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.

Role of ubiquitin-protein ligase UBR5 in the disassembly of mitotic checkpoint complexes.

The mitotic (or spindle assembly) checkpoint system ensures accurate chromosome segregation in mitosis by preventing the onset of anaphase until correct bipolar attachment of sister chromosomes to the mitotic spindle is attained. It acts by promoting the assembly of a mitotic checkpoint complex (MCC), composed of mitotic checkpoint proteins BubR1, Bub3, Mad2, and Cdc20. MCC binds to and inhibits the action of ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome), which targets for degradation regulators of anaphase initiation. When the checkpoint system is satisfied, MCCs are disassembled, allowing the recovery of APC/C activity and initiation of anaphase. Many of the pathways of the disassembly of the different MCCs have been elucidated, but the mode of their regulation remained unknown. We find that UBR5 (ubiquitin-protein ligase N-recognin 5) is associated with the APC/C*MCC complex immunopurified from extracts of nocodazole-arrested HeLa cells. UBR5 binds to mitotic checkpoint proteins BubR1, Bub3, and Cdc20 and promotes their polyubiquitylation in vitro. The dissociation of a Bub3*BubR1 subcomplex of MCC is stimulated by UBR5-dependent ubiquitylation, as suggested by observations that this process in mitotic extracts requires UBR5 and α-ß bond hydrolysis of adenosine triphosphate. Furthermore, a system reconstituted from purified recombinant components carries out UBR5- and ubiquitylation-dependent dissociation of Bub3*BubR1. Immunodepletion of UBR5 from mitotic extracts slows down the release of MCC components from APC/C and prolongs the lag period in the recovery of APC/C activity in the exit from mitotic checkpoint arrest. We suggest that UBR5 may be involved in the regulation of the inactivation of the mitotic checkpoint.

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.

Structural basis of 3'-end poly(A) RNA recognition by LARP1.

La-related proteins (LARPs) comprise a family of RNA-binding proteins involved in a wide range of posttranscriptional regulatory activities. LARPs share a unique tandem of two RNA-binding domains, La motif (LaM) and RNA recognition motif (RRM), together referred to as a La-module, but vary in member-specific regions. Prior structural studies of La-modules reveal they are pliable platforms for RNA recognition in diverse contexts. Here, we characterize the La-module of LARP1, which plays an important role in regulating synthesis of ribosomal proteins in response to mTOR signaling and mRNA stabilization. LARP1 has been well characterized functionally but no structural information exists for its La-module. We show that unlike other LARPs, the La-module in LARP1 does not contain an RRM domain. The LaM alone is sufficient for binding poly(A) RNA with submicromolar affinity and specificity. Multiple high-resolution crystal structures of the LARP1 LaM domain in complex with poly(A) show that it is highly specific for the RNA 3'-end, and identify LaM residues Q333, Y336 and F348 as the most critical for binding. Use of a quantitative mRNA stabilization assay and poly(A) tail-sequencing demonstrate functional relevance of LARP1 RNA binding in cells and provide novel insight into its poly(A) 3' protection activity.

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.

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.

PRL3 pseudophosphatase activity is necessary and sufficient to promote metastatic growth.

Phosphatases of regenerating liver (PRLs) are markers of cancer and promote tumor growth. They have been implicated in a variety of biochemical pathways but the physiologically relevant target of phosphatase activity has eluded 20 years of investigation. Here, we show that PRL3 catalytic activity is not required in a mouse model of metastasis. PRL3 binds and inhibits CNNM4, a membrane protein associated with magnesium transport. Analysis of PRL3 mutants specifically defective in either CNNM-binding or phosphatase activity demonstrate that CNNM binding is necessary and sufficient to promote tumor metastasis. As PRLs do have phosphatase activity, they are in fact pseudo-pseudophosphatases. Phosphatase activity leads to formation of phosphocysteine, which blocks CNNM binding and may play a regulatory role. We show levels of PRL cysteine phosphorylation vary in response to culture conditions and in different tissues. Examination of related protein phosphatases shows the stability of phosphocysteine is a unique and evolutionarily conserved property of PRLs. The demonstration that PRL3 functions as a pseudophosphatase has important ramifications for the design of PRL inhibitors for cancer.

