Structural basis of NF-κB signaling by the p75 neurotrophin receptor interaction with adaptor protein TRADD through their respective death domains

Abstract

The p75 neurotrophin receptor (p75^NTR) is a critical mediator of neuronal death and tissue remodeling and has been implicated in various neurodegenerative diseases and cancers. The death domain (DD) of p75^NTR is an intracellular signaling hub and has been shown to interact with diverse adaptor proteins. In breast cancer cells, binding of the adaptor protein TRADD to p75^NTR depends on nerve growth factor and promotes cell survival. However, the structural mechanism and functional significance of TRADD recruitment in neuronal p75^NTR signaling remain poorly understood. Here we report an NMR structure of the p75^NTR-DD and TRADD-DD complex and reveal the mechanism of specific recognition of the TRADD-DD by the p75^NTR-DD mainly through electrostatic interactions. Furthermore, we identified spatiotemporal overlap of p75^NTR and TRADD expression in developing cerebellar granule neurons (CGNs) at early postnatal stages and discover the physiological relevance of the interaction between TRADD and p75^NTR in the regulation of canonical NF-κB signaling and cell survival in CGNs. Our results provide a new structural framework for understanding how the recruitment of TRADD to p75^NTR through DD interactions creates a membrane-proximal platform, which can be efficiently regulated by various neurotrophic factors through extracellular domains of p75^NTR, to propagate downstream signaling in developing neurons.

Keywords: NMR; TRADD; cell signaling; death domain; p75 neurotrophin receptor; protein structure.

Fig. 1

Solution structure of p75^NTR-DD:TRADD-DD complex.A, superposition of backbone heavy atoms of the ten lowest-energy complex structures of the p75^NTR-DD:TRADD-DD. N- and C-termini of DDs are indicated. B, Ribbon drawing of p75^NTR-DD:TRADD-DD.

Fig. 2

Binding interface of p75^NTR-DD:TRADD-DD complex.A, representative slice from the ¹³C,¹⁵N-filtered 3D NOESY spectrum. ∗, ambiguous NOE peaks. The p75^NTR-DD was labeled with ¹³C and ¹⁵N, and the TRADD-DD was unlabeled. B, detail of binding interface in the p75^NTR-DD:TRADD-DD complex. Key residues at the binding interface are labeled and depicted as stick models. Close distances between nitrogen and oxygen atoms (~5 Å or less) are showed in dash lines.

Fig. 3

Mutagenesis studies.A, ITC binding curves of human TRADD to human p75^NTR. Dissociation constants (K_d) are the mean ± SEM from three independent experiments. B, binding affinities expressed as K_d of WT and point mutants of p75^NTR and TRADD derived from ITC data. Dissociation constants are the mean ± SEM from three independent experiments. C, coimmunoprecipitation of wild-type (WT) and point mutants of HA-tagged human p75^NTR with Myc-tagged human TRADD in transfected HEK 293T cells. The immunoblots shown are representative of three independent experiments. D, coimmunoprecipitation of WT and point mutants of Myc-tagged human TRADD with HA-tagged human p75^NTR in transfected HEK 293T cells. The immunoblots shown are representative of three independent experiments. IB, immunoblotting; IP, immunoprecipitation; WCL, whole cell lysate.

Fig. 4

p75^NTR-DD with specific binding sites for intracellular interactors. Charged surface of the p75^NTR-DD and the binding sites for intracellular interactors. Unstructured regions are not shown. Color code is blue for positive charges, red for negative charges, and white for neutral surface. The patches on the surface of the p75^NTR-DD responsible for binding TRADD, RhoGDI, RIP2, and the p75^NTR-DD itself are circled in pink, cyan, green, and brown, respectively.

Fig. 5

Expression of TRADD in developing cerebellum. Micrographs of a representative mid-sagittal section through the developing cerebellum of P2 (A/B), P5 (C/D), and P7 (E/F) wild-type mouse stained with anti-TRADD (red) and anti-p75^NTR (green) antibodies, and counterstained with DAPI (blue). White outlines in C and E indicate no tissue in these areas. Higher magnification panels of P2, P5, and P7 are shown in B, D, and F, respectively. Scale bars: A, 300 μm; B, 50 μm (upper) and 100 μm (lower); C, 300 μm; D, 100 μm; E, 300 μm; F, 100 μm.

Fig. 6

Functional role of TRADD/p75^NTRinteraction in NF-κB signaling.A, representative micrographs of p75^NTR knockout (KO) CGNs transfected with expression plasmids containing HA-tagged p75^WT (wild-type) and HA-tagged p75^K349A mutant. After 2 days in vitro, cultures were immunostained with anti-P65 NF-κB (green) and anti-HA (magenta) antibodies, and counterstained with DAPI (blue). Transfected cells expressing HA constructs are indicated with white arrows. Scale bar, 20 μm. B, quantification of nuclear p65 NF-κB (expressed as nuclear p65 fluorescence intensity/cytoplasmic p65 fluorescence intensity) in p75^NTR wild-type (WT) and knockout (KO) CGNs transfected with HA-p75^WTand HA-p75^K349A plasmids as indicated. Results are expressed as mean ± SEM from three independent cultures (∗p < 0.05 and ∗∗p < 0.01, one-way ANOVA followed by Turkey's multiple comparison test).

Fig. 7

Structure model of p75^NTRNF-κB signaling via the recruitment of TRADD. p75^NTR model was built based on available structures of individual domains and domain complexes. The domain orientation and interface between TRADD-NTD and TRADD-DD are not defined in this cartoon.