20 YEARS OF LEPTIN: Insights into signaling assemblies of the leptin receptor

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   Figure 1

   Leptin sequence, structure, and position of binding-sites. (A) Pairwise sequence alignment of human (purple) and murine (green) leptin shows high sequence conservation (84.9% identity). Differences are shown in red. Secondary structures (α helices and loops), as well as the intramolecular disulfide bridge, are shown based on the crystal structure of human leptin. (B) X-ray crystallographic structure of human leptin W100E (PDB ID:1AX8), shown as cartoon with rainbow color scheme. Residues of binding sites I (C), II (D), and III (E) are shown as spheres.
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   Figure 2

   Schematic structure of leptin receptor (LR). (A) LR can be divided into three parts: extracellular (LR_ecto), transmembrane (LR_TM), and cytoplasmic tail (LR_cyto). The extracellular part LR_ecto is composed of a N-terminal domain (NTD), two cytokine receptor homology (CRH) domains (CRH1 and CRH2), an immunoglobulin-like domain (IGD), and two membrane-proximal fibronectin type III (FN III) domains. Numbers indicate the predicted domain borders. N-linked glycosylation sites (red), position of free cysteines (purple), and disulfide clusters (green) are shown on the right. (B) Modular structure of gp130 family of cytokine receptors. (C) LR isoforms: at least six different LR isoforms are found in human, namely LRa to LRf. LRb is the longest one with fully functional intracellular parts and thus is the only functional isotype. Short forms (LRa, LRc, LRd, and LRf) have unique C-terminal stretch of amino acids. LRe is a soluble variant.
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   Figure 3

   Structure of (A) the 2:2:2 IL6–IL6 receptor α–gp130 complex and of (B) the 2:2 G-CSF–G-CSF receptor complex.
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   Figure 4

   Crystal structure of the CRH2 domain. The 622-WSNWS-626 motif forms a π-stack, and N624 is glycosylated. Residues or loops that are predicted to interact with leptin are indicated: residues 503-IFLL-506, Y472, and the J-K loop. C604 is not involved in formation of a disulfide bridge with a cysteine of CRH2.
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   Figure 5

   Models of the IGD and CRH1 domains. (A) Model of the IGD, with indication of the residues that presumably interact with binding site III of leptin. (B) Model of the CRH1 domain. The 317-WSXWS-321 motif in the second barrel is involved in a π-stack. The rat Fa/Fa mutation Q269P is adjacent to this π-stack and may affect the CRH1 structure. The human Q223R mutation (Q222 in mouse) is found at the surface of the first barrel.
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   Figure 6

   Model of the membrane-proximal LR FN III domains (rainbow color ribbons) linked to the LR CRH2 (grey ribbons). All domains are linked as in the gp130 crystal structure, used as a template for modeling. Several residues that interact between the two FN III domains are identical or very conserved in gp130 (sticks). C674 in the first FN III domain is automatically modeled as a disulfide with C604 of the CRH2 domain.
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   Figure 7

   Models for leptin–LR interaction and topology of binding site III. (A) A model for a tetrameric 2:2 leptin–LR complex. Only the CRH2 and Ig-like domain of the LR are modeled. Residues S120 and T121 (green spheres) are close to the IGD, while residues 39-LDFI-42 (yellow spheres) do not contact the IGD. (B) A model for a hexameric 2:4 leptin–LR complex. S120 and T121 (green spheres) are close to the Ig-like domain, as in the tetrameric model of Fig. 7A. Residues 39-LDFI-42 (yellow spheres) interact with the CRH2 domain of an additional LR chain. (C) Binding site III residues mapped on the leptin structure. Residues corresponding to mutations that strongly affect LR activation are colored green, red, or yellow. Mutations with a moderate effect on LR activation are colored cyan blue. Residues S117 and 120-STE-122 seem to form a first-site IIIa, residues R128 and 39-LDFI-42 form a second-site IIIb.
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   Figure 8

   3D density maps based on single-particle EM (A) and SAXS (B) of leptin–LR 2:2 complexes. Density maps based on SAXS of the 1:1 complexes of leptin mutants S120A-T121A (Leptin_a1) (C) and 39-AAAA-42 (Leptin_a2) (D) with the LR.

• © 2014 Society for Endocrinology