Phosphatase, pseudo-phosphatase, or both? Understanding PRL oncogenicity.

Phosphatases of regenerating liver (PRL1-3) are among the most oncogenic protein phosphatases but their mechanism of action is poorly understood. Multiple substrates have been proposed as well as a non-catalytic function regulating magnesium transport. Our recent identification of a catalytically inactive PRL mutant that retains oncogenicity in a mouse model promises to resolve the question of whether PRLs act as phosphatases or pseudo-phosphatases in different cancer models.

LARP1 and LARP4: up close with PABP for mRNA 3' poly(A) protection and stabilization.

La-related proteins (LARPs) share a La motif (LaM) followed by an RNA recognition motif (RRM). Together these are termed the La-module that, in the prototypical nuclear La protein and LARP7, mediates binding to the UUU-3'OH termination motif of nascent RNA polymerase III transcripts. We briefly review La and LARP7 activities for RNA 3' end binding and protection from exonucleases before moving to the more recently uncovered poly(A)-related activities of LARP1 and LARP4. Two features shared by LARP1 and LARP4 are direct binding to poly(A) and to the cytoplasmic poly(A)-binding protein (PABP, also known as PABPC1). LARP1, LARP4 and other proteins involved in mRNA translation, deadenylation, and decay, contain PAM2 motifs with variable affinities for the MLLE domain of PABP. We discuss a model in which these PABP-interacting activities contribute to poly(A) pruning of active mRNPs. Evidence that the SARS-CoV-2 RNA virus targets PABP, LARP1, LARP 4 and LARP 4B to control mRNP activity is also briefly reviewed. Recent data suggests that LARP4 opposes deadenylation by stabilizing PABP on mRNA poly(A) tails. Other data suggest that LARP1 can protect mRNA from deadenylation. This is dependent on a PAM2 motif with unique characteristics present in its La-module. Thus, while nuclear La and LARP7 stabilize small RNAs with 3' oligo(U) from decay, LARP1 and LARP4 bind and protect mRNA 3' poly(A) tails from deadenylases through close contact with PABP.Abbreviations: 5'TOP: 5' terminal oligopyrimidine, LaM: La motif, LARP: La-related protein, LARP1: La-related protein 1, MLLE: mademoiselle, NTR: N-terminal region, PABP: cytoplasmic poly(A)-binding protein (PABPC1), Pol III: RNA polymerase III, PAM2: PABP-interacting motif 2, PB: processing body, RRM: RNA recognition motif, SG: stress granule.

The isolated La-module of LARP1 mediates 3' poly(A) protection and mRNA stabilization, dependent on its intrinsic PAM2 binding to PABPC1.

The protein domain arrangement known as the La-module, comprised of a La motif (LaM) followed by a linker and RNA recognition motif (RRM), is found in seven La-related proteins: LARP1, LARP1B, LARP3 (La protein), LARP4, LARP4B, LARP6, and LARP7 in humans. Several LARPs have been characterized for their distinct activity in a specific aspect of RNA metabolism. The La-modules vary among the LARPs in linker length and RRM subtype. The La-modules of La protein and LARP7 bind and protect nuclear RNAs with UUU-3' tails from degradation by 3' exonucleases. LARP4 is an mRNA poly(A) stabilization factor that binds poly(A) and the cytoplasmic poly(A)-binding protein PABPC1 (also known as PABP). LARP1 exhibits poly(A) length protection and mRNA stabilization similar to LARP4. Here, we show that these LARP1 activities are mediated by its La-module and dependent on a PAM2 motif that binds PABP. The isolated La-module of LARP1 is sufficient for PABP-dependent poly(A) length protection and mRNA stabilization in HEK293 cells. A point mutation in the PAM2 motif in the La-module impairs mRNA stabilization and PABP binding in vivo but does not impair oligo(A) RNA binding by the purified recombinant La-module in vitro. We characterize the unusual PAM2 sequence of LARP1 and show it may differentially affect stable and unstable mRNAs. The unique LARP1 La-module can function as an autonomous factor to confer poly(A) protection and stabilization to heterologous mRNAs.

Disrupting the CD95-PLCγ1 interaction prevents Th17-driven inflammation.

