Autoregulation of Parkin activity through its ubiquitin-like domain

Abstract

Parkin is an E3-ubiquitin ligase belonging to the RBR (RING-InBetweenRING-RING family), and is involved in the neurodegenerative disorder Parkinson's disease. Autosomal recessive juvenile Parkinsonism, which is one of the most common familial forms of the disease, is directly linked to mutations in the parkin gene. However, the molecular mechanisms of Parkin dysfunction in the disease state remain to be established. We now demonstrate that the ubiquitin-like domain of Parkin functions to inhibit its autoubiquitination. Moreover pathogenic Parkin mutations disrupt this autoinhibition, resulting in a constitutively active molecule. In addition, we show that the mechanism of autoregulation involves ubiquitin binding by a C-terminal region of Parkin. Our observations provide important molecular insights into the underlying basis of Parkinson's disease, and in the regulation of RBR E3-ligase activity.

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

“…Structures of near full‐length parkin (Kumar et al , 2015; Sauvé et al , 2015) show the C‐terminal RING0–RING1–IBR–RING2(Rcat) domains (termed “R0RBR”) form a compact unit whereby the N‐terminal Ubl domain interacts with the RING1 and IBR domains and portions of the tether region (Fig 1A). This mode of interaction confirmed that the Ubl domain exerted its previously identified autoinhibitory effect (Chaugule et al , 2011) by blocking the expected binding site on the RING1 domain from an E2 conjugating enzyme resulting in negligible ubiquitination activity. Despite the apparent well‐folded, compact nature of parkin in this state, a wealth of flexibility exists within the E3 ligase that is not obvious from the crystal structures.…”

“…Autoinhibited state whereby the Ubl domain masks the E2 binding site on the RING1 (R1) domain as described by Chaugule et al (2011) and supported through crystallographic studies.…”

“…Mutation of these residues renders parkin unreactive with an E2‐based activity probe, consistent with a requirement for pUbl interaction (Pao et al , 2016). Yet other experiments show parkin retains appreciable ubiquitination ability in the absence of its Ubl domain (Chaugule et al , 2011; Kazlauskaite et al , 2014; Kumar et al , 2015), suggesting phosphorylation and the Ubl domain itself are less important. Our NMR data of full‐length pParkin:pUb (Fig 5) indicate the native interaction of the pUbl domain with the RING0 domain is weak in this state in agreement with previous affinity experiments (Kumar et al , 2015; Sauvé et al , 2015).…”

“…Although parkin was originally thought to be a constitutively active enzyme, it is now known to regulate its activity through intramolecular domain–domain interactions and binding to effectors (Chaugule et al , 2011; Kumar et al , 2015; Sauvé et al , 2015). In particular, PINK1 regulates parkin activity through phosphorylation of the Ubl domain, and of ubiquitin itself (Kumar et al , 2015; Sauvé et al , 2015; Wauer et al , 2015; Kumar et al , 2017), which then acts as an effector.…”

“…This E2~Ub arrangement promotes a conformation susceptible to the transthiolation reaction needed to transfer the Ub cargo from the E2 enzyme to the catalytic cysteine of the RING2(Rcat) domain. 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).…”