Structural snapshots of full-length Jak1, a transmembrane gp130/IL-6/IL-6Rα cytokine receptor complex, and the receptor-Jak1 holocomplex

Abstract

The shared cytokine receptor gp130 signals as a homodimer or heterodimer through activation of Janus kinases (Jaks) associated with the receptor intracellular domains. Here, we reconstitute, in parts and whole, the full-length gp130 homodimer in complex with the cytokine interleukin-6 (IL-6), its alpha receptor (IL-6Rα) and Jak1, for electron microscopy imaging. We find that the full-length gp130 homodimer complex has intimate interactions between the trans- and juxtamembrane segments of the two receptors, appearing to form a continuous connection between the extra- and intracellular regions. 2D averages and 3D reconstructions of full-length Jak1 reveal a three lobed structure comprising FERM-SH2, pseudokinase, and kinase modules possessing extensive intersegmental flexibility that likely facilitates allosteric activation. Single-particle imaging of the gp130/IL-6/IL-6Rα/Jak1 holocomplex shows Jak1 associated with the membrane proximal intracellular regions of gp130, abutting the would-be inner leaflet of the cell membrane. Jak1 association with gp130 is enhanced by the presence of a membrane environment.

Figure 1. Assembly and purification of the full-length gp130/IL-6/IL-6Rα complex

a) Components of the gp130/IL-6/IL-6Rα complex in pre- and post-assembly state. A surface rendering of the structure of the signaling hexamer is shown on top. IgD denotes Ig-like domain, CHR denotes cytokine-binding homology region, and FnIII denotes Fibronectin-type III domain. b) Purification of the gp130/IL6/IL6Rα complex by size exclusion chromatography.

Figure 2. 2D EM averages of the full-length gp130/IL-6/IL-6Rα complex

a) Raw image of negatively stained full-length gp130/IL-6/IL-6Rα complex. Representative particles are boxed in yellow. b) 2D class averages of the gp130/hyper IL-6 complex produced by the classification of 6,070 particles. c) Comparison of a 2D class average to a model based on the gp130/IL-6Rα/IL-6 complex (left). The cartoon on the right shows the domain organization of the gp130/IL-6Rα/IL-6 complex. Lines overlaid on the 2D average trace the chains of the receptors and denote the location of the IL-6 cytokine. [The scale bars in a and b-c correspond to 40 nm and 10 nm, respectively]. See also Figure S1.

Figure 3. Purification and 2D projections of recombinant human Jak1

a) Jak1 was purified from 293S cells lysate via a C-terminal 8-Histidine tag using Ni-affinity column. b) Second step, Streptactin-affinity purification of fraction 2 containing Jak1 via its N-terminal Strep-tag. c) Silver stained gel of Streptactin-purified Jak1 after a Superose 6 size exclusion chromatography step. These fractions were selected for negative stain EM. (see Figure S2) d) Representative 2D class averages of purified Jak1 obtained from the classification of 9,258 particles. The marked classes were used independently for 3D reconstructions See also Figures S3, S4, S5.

Figure 4. 3D reconstructions and modeling of full-length human Jak1

3D reconstructions of Jak1 are based on individual classes obtained from 2D classification of untilted specimen projections (marked in Figure 3D, Figure S6). From the top to the bottom of the panel Jak1 particles transition from open to closed conformations. The FERM (green), SH2-like (cyan), pseudokinase (yellow), and kinase (red) domains of Jak1 were modeled into the EM densities. The orientations of the reconstructions in the left column of the panel are viewing the particles face-on the grid (as shown in the 2D class averages of Figure 3D), while the orientations to the right are rotated 90°, reflecting the flatness of the surface on which Jak1 is lying. See also Figures S5, S6.

Figure 5. Purification and EM imagining of the gp130/IL-6/IL-6Rα/Jak1 complex

a) Anion-exchange chromatography of the gp130/Jak1 complex. Fractions from the Mono-Q column were analyzed by SDS-PAGE gel and subjected to western blot with anti-GluGlu (gp130) and anti-Jak1 antibodies (panel underneath). Since two different antibodies were used to detect gp130 (Anti-GluGlu) and Jak1 (Anti-Jak1) the relative ratios of gp130/Jak1 cannot be derived from this experiment. The anti-GluGlu mAb appears to be more sensitive than anti-Jak1. b) Comparison of representative 2D averages for the soluble gp130/IL-6/IL-6Rα extracellular complex (top three panels), full-length gp130/IL-6/IL-6Rα (middle panels, as shown in Figure 2B), and the gp130/IL-6/IL-6Rα/Jak1 complex (bottom three panels). Schematics for each complex are shown in the far right column. The extra fuzzy density in the Jak1 loaded complexes is apparent by comparison to the non-loaded complexes. See also Figures S7, S8.

Figure 6. Reconstitution of the gp130/IL-6/IL-6Rα/Jak1 complex in lipid nanodiscs

The quaternary gp130/IL-6/IL-6Rα/Jak1 complex was prepared and incorporated into lipid “nanodiscs” comprised of DPPC and the MSP1 scaffold protein. Jak1 was added to the nanodiscs containing gp130/gp130/IL-6/IL-6Rα and applied to a Superose 6 size exclusion column. Since full-length Jak1 and gp130 co-migrate on SDS-PAGE, peak fractions were subjected to SDS-PAGE and coomassie staining (top panel) and western blotting with an anti-Jak1 antibody (bottom panel) in order to confirm the presence of both components. Higher resolution SDS-PAGE and coomassie confirms that both proteins are present in roughly equal amounts.