Structure of a putative immature form of a Rieske-type iron-sulfur protein in complex with zinc chloride.

Iron-sulfur clusters are prosthetic groups of proteins involved in various biological processes. However, details of the immature state of the iron-sulfur cluster into proteins have not yet been elucidated. We report here the first structural analysis of the Zn-containing form of a Rieske-type iron-sulfur protein, PetA, from Thermochromatium tepidum (TtPetA) by X-ray crystallography and small-angle X-ray scattering analysis. The Zn-containing form of TtPetA was indicated to be a dimer in solution. The zinc ion adopts a regular tetra-coordination with two chloride ions and two cysteine residues. Only a histidine residue in the cluster-binding site exhibited a conformational difference from the [2Fe-2S] containing form. The Zn-containing structure indicates that the conformation of the cluster binding site is already constructed and stabilized before insertion of [2Fe-2S]. The binding mode of ZnCl2, similar to the [2Fe-2S] cluster, suggests that the zinc ions might be involved in the insertion of the [2Fe-2S] cluster.

Uptake mechanism of iron-phytosiderophore from the soil based on the structure of yellow stripe transporter.

Calcareous soils cover one-third of all land and cause severe growth defects in plants due to the poor water solubility of iron at high pH. Poaceae species use a unique chelation strategy, whereby plants secrete a high-affinity metal chelator, known as phytosiderophores (mugineic acids), and reabsorb the iron-phytosiderophore complex by the yellow stripe 1/yellow stripe 1-like (YS1/YSL) transporter for efficient uptake of iron from the soil. Here, we present three cryo-electron microscopy structures of barley YS1 (HvYS1) in the apo state, in complex with an iron-phytosiderophore complex, Fe(III)-deoxymugineic acid (Fe(III)-DMA), and in complex with the iron-bound synthetic DMA analog (Fe(III)-PDMA). The structures reveal a homodimeric assembly mediated through an anti-parallel ß-sheet interaction with cholesterol hemisuccinate. Each protomer adopts an outward open conformation, and Fe(III)-DMA is bound near the extracellular space in the central cavity. Fe(III)-PDMA occupies the same binding site as Fe(III)-DMA, demonstrating that PDMA can function as a potent fertilizer in an essentially identical manner to DMA. Our results provide a structural framework for iron-phytosiderophore recognition and transport by YS1/YSL transporters, which will enable the rational design of new, high-potency fertilizers.

Molecular bases for HOIPINs-mediated inhibition of LUBAC and innate immune responses.

The NF-κB and interferon antiviral signaling pathways play pivotal roles in inflammatory and innate immune responses. The LUBAC ubiquitin ligase complex, composed of the HOIP, HOIL-1L, and SHARPIN subunits, activates the canonical NF-κB pathway through Met1-linked linear ubiquitination. We identified small-molecule chemical inhibitors of LUBAC, HOIPIN-1 and HOIPIN-8. Here we show that HOIPINs down-regulate not only the proinflammatory cytokine-induced canonical NF-κB pathway, but also various pathogen-associated molecular pattern-induced antiviral pathways. Structural analyses indicated that HOIPINs inhibit the RING-HECT-hybrid reaction in HOIP by modifying the active Cys885, and residues in the C-terminal LDD domain, such as Arg935 and Asp936, facilitate the binding of HOIPINs to LUBAC. HOIPINs effectively induce cell death in activated B cell-like diffuse large B cell lymphoma cells, and alleviate imiquimod-induced psoriasis in model mice. These results reveal the molecular and cellular bases of LUBAC inhibition by HOIPINs, and demonstrate their potential therapeutic uses.

Subquinocin, a small molecule inhibitor of CYLD and USP-family deubiquitinating enzymes, promotes NF-κB signaling.

