LUBAC synthesizes linear ubiquitin chains via a thioester intermediate

Abstract

The linear ubiquitin chain assembly complex (LUBAC) is a RING E3 ligase that regulates immune and inflammatory signalling pathways. Unlike classical RING E3 ligases, LUBAC determines the type of ubiquitin chain being formed, an activity normally associated with the E2 enzyme. We show that the RING-in-between-RING (RBR)-containing region of HOIP--the catalytic subunit of LUBAC--is sufficient to generate linear ubiquitin chains. However, this activity is inhibited by the N-terminal portion of the molecule, an inhibition that is released upon complex formation with HOIL-1L or SHARPIN. Furthermore, we demonstrate that HOIP transfers ubiquitin to the substrate through a thioester intermediate formed by a conserved cysteine in the RING2 domain, supporting the notion that RBR ligases act as RING/HECT hybrids.

Figure 1

The composition of LUBAC and the activity of the RBRs. (A) Domain structure of the LUBAC subunits, with the domains mediating protein interactions indicated. The colour coding used in this figure (HOIP in green and so on) will be maintained throughout the manuscript. (B) Sequence alignment of RING2 domains and structure of HHARI RING2 (1WD2.pdb). Conserved cysteines that are involved in coordination of the Zn²⁺ are indicated by * and the cysteine forming the thioester by A. This corresponds to Cys357 in HHARI and Cys885 in HOIP. (C) The RBR of HOIP catalyses the synthesis of linear ubiquitin chains. In vitro ubiquitination assays with the RBR of HOIP using wt ubiquitin (left) or His₆-ubiquitin (right) as substrate. A star indicates assay components, mostly E2, that have not precipitated during acidification. (D) In vitro ubiquitination assays covering different time scales with HOIL-1L using wt or His₆-ubiquitin as substrate. The reaction conditions used were identical in C and D. Gels were stained with Coomassie Blue. The constructs used in C and D are indicated schematically above the gels with the domains present in the assays highlighted in colour in green for HOIP and blue for HOIL-1L. HOIL-1L, haem-oxidized iron-regulatory protein 2 ubiquitin ligase-1; HOIP, HOIL-1L-interacting protein; LUBAC, linear ubiquitin chain assembly complex; RBR, RING-in-between-RING; RING, really interesting new gene; SHARPIN, Shank-associated RH domain-interacting protein; UBA, ubiquitin-associated; UBL, ubiquitin-like; wt, wild-type.

Figure 2

Inhibition of the catalytic activity of HOIP by adjacent domains and release through complex formation with HOIL-1L. (A) Comparison of ubiquitin chain synthesis by HOIP constructs containing the RBR (as Fig 1C) and (B) UBA-RBR. (C) ITC titration between the UBA of HOIP and UBL of HOIL-1L. (D) Ubiquitination assay with a HOIP UBA-RBR/HOIL-1L UBL complex. (E) Comparison of the activity of a complex between HOIP UBA-RBR and wt HOIL-1L versus (F) a complex with a catalytically dead HOIL-1L mutant (C460A). Stars indicate assay components that have not precipitated during acidification. The colour coding and reaction conditions used are as in Fig 1C,D. HOIL-1L, haem-oxidized iron-regulatory protein 2 ubiquitin ligase-1; HOIP, HOIL-1L-interacting protein; RBR, RING-in-between-RING; UBA, ubiquitin-associated; UBL, ubiquitin-like; wt, wild-type.

Figure 3

Comparison of linear ubiquitin chain synthesis by different LUBAC complexes. The gels show the increase in the rate of chain formation upon addition of SHARPIN or HOIL-1L to a HOIP ZFs-UBA-RBR construct. There is no further increase in the rate of chain formation in the trimeric complex. Stars indicate assay components that have not precipitated during acidification. HOIL-1L, haem-oxidized iron-regulatory protein 2 ubiquitin ligase-1; HOIP, HOIL-1L-interacting protein; LUBAC, linear ubiquitin chain assembly complex; RBR, RING-in-between-RING; SHARPIN, Shank-associated RH domain-interacting protein; UBA, ubiquitin-associated.

Figure 4

Ubiquitination assays using Cy5-labelled ubiquitin show the formation of a HOIP-thioester. The gels shown only monitor the Cy5-labelled ubiquitin; gels stained for total protein content are shown in supplementary Fig S4 online. (A) Assay using wt HOIP-RBR-C: lanes 1/2 ubiquitin+E1 before addition of ATP, lanes 3/4+ATP, lanes 5/6 +E2, lanes 7/8 +E3 and lanes 9/10+diUb substrate. Uneven numbered lanes are −DTT, even numbered +DTT. (B) Assay using HOIP-RBR C885A, same conditions as in (A), no ubiquitin is transferred to E3 in lane 7, indicating that no thioester is formed. (C) Assay using HOIP-RBR C885S. An adduct is formed upon addition of RBR C885S that is not sensitive to treatment with reducing agent and cannot be transferred to a substrate indicating formation of an oxyester. Lanes 1–8 are equivalent to reactions in panels A and B. Incubation with DTT does not reduce the adduct formed with HOIP-RBR-C but does reduce the E2-thioester (lane 8). Lane 9: the reaction mixture is treated with NaAc, which hydrolyses the RBR-adduct. Lanes 10+11: addition of diUb substrate to the reaction mixture. Blue, yellow and red stars indicate the bands representing E1~Ub, RBR∼Ub and E2∼Ub, respectively. DTT, dithiothreitol; HOIP, haem-oxidized iron-regulatory protein 2 ubiquitin ligase-1-interacting protein; RBR, RING-in-between-RING; wt, wild-type.