CD95L is a transmembrane ligand (m-CD95L) that is cleaved by metalloproteases to release a soluble ligand (s-CD95L). Unlike m-CD95L, interaction between s-CD95L and CD95 fails to recruit caspase-8 and FADD to trigger apoptosis and instead induces a Ca2+ response via docking of PLCγ1 to the calcium-inducing domain (CID) within CD95. This signaling pathway induces accumulation of inflammatory Th17 cells in damaged organs of lupus patients, thereby aggravating disease pathology. A large-scale screen revealed that the HIV protease inhibitor ritonavir is a potent disruptor of the CD95-PLCγ1 interaction. A structure-activity relationship approach highlighted that ritonavir is a peptidomimetic that shares structural characteristics with CID with respect to docking to PLCγ1. Thus, we synthesized CID peptidomimetics abrogating both the CD95-driven Ca2+ response and transmigration of Th17 cells. Injection of ritonavir and the CID peptidomimetic into lupus mice alleviated clinical symptoms, opening a new avenue for the generation of drugs for lupus patients.

Sacsin, mutated in the ataxia ARSACS, regulates intermediate filament assembly and dynamics.

Loss of sacsin, a large 520 kDa multidomain protein, causes autosomal recessive spastic ataxia of the Charlevoix-Saguenay, one of the most common childhood-onset recessive ataxias. A prominent feature is abnormal bundling of neurofilaments in many neuronal populations. This study shows the direct involvement of sacsin domains in regulating intermediate filament assembly and dynamics and identifies important domains for alleviating neurofilament bundles in neurons lacking sacsin. Peptides encoding sacsin internal repeat (SIRPT) 1, J-domains, and ubiquitin-like domain modified neurofilament assembly in vivo. The domains with chaperone homology, the SIRPT and the J-domain, had opposite effects, promoting and preventing filament assembly, respectively. In cultured Sacs-/- motor neurons, both the SIRPT1 and J-domain resolved preexisting neurofilament bundles. Increasing expression of heat shock proteins also resolved neurofilament bundles, indicating that this endogenous chaperone system can compensate to some extent for sacsin deficiency.-Gentil, B. J., Lai, G.-T., Menade, M., Larivière, R., Minotti, S., Gehring, K., Chapple, J.-P., Brais, B., Durham, H. D. Sacsin, mutated in the ataxia ARSACS, regulates intermediate filament assembly and dynamics.

Interdomain allostery promotes assembly of the poly(A) mRNA complex with PABP and eIF4G.

Many RNA-binding proteins contain multiple single-strand nucleic acid-binding domains and assemble into large multiprotein messenger ribonucleic acid protein (mRNP) complexes. The mechanisms underlying the self-assembly of these complexes are largely unknown. In eukaryotes, the association of the translation factors polyadenylate-binding protein-1 (PABP) and eIF4G is essential for high-level expression of polyadenylated mRNAs. Here, we report the crystal structure of the ternary complex poly(A)(11)·PABP(1-190)·eIF4G(178-203) at 2.0 Å resolution. Our NMR and crystallographic data show that eIF4G interacts with the RRM2 domain of PABP. Analysis of the interaction by small-angle X-ray scattering, isothermal titration calorimetry, and electromobility shift assays reveals that this interaction is allosterically regulated by poly(A) binding to PABP. Furthermore, we have confirmed the importance of poly(A) for the endogenous PABP and eIF4G interaction in immunoprecipitation experiments using HeLa cell extracts. Our findings reveal interdomain allostery as a mechanism for cooperative assembly of RNP complexes.

Mycobacterium tuberculosis Exploits a Heterohexameric Enoyl-CoA Hydratase Retro-Aldolase Complex for Cholesterol Catabolism.