The tumor suppressor CYLD negatively regulates polyubiquitination-dependent cellular signaling such as nuclear factor (NF)-κB signaling. In addition to CYLD, multiple deubiquitinating enzymes (DUBs) are also involved in the regulation of this signaling pathway, and distinct role of CYLD is yet to be clarified. Here, we identified a small chemical named Subquinocin that inhibited the DUB activity of recombinant CYLD using a wheat cell-free protein synthesis and an AlphaScreen technology. In cells, Subquinocin increased the polyubiquitination of NEMO and RIP1 and enhanced NF-κB activation. Modeling and mutation analyses indicated that Subquinocin interacted with Y940 in CYLD, which locates close to catalytic center of CYLD, and is conserved among the USP-family DUBs. Further biochemical evaluation revealed that Subquinocin inhibited USP-family DUBs, but not other family DUBs including OTU. Although Subquinocin showed a broad specificity toward USP-family DUBs, the inhibitory effect of Subquinocin on NF-κB signaling was negligible in CYLD-KO cells, indicating that CYLD is a major target of Subquinocin on the suppression of NF-κB signaling. In conclusion, Subquinocin identified here is a useful tool to analyze the signal transduction mediated by USP-family DUBs.

Insights into the mechanisms of epilepsy from structural biology of LGI1-ADAM22.

Epilepsy is one of the most common brain disorders, which can be caused by abnormal synaptic transmissions. Many epilepsy-related mutations have been identified in synaptic ion channels, which are main targets for current antiepileptic drugs. One of the novel potential targets for therapy of epilepsy is a class of non-ion channel-type epilepsy-related proteins. The leucine-rich repeat glioma-inactivated protein 1 (LGI1) is a neuronal secreted protein, and has been extensively studied as a product of a causative gene for autosomal dominant lateral temporal lobe epilepsy (ADLTE; also known as autosomal dominant partial epilepsy with auditory features [ADPEAF]). At least 43 mutations of LGI1 have been found in ADLTE families. Additionally, autoantibodies against LGI1 in limbic encephalitis are associated with amnesia, seizures, and cognitive dysfunction. Although the relationship of LGI1 with synaptic transmission and synaptic disorders has been studied genetically, biochemically, and clinically, the structural mechanism of LGI1 remained largely unknown until recently. In this review, we introduce insights into pathogenic mechanisms of LGI1 from recent structural studies on LGI1 and its receptor, ADAM22. We also discuss the mechanism for pathogenesis of autoantibodies against LGI1, and the potential of chemical correctors as novel drugs for epilepsy, with structural aspects of LGI1-ADAM22.

Structural insights into ubiquitin phosphorylation by PINK1.

Mutations of PTEN-induced putative kinase 1 (PINK1) and the E3 ubiquitin (Ub) ligase parkin can cause familial parkinsonism. These two proteins are essential for ubiquitylation of damaged mitochondria and subsequent degradation. PINK1 phosphorylates Ser65 of Ub and the Ub-like (UBL) domain of parkin to allosterically relieve the autoinhibition of parkin. To understand the structural mechanism of the Ub/UBL-specific phosphorylation by PINK1, we determined the crystal structure of Tribolium castaneum PINK1 kinase domain (TcPINK1) in complex with a nonhydrolyzable ATP analogue at 2.5 Å resolution. TcPINK1 consists of the N- and C-terminal lobes with the PINK1-specific extension. The ATP analogue is bound in the cleft between the N- and C-terminal lobes. The adenine ring of the ATP analogue is bound to a hydrophobic pocket, whereas the triphosphate group of the ATP analogue and two coordinated Mg ions interact with the catalytic hydrophilic residues. Comparison with protein kinases A and C (PKA and PKC, respectively) unveils a putative Ub/UBL-binding groove, which is wider than the peptide-binding groove of PKA or PKC to accommodate the globular head of Ub or UBL. Further crosslinking analyses suggested a PINK1-interacting surface of Ub. Structure-guided mutational analyses support the findings from the present structural analysis of PINK1.

Structural insights into two distinct binding modules for Lys63-linked polyubiquitin chains in RNF168.

