Structural insights into the mechanism and E2 specificity of the RBR E3 ubiquitin ligase HHARI

Abstract

RING-in-between-RING (RBR) ubiquitin (Ub) E3 ligases function with Ub E2s through a RING/HECT hybrid mechanism to conjugate Ub to target proteins. Here, we report the crystal structure of the RBR E3, HHARI, in complex with a UbcH7 ~ Ub thioester mimetic which reveals the molecular basis for the specificity of this cognate E2/RBR E3 pair. The structure also reveals mechanistically important conformational changes in the RING1 and UBA-like domains of HHARI that accompany UbcH7 ~ Ub binding and provides a molecular basis by which HHARI recruits E2 ~ Ub in an ‘open’ conformation. In addition to optimally functioning with an E2 that solely performs transthiolation, our data suggests that HHARI prevents spurious discharge of Ub from E2 to lysine residues by: (1) harboring structural elements that block E2 ~ Ub from adopting a ‘closed’ conformation and (2) participating in contacts to ubiquitin that promote an open E2 ~ Ub conformation.

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

“…This interaction with UbcH7‐Ub shows that the donor Ub is in the open conformation favoured by RBRs (Dove et al , 2016). The interaction site of UbcH7 with the RING1 domain in our structure is in agreement with previous crystal structures of HOIP with UbcH5b‐Ub (Lechtenberg et al , 2016) and HHARI with UbcH7‐Ub (Dove et al , 2017; Yuan et al , 2017) although minor orientation differences occur. This likely arises due to differences in the L2 loop regions of the RBR E3 ligases as previously noted (Spratt et al , 2014; Dove et al , 2016), and in particular differences in the linchpin residue that directs the E2~Ub conjugate to its open state during interaction (Dove et al , 2017).…”

“…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). …”

“…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.…”

Zinc finger domain of the human DTX protein adopts a unique RING fold

“…The canonical RING fingers adopt a ββαβ fold and possess a functional α region (active site) for E2‐binding . Although some RING fingers, like RING2 of RING‐in‐between‐RING, are involved in protein ubiquitination, they do not have the active site for E2‐binding . By contrast, PHD, FYVE, and ZZ fingers are not involved in protein ubiquitination .…”