Structural basis for feedforward control in the PINK1/parkin pathway

Abstract

PINK1 and parkin constitute a mitochondrial quality control system mutated in Parkinson's disease. PINK1, a kinase, phosphorylates ubiquitin to recruit parkin, an E3 ubiquitin ligase, to mitochondria. PINK1 controls both parkin localization and activity through phosphorylation of both ubiquitin and the ubiquitin-like (Ubl) domain of parkin. Here, we observe that phospho-ubiquitin can bind to two distinct sites on parkin, a high affinity site on RING1 that controls parkin localization, and a low affinity site on RING0 that releases parkin autoinhibition. Surprisingly, NMR titrations and ubiquitin vinyl sulfone assays show that the RING0 site has higher affinity for phospho-ubiquitin than the phosphorylated Ubl. Parkin could be activated by micromolar concentrations of tetra-phospho-ubiquitin chains that mimic a mitochondrion bearing multiple phosphorylated ubiquitins. A chimeric form of parkin with the Ubl domain replaced by ubiquitin was readily activated by PINK1 phosphorylation. In all cases, mutation of the binding site on RING0 abolished parkin activation. The feedforward mechanism of parkin activation confers robustness and rapidity to the PINK1-parkin pathway and likely represents an intermediate step in its evolutionary development.

Structure of the second phosphoubiquitin–binding site in parkin

“…1 , B and C ). Overlaying of the pUb and pUbl in the two structures shows the largest conformational shifts are in the loop formed by residues 7 to 11, in agreement with previous modeling ( 31 ) ( Fig. 1 D ).…”

“…1 , G and H ). Overall, pUb forms more direct contacts with RING0 than pUbl, which leads to its roughly threefold higher affinity in isothermal titration calorimetry (ITC) experiments ( 31 ).…”

“…Parkin is also involved in an open-cycle or feed-forward mechanism that does not depend on parkin phosphorylation ( 31 ). We recently showed that binding of pUb to the RING0 pUbl-binding site can mediate phosphorylation-independent activation of parkin.…”

Mitophagy: Molecular Mechanisms, New Concepts on Parkin Activation and the Emerging Role of AMPK/ULK1 Axis

“…A new study shows the pUb’s ability to directly activate Parkin in the absence of the phosphorylation of its Ubl domain ( Figure 1 ) [ 133 ]. This secondary mechanism for the activation of Parkin would be dependent on the RING0 pUbl-binding site.…”

“…Similar to the PINK1-mediated activation of Parkin, the association of the second pUb to RING0 ( Figure 1 B-ii), instead of the pUbl domain ( Figure 1 B-i), leads to conformational reorganization, in which the catalytic RING2 domain is released. This mechanism reveals an amazing flexibility of RING0 for binding pUb and pUbl, suggesting the possibility that Parkin localization and activation may be controlled by other phosphoproteins that have the ability to bind RING0 or RING1 domains [ 133 ]. Thus, other activation mechanisms were proposed, but they require the support of structural analysis in future works.…”