The E3 ubiquitin (Ub) ligase RNF168 plays a critical role in the initiation of the DNA damage response to double-strand breaks (DSBs). The recruitment of RNF168 by ubiquitylated targets involves two distinct regions, Ub-dependent DSB recruitment module (UDM) 1 and UDM2. Here we report the crystal structures of the complex between UDM1 and Lys63-linked diUb (K63-Ub2) and that between the C-terminally truncated UDM2 (UDM2ΔC) and K63-Ub2. In both structures, UDM1 and UDM2ΔC fold as a single α-helix. Their simultaneous bindings to the distal and proximal Ub moieties provide specificity for Lys63-linked Ub chains. Structural and biochemical analyses of UDM1 elucidate an Ub-binding mechanism between UDM1 and polyubiquitylated targets. Mutations of Ub-interacting residues in UDM2 prevent the accumulation of RNF168 to DSB sites in U2OS cells, whereas those in UDM1 have little effect, suggesting that the interaction of UDM2 with ubiquitylated and polyubiquitylated targets mainly contributes to the RNF168 recruitment.

Structures of CYLD USP with Met1- or Lys63-linked diubiquitin reveal mechanisms for dual specificity.

The tumor suppressor CYLD belongs to a ubiquitin (Ub)-specific protease (USP) family and specifically cleaves Met1- and Lys63-linked polyubiquitin chains to suppress inflammatory signaling pathways. Here, we report crystal structures representing the catalytic states of zebrafish CYLD for Met1- and Lys63-linked Ub chains and two distinct precatalytic states for Met1-linked chains. In both catalytic states, the distal Ub is bound to CYLD in a similar manner, and the scissile bond is located close to the catalytic residue, whereas the proximal Ub is bound in a manner specific to Met1- or Lys63-linked chains. Further structure-based mutagenesis experiments support the mechanism by which CYLD specifically cleaves both Met1- and Lys63-linked chains and provide insight into tumor-associated mutations of CYLD. This study provides new structural insight into the mechanisms by which USP family deubiquitinating enzymes recognize and cleave Ub chains with specific linkage types.

Structure of TCR and antigen complexes at an immunodominant CTL epitope in HIV-1 infection.

We investigated the crystal structure of an HLA-A*2402-restricted CTL epitope in the HIV-1 nef gene (Nef134-10) before (pHLA) or after TCR docking. The wild type epitope and two escape mutants were included in the study. Y135F was an early-appearing major mutation, while F139L was a late-appearing mutation which was selected in the patients without Y135F. F139 was an eminent feature of the Nef134-10 epitope. Wild type-specific TCR was less fit to F139L mutant suggesting that F139L is an escape from the CTL against the wild type epitope. Although Y135F mutation disrupted the hydrogen bond to HLA-A*2402 His70, newly formed hydrogen bond between T138 and His70 kept the conformation of the epitope in the reconstituted pMHC. TCR from Y135F- or dually-specific CTL had unique mode of binding to the mutant epitope. Y135F has been reported as a processing mutant but CTL carrying structurally adequate TCR can be found in the patients.

Molecular basis of Lys-63-linked polyubiquitination inhibition by the interaction between human deubiquitinating enzyme OTUB1 and ubiquitin-conjugating enzyme UBC13.

UBC13 is the only known E2 ubiquitin (Ub)-conjugating enzyme that produces Lys-63-linked Ub chain with its cofactor E2 variant UEV1a or MMS2. Lys-63-linked ubiquitination is crucial for recruitment of DNA repair and damage response molecules to sites of DNA double-strand breaks (DSBs). A deubiquitinating enzyme OTUB1 suppresses Lys-63-linked ubiquitination of chromatin surrounding DSBs by binding UBC13 to inhibit its E2 activity independently of the isopeptidase activity. OTUB1 strongly suppresses UBC13-dependent Lys-63-linked tri-Ub production, whereas it allows di-Ub production in vitro. The mechanism of this non-canonical OTUB1-mediated inhibition of ubiquitination remains to be elucidated. Furthermore, the atomic level information of the interaction between human OTUB1 and UBC13 has not been reported. Here, we determined the crystal structure of human OTUB1 in complex with human UBC13 and MMS2 at 3.15 Å resolution. The presented atomic-level interactions were confirmed by surface-plasmon resonance spectroscopy with structure-based mutagenesis. The designed OTUB1 mutants cannot inhibit Lys-63-linked Ub chain formation in vitro and histone ubiquitination and 53BP1 assembly around DSB sites in vivo. Finally, we propose a model for how capping of di-Ub by the OTUB1-UBC13-MMS2/UEV1a complex efficiently inhibits Lys-63-linked tri-Ub formation.

