Volume 21, Issue 1, Pages 27-36 (October 2017) Structural Insights into SHARPIN-Mediated Activation of HOIP for the Linear Ubiquitin Chain Assembly  Jianping Liu, Yingli Wang, Yukang Gong, Tao Fu, Shichen Hu, Zixuan Zhou, Lifeng Pan  Cell Reports  Volume 21, Issue 1, Pages 27-36 (October 2017) DOI: 10.1016/j.celrep.2017.09.031 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 21, 27-36DOI: (10.1016/j.celrep.2017.09.031) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 The UBL Domain of SHARPIN Specifically Binds to the UBA Domain of HOIP (A) A schematic diagram showing the domain architectures of SHARPIN, HOIP, and HOIL-1L proteins as well as the interactions between the three subunits of LUBAC. (B) Mapping the binding region of HOIP for the interaction with SHARPIN UBL domain via co-immunoprecipitation assay. (C) Analytical gel filtration chromatography analyses show the direct interaction between SHARPIN UBL domain and HOIP UBA domain. (D) ITC measurements of the binding affinities of HOIP UBA domain with SHARPIN UBL domain and the full-length SHARPIN protein. (E) Ribbon representation showing the overall structure of SHARPINUBL/HOIPUBA complex. In this drawing, the SHARPINUBL domain is shown in blue and HOIPUBA in green. (F) The combined ribbon and surface representation showing the overall architecture of SHARPINUBL/HOIPUBA complex with the same color scheme as in (A). See also Figure S1 and Table S1. Cell Reports 2017 21, 27-36DOI: (10.1016/j.celrep.2017.09.031) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 Detailed Molecular Interface of SHARPINUBL/HOIPUBA Complex and Its Validation by Site-Directed Point Mutations (A) Enlarged stereo view of ribbon-stick model showing the molecular details of the binding interface between SHARPINUBL and HOIPUBA. The salt bridges and hydrogen bonds involved in the binding are indicated as dashed lines. (B) Overlaid ITC data and fitting curves for the titrations between variants of SHARPIN and HOIP proteins. (C) Overlaid ITC data and fitting curves for the titrations between different HOIPUBA mutants and wild-type SHARPINUBL. (D) The measured binding affinities between different HOIP and SHARPIN proteins by ITC assays. N.D., KD value not detectable. (E) GST pull-down assays verify the interaction between full-length SHARPIN and HOIP as well as the key binding interface residue, SHARPIN V271. (F) In vitro linear ubiquitin chain assembly assays showing that the ability of HOIP to assemble linear ubiquitin chains is stimulated by wild-type SHARPIN but not the SHARPIN V271E mutant, which is unable to interact with HOIP. (G) NF-κB luciferase reporter assays using different HOIP, SHARPIN, and HOIL-1L variants. All luciferase activities are normalized to that of the control cells. Error bars denote the standard deviation between three replicates. See also Figures S2 and S3. Cell Reports 2017 21, 27-36DOI: (10.1016/j.celrep.2017.09.031) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 HOIPUBA Domain Adopts Different Conformations to Interact with SHARPIN and HOIL-1L (A) Overlaid structures of SHARPINUBL/HOIPUBA and HOIL-1LUBL/HOIPUBA complexes in the ribbon representation show the different interaction modes of HOIPUBA in binding to SHARPINUBL and HOIL-1LUBL. In this drawing, the HOIL-1LUBL and HOIPUBA in the HOIL-1LUBL/HOIPUBA complex are drawn in orange and in magenta, respectively. (B) Structural comparison of the configurations of HOIPUBA in the SHARPIN-bound and HOIL-1L-bound states shows the overall re-arrangements of HOIPUBA in binding to SHARPINUBL and HOIL-1LUBL. (C) Comparison of the topologies of HOIPUBA domain in the SHARPIN- and HOIL-1L-bound states. SHARPIN-binding induces the re-arrangement of three α helices (α1, α2, and α3) in the N terminus of HOIPUBA, which are collectively referred as Module1. Asterisks indicate the positions of the three critical hydrophobic residues (M484, V492, and I495) involved in SHARPIN binding. See also Figure S4. Cell Reports 2017 21, 27-36DOI: (10.1016/j.celrep.2017.09.031) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 SHARPINUBL and HOIL-1LUBL Can Synergistically Bind to and Cooperatively Activate HOIP by Facilitating Its E2 Loading (A) Analytic gel filtration chromatography analyses of the HOIPUBA/HOIL-1LUBL complex incubated with increasing molar ratio of SHARPINUBL proteins. (B) SDS-PAGE combined with Coomassie blue staining analyses shows the protein components of corresponding fraction 1 and fraction 2 collected from different analytic gel filtration chromatography experiments in (A). (C and D) ITC measurement of the binding affinity of SHARPINUBL to HOIP(480–1,072) (C) and HOIL-1LUBL/HOIP(480–1,072) complex (D). (E) In vitro linear ubiquitin chain assembly assays using purified E1, E2 enzyme UBE2L3, Ub, HOIP(299–1,072), HOIL-1LUBL, and SHARPINUBL proteins show that HOIL-1LUBL and SHARPINUBL can cooperatively activate HOIP to assemble linear ubiquitin chains. Asterisks indicate the bands of degraded E1 and HOIP proteins. (F) Quantitative measurements of the linear ubiquitin chain synthesis activities by quantification of the amount of residual mono-Ub at each time point under different protein complex conditions in (E). The concentration of mono-Ub is represented by the gray-level integration of mono-Ub band in each lane on the gel and normalized by the value at 0 min for each reaction condition. (G–I) ITC measurement of the binding affinity of UBE2L3 to HOIP(480–1,072) (G), the HOIP(480–1,072)/SHARPINUBL complex (H), and the HOIP(480–1,072)/HOIL-1LUBL complex (I). See also Figures S5–S7. Cell Reports 2017 21, 27-36DOI: (10.1016/j.celrep.2017.09.031) Copyright © 2017 The Author(s) Terms and Conditions