Human adiponectin receptor AdipoR1 assumes closed and open structures

Abstract

The human adiponectin receptors, AdipoR1 and AdipoR2, are key anti-diabetic molecules. We previously reported the crystal structures of human AdipoR1 and AdipoR2, revealing that their seven transmembrane helices form an internal closed cavity (the closed form). In this study, we determined the crystal structure of the D208A variant AdipoR1, which is fully active with respect to the major downstream signaling. Among the three molecules in the asymmetric unit, two assume the closed form, and the other adopts the open form with large openings in the internal cavity. Between the closed- and open-form structures, helices IV and V are tilted with their intracellular ends shifted by about 4 and 11 Å, respectively. Furthermore, we reanalyzed our previous wild-type AdipoR1 diffraction data, and determined a 44:56 mixture of the closed and open forms, respectively. Thus, we have clarified the closed-open interconversion of AdipoR1, which may be relevant to its functional mechanism(s).

Fig. 1. Crystal structure of human AdipoR1(A208).

The crystal structure of human AdipoR1(A208) at 3.1-Å resolution: the asymmetric unit contains three molecules, A, B, and C (a–c). Molecules A (green) (a) and B (cyan) (b) are in the closed form, and molecule C (salmon) (c) is in the open form. The structures are viewed parallel to the membrane. The zinc ion is shown as an orange sphere. Two oleic acid molecules in molecule A and two monoolein molecules in molecule C are shown as slate blue stick models. d–i The major cavities of molecules A (d, g, green) and B (e, h, cyan) in the closed form, and molecule C (f, i, salmon) in the open form of AdipoR1(A208). The outside and inside surfaces of the major cavities are colored black and gray, respectively. The major cavities are viewed parallel to the membrane (d–f) and from the intracellular side (g–i). The lipid-bilayer (LB) and cytoplasmic (CP) openings are labeled LB and CP, respectively. Other minor cavities are omitted for clarity. j–l The simulated-annealing F_o–F_c omit maps, contoured at 2.5σ. The electron densities on helices IV and V and ICL2 of molecules A (j, green) and B (k, cyan) in the closed form, and molecule C (l, salmon) in the open form of AdipoR1(A208). For comparison, molecules C and A are shown in gray in j/k and l, respectively.

Fig. 2. Structures of helix V in the closed and open forms of AdipoR1(A208).

a Superimposition of the closed-form (molecule A, green) and open-form (molecule C, salmon) structures. The cytoplasmic ends of helices V are shifted by 11 Å between the two structures. b The amino acid sequences of ICL2 (Trp255–His263) and helix V (Arg264–Ala288), further divided into the N-terminal (NT) region (Arg264–Arg276), the middle (M1 and M2) regions (Ala268–Leu272 and G273–Val280, respectively), and the C-terminal (CT) region (Pro281–Ala288), of AdipoR1. c–e Structure comparison along the M1, M2, and CT regions of helix V among molecules A (green), B (cyan), and C (salmon), by superimposition of the CT region. f–h The distances (Å) of the main-chain amide carbonyl oxygen atom (CO) of residue i with the main-chain amide nitrogen atom (HN) of residues i + 3 (solid gray line) and i + 4 (dashed gray line) in the M2 region (i = 273–277) of helix V of molecules A (f), B (g), and C (h). According to these CO–HN distances and the bond angles in the M2 region (Supplementary Table 1), a 3₁₀-helical conformation, rather than an α-helix, is assumed with four weak hydrogen bonds (3.2–3.5 Å) between the COs of Leu274–Ser277 (residues i) and the HNs of Ser277–Val280 (residues i + 3) in molecule A (f), whereas such 3₁₀-helical hydrogen bonding is weaker in molecule B (g) and almost negligible in molecule C (h). The CO of Gly273 does not form a hydrogen bond in molecules A–C (Supplementary Table 1). i, j Schematic representations of the main chain of helix V, which is bent inward in molecule A (i) and rather straight in molecule C (j). The 3₁₀-helical conformations of Leu276–Ser277–Gly278, colored green in molecule A (i) and salmon in molecule C (j), in the center of the M2 region are thinner than the α-helical conformations in the M1 and CT regions.

Fig. 3. Interactions of the M1 region of helix V with helices III and IV in the closed and open forms of AdipoR1(A208).

Hydrophobic interactions of helix V in the closed form (molecule A) (a–c) and the open form (molecule C) (d–f). Val270 of helix V interacts with helix IV (a, d), and Phe271 of helix V interacts with helix IV (b, e) and helix III (c, f). Overall, the degrees of the hydrophobic interactions are much more intensive in the open form (d–f) than the closed form (a–c).

Fig. 4. Local structures and interactions of the NT region of helix V and ICL2 in the closed and open forms of AdipoR1(A208).

a–g Molecule A (green) in the closed form. h–l Molecule C (salmon) in the open form. In the closed form, Arg267 (the NT region of helix V) hydrogen bonds with Tyr209 (helix III) and Tyr317 (helix VI) as viewed from the intracellular side (a), thereby constricting the major cavity (b). Pro261 (ICL2) and Arg264 (NT) interact with Lys105 and/or Asp106 of NGD (c), Arg264 and Gln265 (NT) interact with Tyr317, Ala318, and Arg320 (helix VI) (d), and Gln254 (helix IV) and Asp256 (ICL2) interact with Lys206 (helix III) (e) in the closed form. In contrast, in the open form, these interactions are completely disrupted (j–l). In both forms, Arg257 and Thr260 interact with His263 within ICL2 (f, h) and Phe258 (ICL2) interacts with Val252 (helix IV) and Thr266 (helix V) (g, i).

Fig. 5. The closed- and open-form structures of AdipoR1(D208).

a, b The closed form (44%) (a) and the open form (56%) (b) of AdipoR1(D208). c, d The SA F_o–F_c omit maps contoured at 2.0σ for the closed (c) and open (d) forms, on the wire models of the dual closed-open conformational regions (residues 250–279), in helix IV, ICL2, and helix V of AdipoR1(A208). The dual-conformational regions are colored salmon, while the remaining parts of the molecules are colored gray. e Summary of the residues with electron densities that were observed, fully or partly, in the SA F_o–F_c omit maps contoured at 2.0σ, highlighted in blue in the sequences of the dual-conformational regions. f–s The SA F_o–F_c omit maps on helix IV, ICL2, and helix V of molecules A (f–l) and C (m–s), contoured at 2.0σ. The SA F_o–Fc omit maps on residues Gln254, Phe255, and Asp256 (f, m), Arg257, Phe258, and Ala259 (g, n), Arg264, Gln265, Thr266, and Arg267 (h, o), Ala268, Gly269, Val270, and Phe271 (i, p), Thr260, Pro261, Lys262, and His263 (j, q), Leu272, Gly273, Leu274, and Gly275 (k, r), and Leu276, Ser277, Gly278, and Val279 (l, s).