Get1 stabilizes an open dimer conformation of get3 ATPase by binding two distinct interfaces.

Tail-anchored (TA) proteins are integral membrane proteins that possess a single transmembrane domain near their carboxy terminus. TA proteins play critical roles in many important cellular processes such as membrane trafficking, protein translocation, and apoptosis. The GET complex mediates posttranslational insertion of newly synthesized TA proteins to the endoplasmic reticulum membrane. The GET complex is composed of the homodimeric Get3 ATPase and its heterooligomeric receptor, Get1/2. During insertion, the Get3 dimer shuttles between open and closed conformational states, coupled with ATP hydrolysis and the binding/release of TA proteins. We report crystal structures of ADP-bound Get3 in complex with the cytoplasmic domain of Get1 (Get1CD) in open and semi-open conformations at 3.0- and 4.5-Å resolutions, respectively. Our structures and biochemical data suggest that Get1 uses two interfaces to stabilize the open dimer conformation of Get3. We propose that one interface is sufficient for binding of Get1 by Get3, while the second interface stabilizes the open dimer conformation of Get3.

Structure and function of a membrane component SecDF that enhances protein export.

Protein translocation across the bacterial membrane, mediated by the secretory translocon SecYEG and the SecA ATPase, is enhanced by proton motive force and membrane-integrated SecDF, which associates with SecYEG. The role of SecDF has remained unclear, although it is proposed to function in later stages of translocation as well as in membrane protein biogenesis. Here, we determined the crystal structure of Thermus thermophilus SecDF at 3.3 Å resolution, revealing a pseudo-symmetrical, 12-helix transmembrane domain belonging to the RND superfamily and two major periplasmic domains, P1 and P4. Higher-resolution analysis of the periplasmic domains suggested that P1, which binds an unfolded protein, undergoes functionally important conformational changes. In vitro analyses identified an ATP-independent step of protein translocation that requires both SecDF and proton motive force. Electrophysiological analyses revealed that SecDF conducts protons in a manner dependent on pH and the presence of an unfolded protein, with conserved Asp and Arg residues at the transmembrane interface between SecD and SecF playing essential roles in the movements of protons and preproteins. Therefore, we propose that SecDF functions as a membrane-integrated chaperone, powered by proton motive force, to achieve ATP-independent protein translocation.

Structural insight into the membrane insertion of tail-anchored proteins by Get3.

Tail anchored (TA) proteins, which are important for numerous cellular processes, are defined by a single transmembrane domain (TMD) near the C-terminus. The membrane insertion of TA proteins is mediated by the highly conserved ATPase Get3. Here we report the crystal structures of Get3 in ADP-bound and nucleotide-free forms at 3.0 A and 2.8 A resolutions, respectively. Get3 consists of a nucleotide binding domain and a helical domain. Both structures exhibit a Zn(2+)-mediated homodimer in a head-to-head orientation, representing an open dimer conformation. Our cross-link experiments indicated the closed dimer-stimulating ATP hydrolysis, which might be coupled with TA-protein release. Further, our coexpression-based binding assays using a model TA protein Sec22p revealed the direct interaction between the helical domain of Get3 and the Sec22p TMD. This interaction is independent of ATP and dimer formation. Finally, we propose a structural mechanism that links ATP hydrolysis with the TA-protein insertion mediated by the conserved DTAPTGH motif.

Mechanism for the definition of elongation and termination by the class II CCA-adding enzyme.

