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

Abstract

The E3 ligase parkin ubiquitinates outer mitochondrial membrane proteins during oxidative stress and is linked to early‐onset Parkinson's disease. Parkin is autoinhibited but is activated by the kinase PINK1 that phosphorylates ubiquitin leading to parkin recruitment, and stimulates phosphorylation of parkin's N‐terminal ubiquitin‐like (pUbl) domain. How these events alter the structure of parkin to allow recruitment of an E2~Ub conjugate and enhanced ubiquitination is an unresolved question. We present a model of an E2~Ub conjugate bound to the phospho‐ubiquitin‐loaded C‐terminus of parkin, derived from NMR chemical shift perturbation experiments. We show the UbcH7~Ub conjugate binds in the open state whereby conjugated ubiquitin binds to the RING1/IBR interface. Further, NMR and mass spectrometry experiments indicate the RING0/RING2 interface is re‐modelled, remote from the E2 binding site, and this alters the reactivity of the RING2(Rcat) catalytic cysteine, needed for ubiquitin transfer. Our experiments provide evidence that parkin phosphorylation and E2~Ub recruitment act synergistically to enhance a weak interaction of the pUbl domain with the RING0 domain and rearrange the location of the RING2(Rcat) domain to drive parkin activity.

Programmed ubiquitin acetylation using genetic code expansion reveals altered ubiquitination patterns

“…Recent studies of the phosphorylation of Ub and the E3 ligase parkin have identified PINK1 as the requisite protein kinase . Purified versions of this kinase have been successfully used to enzymatically phosphorylate purified ubiquitin . However, other serine residues have been observed to be phosphorylated in Ub and are only reachable using orthogonal methods until the corresponding kinase proteins have been identified.…”

“…Human Uba1 (E1), UBE2D1 or UBE2N/UBE2V2 (E2), and RNF8 345–485 or IpaH3CT (invasion plasmid antigen H3) (E3) enzymes used in ubiquitination assays were produced and purified to homogeneity as described previously . Ubiquitination assays were conducted using 0.2 μ m Uba1, 1 μ m E2, 2 μ m E3, 8 μ m Ub, or acUbKx (where x denotes the acetylated residue), 5 m m MgATP, and 50 m m HEPES at pH 7.4.…”

Quo Vadis Biomolecular NMR Spectroscopy?

“…No other atomic-resolution method can unravel these scenarios at comparable levels of resolution and with similar ease. Indeed, several publications pay tribute to the great power of NMR in such structure–function analyses [94,100,105,149,150,151,152,153,154,155,156,157,158,159]. In the following paragraphs, I discuss three examples that illustrate the scope of phosphorylation-induced structural rearrangements and NMR’s excellent ability to decipher them, and their resulting architectures.…”

Post-translational regulation of ubiquitin signaling

“…4). This modification causes the movement of the Ubl domain and the release of the catalytic RING2, one of the four zinc-binding domains in the E3 ligase (Condos et al, 2018; Sauvé et al, 2018). PINK1 also phosphorylates Ser 65 in poly-Ub chains associated with mitochondria, which further promotes the tethering of Parkin to the organelle (Shiba-Fukushima et al, 2014; Wauer et al, 2015b).…”