Activation of parkin by a molecular glue.

Mutations in parkin and PINK1 cause early-onset Parkinson's disease (EOPD). The ubiquitin ligase parkin is recruited to damaged mitochondria and activated by PINK1, a kinase that phosphorylates ubiquitin and the ubiquitin-like domain of parkin. Activated phospho-parkin then ubiquitinates mitochondrial proteins to target the damaged organelle for degradation. Here, we present the mechanism of activation of a new class of small molecule allosteric modulators that enhance parkin activity. The compounds act as molecular glues to enhance the ability of phospho-ubiquitin (pUb) to activate parkin. Ubiquitination assays and isothermal titration calorimetry with the most active compound (BIO-2007817) identify the mechanism of action. We present the crystal structure of a closely related compound (BIO-1975900) bound to a complex of parkin and two pUb molecules. The compound binds next to pUb on RING0 and contacts both proteins. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments confirm that activation occurs through release of the catalytic Rcat domain. In organello and mitophagy assays demonstrate that BIO-2007817 partially rescues the activity of parkin EOPD mutants, R42P and V56E, offering a basis for the design of activators as therapeutics for Parkinson's disease.

Naturally occurring hyperactive variants of human parkin.

Parkinson's disease (PD) is the second most common neurodegenerative disease in the world. Although most cases are sporadic and occur later in life, 10-15% of cases are genetic. Loss-of-function mutations in the ring-between-ring E3 ubiquitin ligase parkin, encoded by the PRKN gene, cause autosomal recessive forms of early onset PD. Together with the kinase PINK1, parkin forms a mitochondrial quality control pathway that tags damaged mitochondria for clearance. Under basal conditions, parkin is inhibited and compounds that increase its activity have been proposed as a therapy for PD. Recently, several naturally occurring hyperactive parkin variants were identified, which increased mitophagy in cultured cells. Here, we validate the hyperactivities of these variants in vitro and compare the levels of activity of the variants to those of the wild-type and the well-characterized hyperactive variant, W403A. We also study the effects of mutating the parkin ACT (activating element) on parkin activity in vitro. This work advances our understanding of the pathogenicity of parkin variants and is an important first step in the design of molecules to increase parkin activity.

Parkinson's disease-linked parkin mutation disrupts recycling of synaptic vesicles in human dopaminergic neurons.

Parkin-mediated mitophagy has been studied extensively, but whether mutations in parkin contribute to Parkinson's disease pathogenesis through alternative mechanisms remains unexplored. Using patient-derived dopaminergic neurons, we found that phosphorylation of parkin by Ca2+/calmodulin-dependent protein kinase 2 (CaMK2) at Ser9 leads to activation of parkin in a neuronal-activity-dependent manner. Activated parkin ubiquitinates synaptojanin-1, facilitating its interaction with endophilin A1 and synaptic vesicle recycling. Neurons from PD patients with mutant parkin displayed defective recycling of synaptic vesicles, leading to accumulation of toxic oxidized dopamine that was attenuated by boosting endophilin A1 expression. Notably, combined heterozygous parkin and homozygous PTEN-induced kinase 1 (PINK1) mutations led to earlier disease onset compared with homozygous mutant PINK1 alone, further underscoring a PINK1-independent role for parkin in contributing to disease. Thus, this study identifies a pathway for selective activation of parkin at human dopaminergic synapses and highlights the importance of this mechanism in the pathogenesis of Parkinson's disease.

Burst kinetics and CNNM binding are evolutionarily conserved properties of phosphatases of regenerating liver.

Phosphatases of regenerating liver (PRL or PTP4A) are a family of enigmatic protein phosphatases implicated in cell growth and metabolism. Despite their relevance in metastatic cancer, much remains unknown about the PRL family. They act as pseudophosphatases to regulate the CNNM family of magnesium transporters yet also have enzymatic activity on unknown substrates. In mammals, PRLs are mostly found trapped in an intermediate state that regulates their pseudophosphatase activity. Phosphocysteine, which is formed as an intermediate in the phosphatase catalytic cycle, is inefficiently hydrolyzed leading to burst enzyme kinetics and turnover numbers of less than one per hour. In flies, PRLs have recently been shown to have neuroprotective and neurodevelopmental roles raising the question whether they act as phosphatases, pseudophosphatases, or both. Here, we characterize the evolutionary development of PRLs and ask whether their unique structural and functional properties are conserved. We purified recombinant PRL proteins from 15 phylogenetically diverse organisms and characterized their catalytic activities and ability to bind CNNM proteins. We observed PRLs from humans to amoebae form a stable phosphocysteine intermediate and exhibit burst kinetics. Isothermal titration calorimetry experiments confirmed that the PRL-CNNM interaction is broadly conserved with nanomolar affinity in vertebrates. Lastly, we determined the crystal structure of the Drosophila melanogaster PRL-CNNM complex and identified mutants that specifically impair either phosphatase activity or CNNM binding. Our results reveal the unique properties of PRLs are conserved throughout the animal kingdom and open the door to using model organisms to dissect PRL function in cell signaling.