The CCA-adding enzyme synthesizes the CCA sequence at the 3' end of tRNA without a nucleic acid template. The crystal structures of class II Thermotoga maritima CCA-adding enzyme and its complexes with CTP or ATP were determined. The structure-based replacement of both the catalytic heads and nucleobase-interacting neck domains of the phylogenetically closely related Aquifex aeolicus A-adding enzyme by the corresponding domains of the T. maritima CCA-adding enzyme allowed the A-adding enzyme to add CCA in vivo and in vitro. However, the replacement of only the catalytic head domain did not allow the A-adding enzyme to add CCA, and the enzyme exhibited (A, C)-adding activity. We identified the region in the neck domain that prevents (A, C)-adding activity and defines the number of nucleotide incorporations and the specificity for correct CCA addition. We also identified the region in the head domain that defines the terminal A addition after CC addition. The results collectively suggest that, in the class II CCA-adding enzyme, the head and neck domains collaboratively and dynamically define the number of nucleotide additions and the specificity of nucleotide selection.

Tuberous sclerosis tumor suppressor complex-like complexes act as GTPase-activating proteins for Ral GTPases.

The small GTPases RalA and RalB are multifunctional proteins regulating a variety of cellular processes. Like other GTPases, the activity of Ral is regulated by the opposing effects of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Although several RalGEFs have been identified and characterized, the molecular identity of RalGAP remains unknown. Here, we report the first molecular identification of RalGAPs, which we have named RalGAP1 and RalGAP2. They are large heterodimeric complexes, each consisting of a catalytic alpha1 or alpha2 subunit and a common beta subunit. These RalGAP complexes share structural and catalytic similarities with the tuberous sclerosis tumor suppressor complex, which acts as a GAP for Rheb. In vitro GTPase assays revealed that recombinant RalGAP1 accelerates the GTP hydrolysis rate of RalA by 280,000-fold. Heterodimerization was required for this GAP activity. In PC12 cells, knockdown of the beta subunit led to sustained Ral activation upon epidermal growth factor stimulation, indicating that the RalGAPs identified here are critical for efficient termination of Ral activation induced by extracellular stimuli. Our identification of RalGAPs will enable further understanding of Ral signaling in many biological and pathological processes.

Structural basis for specific recognition of Lys 63-linked polyubiquitin chains by tandem UIMs of RAP80.

RAP80 has a key role in the recruitment of the Abraxas-BRCC36-BRCA1-BARD1 complex to DNA-damage foci for DNA repair through specific recognition of Lys 63-linked polyubiquitinated proteins by its tandem ubiquitin-interacting motifs (UIMs). Here, we report the crystal structure of the RAP80 tandem UIMs (RAP80-UIM1-UIM2) in complex with Lys 63-linked di-ubiquitin at 2.2 A resolution. The two UIMs, UIM1 and UIM2, and the alpha-helical inter-UIM region together form a continuous 60 A-long alpha-helix. UIM1 and UIM2 bind to the proximal and distal ubiquitin moieties, respectively. Both UIM1 and UIM2 of RAP80 recognize an Ile 44-centered hydrophobic patch on ubiquitin but neither UIM interacts with the Lys 63-linked isopeptide bond. Our structure suggests that the inter-UIM region forms a 12 A-long alpha-helix that ensures that the UIMs are arranged to enable specific binding of Lys 63-linked di-ubiquitin. This was confirmed by pull-down analyses using RAP80-UIM1-UIM2 mutants of various length inter-UIM regions. Further, we show that the Epsin1 tandem UIM, which has an inter-UIM region similar to that of RAP80-UIM1-UIM2, also selectively binds Lys 63-linked di-ubiquitin.

Crystal structure of the MgtE Mg2+ transporter.

