Structure of a HOIP/E2~ubiquitin complex reveals RBR E3 ligase mechanism and regulation

Abstract

Ubiquitination is a central process affecting all facets of cellular signaling and function1. A critical step in ubiquitination is the transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to a substrate or a growing ubiquitin chain, which is mediated by E3 ubiquitin ligases. RING-type E3 ligases typically facilitate the transfer of ubiquitin from the E2 directly to the substrate2,3. The RBR family of RING-type E3 ligases, however, breaks this paradigm by forming a covalent intermediate with ubiquitin similarly to HECT-type E3 ligases4–6. The RBR family includes Parkin4 and HOIP, the central catalytic factor of the linear ubiquitin chain assembly complex (LUBAC)7. While structural insights into the RBR E3 ligases Parkin and HHARI in their overall autoinhibited forms are available8–13, no structures exist of intact fully active RBR E3 ligases or any of their complexes. Thus, the RBR mechanism of action has remained largely enigmatic. Here we present the first structure of the fully active HOIP-RBR in its transfer complex with an E2~ubiquitin conjugate, which elucidates the intricate nature of RBR E3 ligases. The active HOIP-RBR adopts a conformation markedly different from that of autoinhibited RBRs. HOIP-RBR binds the E2~ubiquitin conjugate in an elongated fashion, with the E2 and E3 catalytic centers ideally aligned for ubiquitin transfer, which structurally both requires and enables a HECT-like mechanism. In addition, surprisingly, three distinct helix–IBR-fold motifs inherent to RBRs form ubiquitin-binding regions that engage the activated ubiquitin of the E2~Ub conjugate as well as an additional regulatory ubiquitin molecule. The features uncovered reveal critical states of the HOIP-RBR E3 ligase cycle, and comparison with Parkin and HHARI suggests a general mechanism for RBR E3 ligases.

Synergistic recruitment of UbcH7~Ub and phosphorylated Ubl domain triggers parkin activation

“…We interpreted the chemical shift changes within the tether region to result from both direct E2 binding and an altering of the tether position to accommodate the E2 enzyme. The open arrangement of the E2‐Ub bound to R0RBR parkin more closely resembles the interaction of UbcH5b‐Ub with HOIP (Lechtenberg et al , 2016) than either of the structures for UbcH7‐Ub with HHARI (Dove et al , 2017; Yuan et al , 2017; Fig EV4). Ub binding is governed predominantly by contacts from β1‐L1‐β2 (K6, L8, K11, I13‐T14), the linker following helix α1 (Q31‐D32) and C‐terminus (L73, R74) to an R0RBR surface including β1 (P333, P335) and the C‐terminus of the IBR domain (E370) and adjacent tether (V380, F381, S384, T386), RING1 helix H1 (N273) and the straightened RING1 helix H3 (R314, Y318).…”

“…These observations show the pUbl domain is no longer bound at the IBR/RING1 interface and, consistent with previous NMR, sedimentation velocity and computational experiments, indicates the pUbl domain adopts a range of bound/free conformations in the pParkin:pUb state (Fig 1D; Caulfield et al , 2014; Aguirre et al , 2017). In this scenario, release of the pUbl domain exposes the predicted E2~Ub binding site, based on other RING/E2 complexes (Lechtenberg et al , 2016; Dove et al , 2017; Yuan et al , 2017). …”

“…The Ub conjugate is positioned such that two hydrophobic regions including the I44 patch and the C‐terminus (V70, L71) of Ub are pointed away from the RING1 domain and exposed to solvent. Although not identical, the activated Ub in the HOIP/UbcH5b complex and donor Ub in the RING2L transfer complex are positioned similarly (Stieglitz et al , 2013; Lechtenberg et al , 2016). These hydrophobic regions (I44, V70, L71) are used to recruit helix h L2 from the catalytic RING2L domain in the domain‐swapped dimer structure (Lechtenberg et al , 2016).…”

“…In particular, RBR E3 ligases incorporate a hybrid ubiquitination mechanism (Wenzel et al , 2011) whereby an E2 conjugating enzyme is recruited to the RING1 domain (similar to RING E3 ligases) and ubiquitin (Ub) is transferred from the E2~Ub conjugate to a catalytic cysteine in the RING2(Rcat) domain (similar to HECT E3 mechanisms) prior to labelling of a substrate lysine. RBR E3 ligases and RING E3 ligases have RING domains that are structurally similar and are expected to recruit E2 enzymes in a similar fashion (Budhidarmo et al , 2012), as recently shown in crystal structures of the RBR E3 ligases HHARI with UbcH7‐Ub (Dove et al , 2017; Yuan et al , 2017) and HOIP with UbcH5b‐Ub (Lechtenberg et al , 2016). However, a distinguishing feature of the HHARI and HOIP RBR E3 ligases is their ability to recognize an extended (“open”) form of the E2~Ub conjugate similar to that used by HECT E3 enzymes.…”

“…A similar dilemma arises from recent structures of HHARI in complex with UbcH7‐Ub that show the ubiquitin molecule is 47–53 Å from the catalytic site (Dove et al , 2017; Yuan et al , 2017). Alternatively, structures of HOIP:E2‐Ub and parkin:pUb complexes assembled from domain‐swapping or symmetry‐related molecules raise the possibility of co‐operation between multiple E3 ligase molecules to promote ubiquitin transfer (Lechtenberg et al , 2016; Kumar et al , 2017). …”