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. 2012 Nov 30;48(4):655-61.
doi: 10.1016/j.molcel.2012.09.003. Epub 2012 Oct 11.

Ligand-induced architecture of the leptin receptor signaling complex

Liliya V Mancour  1 , Hikmat N Daghestani, Somnath Dutta, Gerwin H Westfield, Justin Schilling, Austin N Oleskie, Jeffrey F Herbstman, Steven Z Chou, Georgios Skiniotis
Affiliations

Ligand-induced architecture of the leptin receptor signaling complex

Liliya V Mancour et al. Mol Cell. .

Abstract

Despite the crucial impact of leptin signaling on metabolism and body weight, little is known about the structure of the liganded leptin receptor (LEP-R) complex. Here, we applied single-particle electron microscopy (EM) to characterize the architecture of the extracellular region of LEP-R alone and in complex with leptin. We show that unliganded LEP-R displays significant flexibility in a hinge region within the cytokine homology region 2 (CHR2) that is connected to rigid membrane-proximal FnIII domains. Leptin binds to CHR2 in order to restrict the flexible hinge and the disposition of the FnIII "legs." Through a separate interaction, leptin engages the Ig-like domain of a second liganded LEP-R, resulting in the formation of a quaternary signaling complex. We propose that the membrane proximal domain rigidification in the context of a liganded cytokine receptor dimer is a key mechanism for the transactivation of Janus kinases (Jaks) bound at the intracellular receptor region.

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Figures

Figure 1
Figure 1. Purification and thermodynamic analysis of the assembly of Leptin/LEP-R complexes
A) Isothermal titration calorimetry for the assembly of Leptin and LEP-R. The inset table lists the thermodynamic parameters for the binding isotherm. B) Size exclusion chromatography profile and SDS-PAGE analysis of purified liganded LEP-R[D1-D7].
Figure 2
Figure 2. Conformational dynamics of LEP-R in unliganded and liganded states
A) Representative 2D class averages of unliganded LEP-R[D1-D7] reveal significant flexibility in a hinge between D4 and a rigid D5-D7 module (white arrowheads). B) 2D class averages of unliganded LEP-R[D1-D5] confirm the domain assignments and the variable disposition of D5 (white arrowheads) in regards to D4. C) Comparison of the crystal structures from LEP-R, gp130 and LIF-R extracellular domains and homology model for LEP-R[D1-D7]. D) Representative 2D class average of the binary leptin/LEP-R[D1-D7] complex. The cytokine binds to CHR2 resulting in the stabilization of the rigid D5-D7 module in a single conformation. E) Representative 2D class average the binary leptin/LEP-R[D1-D5] complex. The orange and white arrowheads point to the leptin density and the LEP-R C-terminus, respectively. F) 3D reconstruction of the binary leptin/LEP-R[D1-D7] complex with docked leptin/LEP-R homology model. G) The double mutation L503S/L504S on D4 of Lep-R abolishes leptin binding via epitope II. EM class averages of this mutant after incubation with leptin reveal only monomeric receptor chains with no ligand bound at CHR2 (compare to A and D). All scale bars correspond to 5 nm.
Figure 3
Figure 3. Architecture of the quaternary leptin/LEP-R signaling complex
A) 2D class averages of side-view projections reveal the cross-over configuration of two LEP-R extracellular chains. The two chains connect at the CHR2 level, while the membrane-proximal domains (white arrowheads) point towards each other and arrive in close proximity at their C-terminal tips. B) 3D reconstruction of the leptin/LEP-R side-view projections shown in (A). Flexible docking of two liganded LEP-R chains into the 3D density map shows the complex configuration (right). C) Representative 2D class average of a top view of the quaternary leptin/LEP-R[D1-D7] complex. In this type of projections the N-terminal CHR1 and C-terminal FnIII domains are collapsed on the carbon support (black arrowheads). The boxed area shows the rectangular formation that is reminiscent of the anti-parallel gp130/IL-6 or GCSF/GCSF-R interaction. D) Representative 2D class average of a top view of the quaternary leptin/LEP-R[D1-D5] complex, as in (C). The dashed white arrowheads point to missing densities from the omitted C-terminal domains of the truncated construct, as compared to (C). E) 3D reconstruction from top-view leptin/LEP-R[D1-D7] projections shown in (C), and docked gp130/IL-6 crystal structure into the central rectangular density of the 3D map (right). F) Mutation L370A on the IgD (D3) of LEP-R abolishes leptin binding via epitope III. EM class averages of this mutant after incubation with leptin reveal only monomeric receptor chains with ligand bound at CHR2 (orange arrows). All scale bars correspond to 5 nm.
Figure 4
Figure 4. Signaling architecture of tall cytokine receptors
Receptor organization and ligand epitope usage in the signaling complexes of gp130/IL-6/IL-6Rα (A; left), gp130/LIF-R/CNTF/CNTF-Rα (A; right), and leptin/LEP-R (B; left). Leptin employs only epitopes II and III to engage the CHR2 and IgD of LEP-R, respectively. Leptin-induced stabilization of each CHR2 in the quaternary complex results in the constrained and close disposition of the membrane-proximal domains, which likely favors intracellular Jak2 trans-phosphorylation (B; right).
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