The Structure of Miro and its Ubiquitination Mechanism by Parkin

The outer mitochondrial membrane protein Miro (also called RhoT) is a highly conserved calcium (Ca2+)-binding GTPase at the regulatory nexus of a diverse set of mitochondrial processes, regulating mitochondrial shape, movement, organelle interactions and degradation. Miro is essential for the axonal transport of mitochondria, as it attaches mitochondria to the microtubule-based motor protein kinesin-1 through the cargo-adaptor protein Milton. Miro is also a substrate of the Parkinson's disease related protein Parkin E3 ubiquitin ligase in a signaling cascade that facilitates degradation of damaged mitochondria. However, relatively little is known about the molecular underpinnings of these processes and a structural understanding of the relevant protein machinery is lacking.;In this work, we present the first crystal structures of Miro, comprising the tandem EF hand and C-terminal GTPase (cGTPase) domains of Drosophila Miro. The structures reveal two previously unidentified "hidden" EF hands, each paired with a canonical EF hand. Each EF hand pair is bound to a helix that structurally mimics an EF hand ligand. A key nucleotide-sensing element lies within an extensive EF hand/cGTPase interface. These findings suggest structural mechanisms for calcium and nucleotide-dependent regulation of mitochondrial function by Miro.;To gain insight into the mechanistic principles underlying substrate ubiquitination by Parkin, we undertook an extensive structural and biochemical characterization of its substrates hMiro1 and hMiro2. Using an in vitro reconstitution system, we show that both are direct substrates of PINK1-phosphorylated Parkin (pParkin), and that hMiro1 ubiquitination proceeds with greater efficiency that ubiquitination of hMiro2. Mapping of ubiquitinated lysines identified by mass spectrometry onto a crystal structure we have determined of the hMiro1 EF hand and C-terminal GTPase (cGTPase) domains uncovers a preference for lysines in alpha-helical segments. By systematically testing for ubiquitination with isolated hMiro1 domains we show that the cGTPase domain is necessary and sufficient for efficient ubiquitination by pParkin, with a similar pattern observed for hMiro2. Moreover, the isolated nGTPase domains of hMiro1 and hMiro2 are not appreciably ubiquitinated by pParkin, despite harboring modified lysines identified by mass spectrometry from the ubiquitinated, full-length proteins.;Additionally, we find that the isolated cGTPase domains of hMiro1 and hMiro2 dimerize in solution, and high-resolution crystal structures of both reveal a hydrophobic dimerization interface that is mutually exclusive with the EF hand/cGTPase interface. While the hMiro2 cGTPase dimer is weakly ubiquitinated at best, the hMiro1 cGTPase dimer is ubiquitinated efficiently at multiple sites, including lysines not modified in full-length hMiro1. Surprisingly, disruption of the hMiro1 cGTPase dimer alters its ubiquitination pattern, yielding a mostly single-lysine substrate ubiquitinated at K572. Using this unique substrate, we show that phosphorylation of Parkin not only increases its ubiquitination activity but also confers the ability to specifically target K572, since an un-phosphorylated, N-terminally tagged Parkin shows no preference for target lysines in addition to lower overall activity. Furthermore, using a ubiquitin chemically modified at its C-terminus (Ub-MES) that bypasses the requirement for E1 and E2 activation, we show that K572 lysine prioritization by pParkin is a property intrinsic to the E3 and does not require E2 binding. Based on our findings identifying phosphorylation of Parkin as a pre-requisite for K572 targeting in the hMiro1 cGTPase domain, we propose a dual role for Parkin Ubl phosphorylation, namely stimulating catalytic activity and priming the ligase to specifically recognize and orient substrates for targeted lysine modification.;Our study is the first to provide a detailed analysis of substrate ubiquitination by the Parkin RBR ligase, outlining mechanistic principles underlying Parkin ubiquitination of Miro that are likely applicable to other Parkin substrates.