Photon Factory Activity Report 2017 #35 (2018)

BL-1A, BL-17A/2015G703, 2016G170

Crystal structure of hetero-trimeric core of LUBAC, a unique ubiquitin ligase forlinear ubiquitination
Akira Tokunaga*1, Hiroaki Fujita2, Erik Walinda2, Izuru Ohki1, Mariko Ariyoshi3, Kazuhiro Iwai2,
Hidehito Tochio4, and Masahiro Shirakawa1
1

Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto
615-8510, Japan2
Department of Molecular and Cellular Physiology, Graduate School of Medicine, Kyoto University,Kyoto 606-8501, Japan
3
Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan
4
Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan
1 Introduction
Linear ubiquitin chain assembly complex (LUBAC) is aunique ligase synthesizing a linear ubiquitin chain, in
which the amino group of the N-terminal Met of one
ubiquitin and the C-terminal carboxyl group of another areconjugated. In nuclear factor-kappa B (NF-κB) signaling
pathway, LUBAC attaches the linear ubiquitin to NF-κB
essential modulator (NEMO) and promotes transcriptionof genes encoding several cytokines, chemokines, or celladhesion molecules, activating the immune response orcell death repression. LUBAC is composed of the catalytic
subunit HOIP and the accessory subunits HOIL-1L andSHARPIN. It has been reported that mice lacking
SHARPIN suffer from auto-inflammation andimmunodeficiency, most likely due to destabilization ofLUBAC and consequential dysregulation of NF-κB
signaling. Although this implies that the integrity of thethree subunits is needed for the catalytic activity ofLUBAC, the structural details of simultaneous trimericinteractions remain elusive.
Here we report a crystal structure of the core region ofLUBAC at 2.40 Å resolution, which explains the ternaryintermolecular interactions essential to functional LUBAC
formation [1].
2 ExperimentSingle-wavelength (0.98Å) data for the core region ofLUBAC was collected on the beamline 1A at Photon
Factory. The data was processed using HKL2000 and theCCP4 program suite.

Fig: (Top) Overall structure of the core region of LUBAC.
The structure of HOIP UBA, SHARPIN LTM-UBL, andHOIL-1L LTM-UBL are shown in blue, pink, and yellow,
respectively. (Bottom) The structure of the tethering
domain is shown in the left panel. The right panel displays
a topology diagram of the tethering domain.

3 Results and Discussion
The structure of the trimeric core of LUBAC wasdetermined using molecular replacement and refined at aresolution of 2.40 Å to a crystallographic R-factor of24.4%. The final model was comprised of amino acidresidues 2-136 (A chain; HOIL-1L), 473-629 (B chain;HOIP), 169-300 (C chain; SHARPIN), a glycerol molecule,and 21 waters (Fig).
The three subunits form a trimeric complex with a 1:1:1
stoichiometry. HOIP constitutes the ubiquitin-associated(UBA) region comprising seven a helices and two 310
helices, in which two UBA-like modules are tandemly

linked via a middle a4 helix. HOIL-1L and SHARPIN
share the serial motifs of a LUBAC tethering motif (LTM)
and a ubiquitin-like (UBL) domain. HOIP UBA binds theUBL domains of HOIL-1L and SHARPIN mainly using its
N- and C-terminal UBA modules, respectively, though thebinding modes are completely different from each other. Inaddition, the structure demonstrates a novel interaction
between the LTMs of the HOIL-1L and SHARPIN, inwhich the helix-helix-strand motifs of LTMs are swapped
to fold into a domain-like architecture, denominated as thetethering domain. The structural basis of LUBAC

Photon Factory Activity Report 2017 #35 (2018)

formation established by the findings of the simultaneoustrimeric interactions would be valuable not only forunderstanding the molecular mechanism of LUBAC
destabilization and dysregulation, but also for targeting
LUBAC in therapeutics.
References[1] Fujita, H., Tokunaga, A., et al. (2018) Cell Rep. 23(4),1192–1204.
* tokunaga.akira.38w@st.kyoto-u.ac.jp