Evidence for M1-Linked Polyubiquitin-Mediated Conformational Change in NEMO

Abstract

The NF-κB Essential Modulator (NEMO) is the scaffolding subunit of the inhibitor of κB kinase (IKK) holocomplex and is required for the activation of the catalytic IKK subunits, IKKα and IKKβ, during the canonical inflammatory response. Though structures of shorter constructs of NEMO have been solved, efforts to elucidate the full-length structure of NEMO have proven difficult due to its apparent high conformational plasticity. To better characterize the gross dimensions of full-length NEMO, we employed in-line size exclusion chromatography – small angle X-ray scattering (SEC-SAXS). We show that NEMO adopts a more compact conformation (Dmax=320 Å) than predicted for a fully extended coiled-coil structure (>500 Å). Additionally, we map a region of NEMO (residues 112-150) in its CC1 domain that impedes the binding of linear (M1-linked) di-ubiquitin to its CC2-LZ ubiquitin binding domain. This ubiquitin binding inhibition can be overcome by a longer chain of linear, but not K63-linked polyubiquitin. Collectively, these observations suggest that NEMO may be auto-inhibited in the resting state by intramolecular interactions, and that during signaling NEMO may be allosterically activated by binding to long M1-linked polyubiquitin chains.

Necroptosis in development and diseases

“…NEMO is a ubiquitin-binding protein with a UBAN domain and can bind M1 ubiquitin chains (Hauenstein et al 2017). While the well-established role of NEMO is to facilitate the efficient recruitment of IKKα/β into complex I and support their activation in mediating NF-κB activation (Ea et al 2006;Wu et al 2006), recent studies suggest that NEMO is an important suppressor of RIPK1 (Legarda-Addison et al 2009;Kondylis et al 2015;Pescatore et al 2016;Vlantis et al 2016).…”

Auxiliary-assisted chemical ubiquitylation of NEMO and linear extension by HOIP

“…9). The increased structural rigidity of the CoZi domain might contribute to the conformational rearrangement, which takes place upon activation by M1-linked polyubiquitin 46 .…”

GIT1 regulates angiogenic factor secretion in bone marrow mesenchymal stem cells via NF‐κB/Notch signalling to promote angiogenesis

“…One is composed of the CC2 and leucine zipper (LZ) domains, together called the UBAN domain, while the other is composed of the C-terminal zinc finger (ZF) motif. 47 It has been shown that NEMO binds to the polyubiquitin chains, such as K11-linked and K63-linked chains, via its UBDs for the NF-κB activation. [21][22][23][24][25] To investigate whether NEMO un- Figure 6A).…”