Feedforward activation of PRKN/parkin.

Parkinson disease is a neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the midbrain. The majority of early onset forms of Parkinson disease are a result of autosomal mutations in PRKN (parkin RBR E3 ubiquitin protein ligase) and PINK1 (PTEN induced kinase 1), which together regulate the clearance of damaged mitochondria from cells through selective autophagy of mitochondria (mitophagy). In a pair of recent papers, we characterized a secondary mechanism of activation of PRKN by PINK1 that is responsible for approximately a quarter of mitophagy in a cellular model. Our deepening understanding of PRKN-PINK1 signaling affords hope for the development of small molecule therapeutics for the treatment of Parkinson disease.

Structure of the second phosphoubiquitin-binding site in parkin.

Parkin and PINK1 regulate a mitochondrial quality control system that is mutated in some early onset forms of Parkinson's disease. Parkin is an E3 ubiquitin ligase and regulated by the mitochondrial kinase PINK1 via a two-step cascade. PINK1 first phosphorylates ubiquitin, which binds a recruitment site on parkin to localize parkin to damaged mitochondria. In the second step, PINK1 phosphorylates parkin on its ubiquitin-like domain (Ubl), which binds a regulatory site to release ubiquitin ligase activity. Recently, an alternative feed-forward mechanism was identified that bypasses the need for parkin phosphorylation through the binding of a second phosphoubiquitin (pUb) molecule. Here, we report the structure of parkin activated through this feed-forward mechanism. The crystal structure of parkin with pUb bound to both the recruitment and regulatory sites reveals the molecular basis for differences in specificity and affinity of the two sites. We use isothermal titration calorimetry measurements to reveal cooperativity between the two binding sites and the role of linker residues for pUbl binding to the regulatory site. The observation of flexibility in the process of parkin activation offers hope for the future design of small molecules for the treatment of Parkinson's disease.

Structural basis for feedforward control in the PINK1/Parkin pathway.

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 observed 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, ubiquitin vinyl sulfone assays, ITC, and NMR titrations showed that the RING0 site has higher affinity for phospho-ubiquitin than phosphorylated Ubl in trans. We observed parkin activation by micromolar concentrations of tetra-phospho-ubiquitin chains that mimic mitochondria 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.

The double lives of phosphatases of regenerating liver: A structural view of their catalytic and noncatalytic activities.

Phosphatases of regenerating liver (PRLs) are protein phosphatases involved in the control of cell growth and migration. They are known to promote cancer metastasis but, despite over 20 years of study, there is still no consensus about their mechanism of action. Recent work has revealed that PRLs lead double lives, acting both as catalytically active enzymes and as pseudophosphatases. The three known PRLs belong to the large family of cysteine phosphatases that form a phosphocysteine intermediate during catalysis. Uniquely to PRLs, this intermediate is stable, with a lifetime measured in hours. As a consequence, PRLs have very little phosphatase activity. Independently, PRLs also act as pseudophosphatases by binding CNNM membrane proteins to regulate magnesium homeostasis. In this function, an aspartic acid from CNNM inserts into the phosphatase catalytic site of PRLs, mimicking a substrate-enzyme interaction. The delineation of PRL pseudophosphatase and phosphatase activities in vivo was impossible until the recent identification of PRL mutants defective in one activity or the other. These mutants showed that CNNM binding was sufficient for PRL oncogenicity in one model of metastasis, but left unresolved its role in other contexts. As the presence of phosphocysteine prevents CNNM binding and CNNM-binding blocks catalytic activity, these two activities are inherently linked. Additional studies are needed to untangle the intertwined catalytic and noncatalytic functions of PRLs. Here, we review the current understanding of the structure and biophysical properties of PRL phosphatases.

Crystal structure of an archaeal CorB magnesium transporter.

