Cooperative Domain Formation by Homologous Motifs in HOIL-1L and SHARPIN Plays A Crucial Role in LUBAC Stabilization

Abstract

The linear ubiquitin chain assembly complex (LUBAC) participates in inflammatory and oncogenic signaling by conjugating linear ubiquitin chains to target proteins. LUBAC consists of the catalytic HOIP subunit and two accessory subunits, HOIL-1L and SHARPIN. Interactions between the ubiquitin-associated (UBA) domains of HOIP and the ubiquitin-like (UBL) domains of two accessory subunits are involved in LUBAC stabilization, but the precise molecular mechanisms underlying the formation of stable trimeric LUBAC remain elusive. We solved the co-crystal structure of the binding regions of the trimeric LUBAC complex and found that LUBAC-tethering motifs (LTMs) located N terminally to the UBL domains of HOIL-1L and SHARPIN heterodimerize and fold into a single globular domain. This interaction is resistant to dissociation and plays a critical role in stabilizing trimeric LUBAC. Inhibition of LTM-mediated HOIL-1L/SHARPIN dimerization profoundly attenuated the function of LUBAC, suggesting LTM as a superior target of LUBAC destabilization for anticancer therapeutics.

The linear ubiquitin chain assembly complex LUBAC generates heterotypic ubiquitin chains

“…Purifications of the three LUBAC components expressed individually in E. coli consistently gave low yields and isolated proteins were co-purified with several contaminants; this was particularly severe in purifications of full length HOIP ( Figure 1A). Given that HOIP is destabilized in cells lacking SHARPIN or HOIL-1L (Fujita et al, 2018, Gerlach et al, 2011, Ikeda et al, 2011, Peltzer et al, 2018, we conjectured that HOIP could be unstable when recombinantly expressed in the absence of its interaction partners. To this end, we expressed HOIP (119.8 kDa), N-terminally His-tagged HOIL-1L (59.2 kDa), and N-terminally Strep(II)-tagged SHARPIN (43.0 kDa) in insect cells in order to co-purify the LUBAC holoenzyme by tandem affinity chromatography.…”

“…HOIP has a catalytic center in its RING2 domain responsible for assembly of linear ubiquitin chains, while HOIL-1L and SHARPIN have been recognized as accessory proteins for the process. It is more recent that HOIL-1L has been shown to catalyse ubiquitination (Pao et al, 2018, Smit et al, 2013, Stieglitz et al, 2012.Linear ubiquitin chains and the three LUBAC components are essential components in biological functions including immune signalling (Gerlach et al, 2011, Ikeda, 2015, Rahighi et al, 2009, Rittinger & Ikeda, 2017, development in mice (Fujita et al, 2018, Peltzer et al, 2018, Peltzer et al, 2014, protein quality control (van Well et al, 2019), Wnt signalling (Rivkin et al, 2013), and xenophagy (Noad et al, 2017, van Wijk et al, 2017.…”

“…The precise stoichiometry and oligomerization state of the LUBAC complex have not been established although recent structural work has suggested a 1:1:1 stoichiometry between the three core components (Fujita et al, 2018). To determine the stoichiometry and oligomerization state of the LUBAC complex we used mass photometry (MP), a technique that enables accurate mass measurements based on the scattering of light by single macromolecules in solution (Sonn-Segev et al, 2019, Young et al, 2018 ( Figure 2D and Figure EV4).…”

“…Structural work of protein fragments has shown that HOIL-1L and SHARPIN interact with HOIP through their respective Ubiquitin-Like (UBL) domains, which bind cooperatively to the HOIP Ubiquitin-Associated (UBA) domain (Fujita et al, 2018, Liu et al, 2017, Yagi et al, 2012 ( Figure 3A). By using truncation mutants, it has also been shown that the SHARPIN UBL domain interacts with the Npl Zinc Finger 2 (NZF2) domain of HOIP (Ikeda et al, 2011).…”

“…By using truncation mutants, it has also been shown that the SHARPIN UBL domain interacts with the Npl Zinc Finger 2 (NZF2) domain of HOIP (Ikeda et al, 2011). More recently, structural work has revealed that SHARPIN and HOIL-1L interact with each other through their respective LUBAC-Tethering Motifs (LTMs) (Fujita et al, 2018). Otherwise, there is no further information available about the overall spatial arrangement of the domains of HOIP, HOIL-1L, and SHARPIN.…”