New Perspectives on Phosphorylation State in the Parkin Ubiquitination Cascade
Abstract
The RBR E3 ligase parkin is recruited to the outer mitochondrial membrane (OMM) during oxidative stress where it becomes activated and ubiquitinates numerous proteins. Parkin activation involves binding of a phosphorylated ubiquitin (pUb), followed by phosphorylation of parkin itself, both mediated by the OMM kinase, PINK1. However, targeted mitochondrial proteins have little structural or sequence similarity, with the commonality between substrates being proximity to the OMM. The objective of this thesis was to identify the molecular consequences of parkin phosphorylation, interaction with pUb and how this promotes ubiquitination activity of known Ub-acceptor proteins and parkin itself.
The three-dimensional structure of the isolated pUbl domain revealed structural changes that alter the surfaces charges to disrupt the autoinhibitory interaction. In the presence of pUb, this releases pUbl and promotes the active form of parkin. Parkinson’s disease related substitutions throughout the Ubl domain reduce the rate of phosphorylation by PINK1, destabilize the domain and alter autoubiquitination.
Also shown is a model of activated parkin bound to pUb and an E2-Ub conjugate established from NMR chemical shift perturbations. The RING0/Rcat interface is distant from the E2-Ub binding site but it remodelled to increase exposure to the catalytic cysteine. These 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 Rcat domain to drive parkin activity.
Finally, parkin was shown to efficiently ubiquitinate acceptor proteins pre-ligated to pUb and phosphorylation of parkin triggers autoubiquitination activity. Parkin phosphorylation is not required for acceptor-pUb ubiquitination. In fact, only phospho-parkin induced self-ubiquitination and deletion of Ubl or mutation at K211N inhibited self-ubiquitination. We propose divergent parkin mechanisms whereby parkin-mediated ubiquitination of acceptor proteins is driven by binding to pre-existing pUb and subsequent parkin phosphorylation triggers autoubiquitination. This finding is critical for understanding parkin’s role in mitochondrial homeostasis and has implications on targets for therapeutics.