Phosphorylation by PINK1 Releases the UBL Domain and Initializes the Conformational Opening of the E3 Ubiquitin Ligase Parkin

Abstract

Loss-of-function mutations in PINK1 or PARKIN are the most common causes of autosomal recessive Parkinson's disease. Both gene products, the Ser/Thr kinase PINK1 and the E3 Ubiquitin ligase Parkin, functionally cooperate in a mitochondrial quality control pathway. Upon stress, PINK1 activates Parkin and enables its translocation to and ubiquitination of damaged mitochondria to facilitate their clearance from the cell. Though PINK1-dependent phosphorylation of Ser65 is an important initial step, the molecular mechanisms underlying the activation of Parkin's enzymatic functions remain unclear. Using molecular modeling, we generated a complete structural model of human Parkin at all atom resolution. At steady state, the Ub ligase is maintained inactive in a closed, auto-inhibited conformation that results from intra-molecular interactions. Evidently, Parkin has to undergo major structural rearrangements in order to unleash its catalytic activity. As a spark, we have modeled PINK1-dependent Ser65 phosphorylation in silico and provide the first molecular dynamics simulation of Parkin conformations along a sequential unfolding pathway that could release its intertwined domains and enable its catalytic activity. We combined free (unbiased) molecular dynamics simulation, Monte Carlo algorithms, and minimal-biasing methods with cell-based high content imaging and biochemical assays. Phosphorylation of Ser65 results in widening of a newly defined cleft and dissociation of the regulatory N-terminal UBL domain. This motion propagates through further opening conformations that allow binding of an Ub-loaded E2 co-enzyme. Subsequent spatial reorientation of the catalytic centers of both enzymes might facilitate the transfer of the Ub moiety to charge Parkin. Our structure-function study provides the basis to elucidate regulatory mechanisms and activity of the neuroprotective Parkin. This may open up new avenues for the development of small molecule Parkin activators through targeted drug design.

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

“…This is likely a direct effect of phosphorylation only since several residues in this region (I44, A46, G47) have decreased amide exchange in the isolated Ubl domain (Aguirre et al , 2017). These observations show the pUbl domain is no longer bound at the IBR/RING1 interface and, consistent with previous NMR, sedimentation velocity and computational experiments, indicates the pUbl domain adopts a range of bound/free conformations in the pParkin:pUb state (Fig 1D; Caulfield et al , 2014; Aguirre et al , 2017). In this scenario, release of the pUbl domain exposes the predicted E2~Ub binding site, based on other RING/E2 complexes (Lechtenberg et al , 2016; Dove et al , 2017; Yuan et al , 2017).…”

Structure of phosphorylated UBL domain and insights into PINK1-orchestrated parkin activation

“…In contrast, the most compact model observed was autoinhibited pParkin/pUb (4.56 S, R G = 27Å). These observations are consistent with molecular dynamics calculations for compact and extended forms of parkin (24). Taking all calculated structures into account, the observed sedimentation coefficient (4.02 S) for pParkin/pUb is most consistent with an average structure whereby pUBL is displaced from R0RBR with R G ≈ 32Å, occupying a range of positions dictated by the configuration of the disordered linker.…”

“…Parkin's UBL shares high sequence and structural conservation with Ub, yet is significantly less thermodynamically stable (27). We show PINK1 phosphorylation further destabilizes the UBL by modifying its secondary structure, hydrogen bonding, and hydrophobicity, creating a new secondary structural element (α2-L) near the phosphorylation site proposed to be important for interactions with the linker based on computational studies (24,37). The significant structural differences in pUBL compared with pUb show why it cannot compete for the pUb-binding site, yet can independently increase parkin activity, if only partially.…”

“…S6). The movement of pSer65 into the bulk solvent uncovers a portion of this hydrophobic core close to the region suggested to be important for cleft widening with the linker region in parkin during activation (24). Whereas 1 H-15 N heteronuclear singlequantum coherence (HSQC) data report several large chemical shift changes upon phosphorylation (22), these do not report this change to the hydrophobic core (Fig.…”

“…Phosphorylation of parkin at S65 increases parkin's affinity for phosphoubiquitin (pUb) (14,15,22,23). Three-dimensional structures show this optimizes a pUb binding site, remote from the UBL site (15), and computational models predict local structural changes occur in the UBL domain upon phosphorylation (24). Further, binding of pUb allosterically induces a conformational change in parkin, liberating parkin from its autoinhibited state (14,15,22,25).…”