Parkin–phosphoubiquitin complex reveals cryptic ubiquitin-binding site required for RBR ligase activity

Abstract

RING-BETWEENRING-RING (RBR) E3 ligases are a class of ubiquitin ligases distinct from RING or HECT E3 ligases. An important RBR is Parkin, mutations in which lead to early onset hereditary Parkinsonism. Parkin and other RBRs share a catalytic RBR module, but are usually autoinhibited and activated via distinct mechanisms. Recent insights into Parkin regulation predict large, unknown conformational changes during activation of Parkin. However, current data on active RBRs are in the absence of regulatory domains. Therefore, how individual RBRs are activated, and whether they share a common mechanism remains unclear. We now report the crystal structure of a human Parkin-phosphoubiquitin complex, which shows that phosphoubiquitin binding induces a movement in the IBR domain to reveal a cryptic ubiquitin binding site. Mutation of this site negatively impacts on Parkin’s activity. Furthermore, ubiquitin binding promotes cooperation between Parkin molecules, suggesting a role for interdomain association in RBR ligase mechanism.

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

“…NMR dynamics experiments show significant mobility for the IBR domain and segments on either side of a short helix within the tether region where poor electron density is frequently observed in crystal data (see fig S4 in Kumar et al , 2015). Details of the partial E3 ligase activation of parkin have been shown in complexes with phosphorylated ubiquitin (pUb; Kumar et al , 2015, 2017; Wauer et al , 2015), where pUb binds to a broad crevasse between the RING0 and RING1 domains (Fig 1A). The association of pUb causes rearrangement of the IBR domain and results in the formation of a large gap between the IBR and RING1 domains.…”

“…Parkin on the other hand has been shown to function with a variety of E2 enzymes including UbcH7 and UbcH5b (Chaugule et al , 2011; Wenzel et al , 2011) although it appears that UbcH7 is the optimal E2 enzyme owing to its preference for ubiquitin transfer to cysteine, a requirement for RBR E3 ligases. While the conformation of the UbcH7~Ub conjugate during recruitment by parkin is unknown, it has been established that a cryptic Ub binding site within the RING1–IBR interface is only uncovered upon pUb binding to parkin and this has been proposed to help coordinate E2~Ub recruitment (Kumar et al , 2017). …”

“…Under oxidative stress conditions, PINK1 is activated and phosphorylates ubiquitin (pUb) near the outer mitochondrial membrane. This in turn helps recruit parkin to the membrane through binding of pUb to the RING1–IBR region of the E3 ligase (Sauvé et al , 2015; Wauer et al , 2015; Kumar et al , 2017) and subsequent phosphorylation of parkin's Ubl (pUbl) domain (Ordureau et al , 2014). These two events greatly stimulate ubiquitination activity (Kondapalli et al , 2012; Shiba‐Fukushima et al , 2012; Kane et al , 2014; Kazlauskaite et al , 2014; Koyano et al , 2014) through an allosteric displacement mechanism of the pUbl domain from parkin (Kumar et al , 2015; Sauvé et al , 2015).…”

“…What is less clear is how parkin positions the E2~Ub conjugate to enable transfer of the Ub molecule to the RING2(Rcat) domain as a necessary step for catalysis. Current crystal structures of parkin show the proposed E2 binding site on the RING1 domain is > 50 Å from the catalytic site (C431) in the RING2(Rcat) domain suggesting a significant conformational rearrangement is needed (Riley et al , 2013; Trempe et al , 2013; Wauer & Komander, 2013; Kumar et al , 2015, 2017; Sauvé et al , 2015; Wauer et al , 2015). 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).…”

“…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). …”