CNNM/CorB proteins are a broadly conserved family of integral membrane proteins with close to 90,000 protein sequences known. They are associated with Mg2+ transport but it is not known if they mediate transport themselves or regulate other transporters. Here, we determine the crystal structure of an archaeal CorB protein in two conformations (apo and Mg2+-ATP bound). The transmembrane DUF21 domain exists in an inward-facing conformation with a Mg2+ ion coordinated by a conserved π-helix. In the absence of Mg2+-ATP, the CBS-pair domain adopts an elongated dimeric configuration with previously unobserved domain-domain contacts. Hydrogen-deuterium exchange mass spectrometry, analytical ultracentrifugation, and molecular dynamics experiments support a role of the structural rearrangements in mediating Mg2+-ATP sensing. Lastly, we use an in vitro, liposome-based assay to demonstrate direct Mg2+ transport by CorB proteins. These structural and functional insights provide a framework for understanding function of CNNMs in Mg2+ transport and associated diseases.

Mg2+-ATP Sensing in CNNM, a Putative Magnesium Transporter.

The family of cystathionine-ß-synthase (CBS)-pair domain divalent metal cation transport mediators (CNNMs) is composed of four integral membrane proteins associated with Mg2+ transport. Structurally, CNNMs contain large cytosolic regions composed of a CBS-pair and a cyclic nucleotide-binding homology (CNBH) domain. How these regulate Mg2+ transport activity is unknown. Here, we determined the crystal structures of cytosolic fragments in two conformations: Mg2+-ATP-analog bound and ligand free. The structures reveal open and closed conformations with functionally important contacts not observed in structures of the individual domains. We also identified a second Mg2+-binding region in the CBS-pair domain and a different dimerization interface for the CNBH domain. Analytical ultracentrifugation and isothermal titration calorimetry experiments revealed a tight correlation between Mg2+-ATP binding and protein dimerization. Mutations that blocked either function prevented cellular Mg2+ efflux activity. The results suggest Mg2+ efflux is regulated by conformational changes associated with Mg2+-ATP binding to CNNM CBS-pair domains.

Cryo-electron microscopy structures of ArnA, a key enzyme for polymyxin resistance, revealed unexpected oligomerizations and domain movements.

Gram-negative bacteria evade the attack of cationic antimicrobial peptides through modifying their lipid A structure in their outer membranes with 4-amino-4-deoxy-L-arabinose (Ara4N). ArnA is a crucial enzyme in the lipid A modification pathway and its deletion abolishes the polymyxin resistance of gram-negative bacteria. Previous studies by X-ray crystallography have shown that full-length ArnA forms a three-bladed propeller-shaped hexamer. Here, the structures of ArnA determined by cryo-electron microscopy (cryo-EM) reveal that ArnA exists in two 3D architectures, hexamer and tetramer. This is the first observation of a tetrameric ArnA. The hexameric cryo-EM structure is similar to previous crystal structures but shows differences in domain movements and conformational changes. We propose that ArnA oligomeric states are in a dynamic equilibrium, where the hexamer state is energetically more favorable, and its domain movements are important for cooperating with downstream enzymes in the lipid A-Ara4N modification pathway. The results provide us with new possibilities to explore inhibitors targeting ArnA.

The cyclic nucleotide-binding homology domain of the integral membrane protein CNNM mediates dimerization and is required for Mg2+ efflux activity.

Proteins of the cyclin M family (CNNMs; also called ancient conserved domain proteins, or ACDPs) are represented by four integral membrane proteins that have been proposed to function as Mg2+ transporters. CNNMs are associated with a number of genetic diseases affecting ion movement and cancer via their association with highly oncogenic phosphatases of regenerating liver (PRLs). Structurally, CNNMs contain an N-terminal extracellular domain, a transmembrane domain (DUF21), and a large cytosolic region containing a cystathionine-ß-synthase (CBS) domain and a putative cyclic nucleotide-binding homology (CNBH) domain. Although the CBS domain has been extensively characterized, little is known about the CNBH domain. Here, we determined the first crystal structures of the CNBH domains of CNNM2 and CNNM3 at 2.6 and 1.9 Å resolutions. Contrary to expectation, these domains did not bind cyclic nucleotides, but mediated dimerization both in crystals and in solution. Analytical ultracentrifugation experiments revealed an inverse correlation between the propensity of the CNBH domains to dimerize and the ability of CNNMs to mediate Mg2+ efflux. CNBH domains from active family members were observed as both dimers and monomers, whereas the inactive member, CNNM3, was observed only as a dimer. Mutational analysis revealed that the CNBH domain was required for Mg2+ efflux activity of CNNM4. This work provides a structural basis for understanding the function of CNNM proteins in Mg2+ transport and associated diseases.