Cholesterol catabolism plays an important role in Mycobacterium tuberculosis's (Mtb's) survival and persistence in the host. Mtb exploits three ß-oxidation cycles to fully degrade the side chain of cholesterol. Five cistronic genes in a single operon encode three enzymes, 3-oxo-4-pregnene-20-carboxyl-CoA dehydrogenase (ChsE1-ChsE2), 3-oxo-4,17-pregnadiene-20-carboxyl-CoA hydratase (ChsH1-ChsH2), and 17-hydroxy-3-oxo-4-pregnene-20-carboxyl-CoA retro-aldolase (Ltp2), to perform the last ß-oxidation cycle in this pathway. Among these three enzymes, ChsH1-ChsH2 and Ltp2 form a protein complex that is required for the catalysis of carbon-carbon bond cleavage. In this work, we report the structure of the full length ChsH1-ChsH2-Ltp2 complex based on small-angle X-ray scattering and single-particle electron microscopy data. Mutagenesis experiments confirm the requirement for Ltp2 to catalyze the retro-aldol reaction. The structure illustrates how acyl transfer between enzymes may occur. Each protomer of the ChsH1-ChsH2-Ltp2 complex contains three protein components: a chain of ChsH1, a chain of ChsH2, and a chain of Ltp2. Two protomers dimerize at the interface of Ltp2 to form a heterohexameric structure. This unique heterohexameric structure of the ChsH1-ChsH2-Ltp2 complex provides entry to further understand the mechanism of cholesterol catabolism in Mtb.

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.

A PH-like domain of the Rab12 guanine nucleotide exchange factor DENND3 binds actin and is required for autophagy.

Rab GTPases are key regulators of membrane trafficking, and many are activated by guanine nucleotide exchange factors bearing a differentially expressed in normal and neoplastic cells (DENN) domain. By activating the small GTPase Rab12, DENN domain-containing protein 3 (DENND3) functions in autophagy. Here, we identified a structural domain (which we name PHenn) containing a pleckstrin homology subdomain that binds actin and is required for DENND3 function in autophagy. We found that a hydrophobic patch on an extended ß-turn of the PHenn domain mediates an intramolecular interaction with the DENN domain of DENND3. We also show that DENND3 binds actin through a surface of positively charged residues on the PHenn domain. Substitutions that blocked either DENN or actin binding compromised the role of DENND3 in autophagy. These results provide new mechanistic insight into the structural determinants regulating DENND3 in autophagy and lay the foundation for future investigations of the DENN protein family.

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.

Structures of ubiquitin-like (Ubl) and Hsp90-like domains of sacsin provide insight into pathological mutations.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a neurodegenerative disease that is caused by mutations in the SACS gene. The product of this gene is a very large 520-kDa cytoplasmic protein, sacsin, with a ubiquitin-like (Ubl) domain at the N terminus followed by three large sacsin internal repeat (SIRPT) supradomains and C-terminal J and HEPN domains. The SIRPTs are predicted to contain Hsp90-like domains, suggesting a potential chaperone activity. In this work, we report the structures of the Hsp90-like Sr1 domain of SIRPT1 and the N-terminal Ubl domain determined at 1.55- and 2.1-Å resolutions, respectively. The Ubl domain crystallized as a swapped dimer that could be relevant in the context of full-length protein. The Sr1 domain displays the Bergerat protein fold with a characteristic nucleotide-binding pocket, although it binds nucleotides with very low affinity. The Sr1 structure reveals that ARSACS-causing missense mutations (R272H, R272C, and T201K) disrupt protein folding, most likely leading to sacsin degradation. This work lends structural support to the view of sacsin as a molecular chaperone and provides a framework for future studies of this protein.

A Ubl/ubiquitin switch in the activation of Parkin.

Mutations in Parkin and PINK1 cause an inherited early-onset form of Parkinson's disease. The two proteins function together in a mitochondrial quality control pathway whereby PINK1 accumulates on damaged mitochondria and activates Parkin to induce mitophagy. How PINK1 kinase activity releases the auto-inhibited ubiquitin ligase activity of Parkin remains unclear. Here, we identify a binding switch between phospho-ubiquitin (pUb) and the ubiquitin-like domain (Ubl) of Parkin as a key element. By mutagenesis and SAXS, we show that pUb binds to RING1 of Parkin at a site formed by His302 and Arg305. pUb binding promotes disengagement of the Ubl from RING1 and subsequent Parkin phosphorylation. A crystal structure of Parkin Δ86-130 at 2.54 Å resolution allowed the design of mutations that specifically release the Ubl domain from RING1. These mutations mimic pUb binding and promote Parkin phosphorylation. Measurements of the E2 ubiquitin-conjugating enzyme UbcH7 binding to Parkin and Parkin E3 ligase activity suggest that Parkin phosphorylation regulates E3 ligase activity downstream of pUb binding.