane core, and induce local adjustments of helices TM2–TM6 to accommodate their distinct chemical scaffolds. (C) Both NPFF and AR234958 reside in a binding pocket formed by a similar set of conserved residues, including Y1684.61, Y912.64, Y95ECL1, I172ECL2, Y2516.58, H361.36, and H2627.35. Despite ligand differences, these residues delineate a shared structural cavity supporting promiscuous yet selective binding. (D) Overlay of H2627.35 and H2637.36 shows their distinct conformational positions in the two complexes. Although both appear to contribute to ligand interaction structurally, complementary functional data (see Appendix Fig. S6) identify H2627.35 as the principal hydrophobic anchoring residue essential for ligand-induced activation. (E) A conserved hydrophobic surface formed by Y2486.55, L2667.39, L872.60, and I842.57 is…

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  Figure 6. Comparative analysis of Mas1 and MRGPR family reveals conserved hydrophobic cleft and functional divergence in ligand recognition. (A) Surface representations of the ligand-binding pockets in Mas1 (NPFF-bound and AR234958-bound), MRGPRX2 (C14-bound), MRGPRX1 (BAM8-22-bound), and dopamine receptor D3R (rotigotine-bound). Although Mas1 and MRGPRX family receptors share a conserved deep hydrophobic cavity shaped around position 6.48 (e.g., A2416.48 in Mas1, G2366.48 in MRGPRX2), their activation appears to be mediated indirectly through ligand engagement with residues surrounding the cavity, rather than via direct interaction with 6.48. In contrast, rotigotine in D3R makes direct contact with W3416.48, exemplifying a canonical class A GPCR activation mechanism. (B) Left: Structural overlay of Mas1 in different states—AlphaFold3 inactive model (gray), NPFF (orange), and AR234958 (purple)—highlighting a 16.8° outward displacement of TM6 in ligand-bound states relative to AlphaFold3. Right: Side view of TM3–TM6 swing in Mas1 showing the reorientation of A2416.48, L1203.40, and F2376.44. (C) Structural comparison of Mas1 (orange) and MRGPRX1 (green) reveals conserved core residues…