Fig. 3: Architecture of the Egr:Grnd complex and mapping of the interaction interface.
A Sequence of the TNF-homology domain of Egr coloured by sequence identity with TNF ligands interacting with single-CRD TNFR (see Methods for details). Residues implicated in Grnd binding are marked by red rhombuses. B Surface representation of the Egr:Grnd hexamer in the same orientation as in (D). C, D Cartoon representation of the Egr:Grnd complex at the indicated orientations. The three Egr-TNF copies are shown in shades of yellow, and Grnd monomers in purple. The threefold axis is indicated as a triangle. E–G Enlarged views of three patches of the Egr269–409:Grnd30–81 interface. H Strep pull-down assay with 2 μM Egr146–409 absorbed on strep-tactin beads and 10 μM Grnd30–97 in solution, either wild-type or carrying the indicated alanine substitutions, visualized by Coomassie staining. Two parts of the SDS-PAGE corresponding to the molecular weight of Egr146–409 and Grnd30–97 are shown. The Grnd inputs used in the pull-down experiment are shown in the bottom SDS-PAGE. Grnd mutations fully or partially impairing binding to Egr are labelled in red and orange, respectively. I Table of binding affinities (dissociation constants KD) between Egr146–409 and wild-type or mutated Grnd30–97 measured by ITC. J Co-IP experiments between  HA-tagged Eiger and Flag-tagged Grnd, wild-type or mutated as indicated, from S2 cell lysates. Bound molecules are visualized by anti-HA and anti-Flag antibodies. Immunoprecipitation of V5 was used as specificity control. K Strep pull-down assays analogous to the ones described in (H), performed with the indicated Egr146–409 mutants and wild-type Grnd30–97. Results of experiments presented in panels H–J, K were confirmed by successful replicate experiments. Uncropped images of SDS-PAGE gels and immunoblots are provided in Supplementary Fig. 6.