The magnesium ion Mg2+ is a vital element involved in numerous physiological processes. Mg2+ has the largest hydrated radius among all cations, whereas its ionic radius is the smallest. It remains obscure how Mg2+ transporters selectively recognize and dehydrate the large, fully hydrated Mg2+ cation for transport. Recently the crystal structures of the CorA Mg2+ transporter were reported. The MgtE family of Mg2+ transporters is ubiquitously distributed in all phylogenetic domains, and human homologues have been functionally characterized and suggested to be involved in magnesium homeostasis. However, the MgtE transporters have not been thoroughly characterized. Here we determine the crystal structures of the full-length Thermus thermophilus MgtE at 3.5 A resolution, and of the cytosolic domain in the presence and absence of Mg2+ at 2.3 A and 3.9 A resolutions, respectively. The transporter adopts a homodimeric architecture, consisting of the carboxy-terminal five transmembrane domains and the amino-terminal cytosolic domains, which are composed of the superhelical N domain and tandemly repeated cystathionine-beta-synthase domains. A solvent-accessible pore nearly traverses the transmembrane domains, with one potential Mg2+ bound to the conserved Asp 432 within the pore. The transmembrane (TM)5 helices from both subunits close the pore through interactions with the 'connecting helices', which connect the cystathionine-beta-synthase and transmembrane domains. Four putative Mg2+ ions are bound at the interface between the connecting helices and the other domains, and this may lock the closed conformation of the pore. A structural comparison of the two states of the cytosolic domains showed the Mg2+-dependent movement of the connecting helices, which might reorganize the transmembrane helices to open the pore. These findings suggest a homeostasis mechanism, in which Mg2+ bound between cytosolic domains regulates Mg2+ flux by sensing the intracellular Mg2+ concentration. Whether this presumed regulation controls gating of an ion channel or opening of a secondary active transporter remains to be determined.

Crystallization and preliminary X-ray diffraction analysis of the cytosolic domain of the Mg2+ transporter MgtE.

The MgtE family of Mg(2+) transporters is ubiquitously conserved in all domains of life. The cytosolic domains of the MgtE Mg(2+) transporters include a cystathionine-beta-synthase (CBS) domain which is known to play a regulatory function in transporter proteins. The cytosolic domain of MgtE from Thermus thermophilus was overexpressed, purified and crystallized in the presence and absence of Mg(2+). The crystals formed in the presence of Mg(2+) diffracted X-rays to 2.3 A resolution using synchrotron radiation, belong to space group P6(5)22 with unit-cell parameters a = b = 57.7, c = 317.6 A and are expected to contain one molecule in the asymmetric unit. The crystals formed in the absence of Mg(2+) diffracted X-rays to 3.5 A resolution using synchrotron radiation, belong to space group P2(1)2(1)2(1) with unit-cell parameters a = 77.0, b = 100.3, c = 100.3 A and are expected to contain two molecules in the asymmetric unit.

Complete crystallographic analysis of the dynamics of CCA sequence addition.

CCA-adding polymerase matures the essential 3'-CCA terminus of transfer RNA without any nucleic-acid template. However, it remains unclear how the correct nucleotide triphosphate is selected in each reaction step and how the polymerization is driven by the protein and RNA dynamics. Here we present complete sequential snapshots of six complex structures of CCA-adding enzyme and four distinct RNA substrates with and without CTP (cytosine triphosphate) or ATP (adenosine triphosphate). The CCA-lacking RNA stem extends by one base pair to force the discriminator nucleoside into the active-site pocket, and then tracks back after incorporation of the first cytosine monophosphate (CMP). Accommodation of the second CTP clamps the catalytic cleft, inducing a reorientation of the turn, which flips C74 to allow CMP to be accepted. In contrast, after the second CMP is added, the polymerase and RNA primer are locked in the closed state, which directs the subsequent A addition. Between the CTP- and ATP-binding stages, the side-chain conformation of Arg 224 changes markedly; this is controlled by the global motion of the enzyme and position of the primer terminus, and is likely to achieve the CTP/ATP discrimination, depending on the polymerization stage. Throughout the CCA-adding reaction, the enzyme tail domain firmly anchors the TPsiC-loop of the tRNA, which ensures accurate polymerization and termination.

Functional categorization of the conserved basic amino acid residues in TrmH (tRNA (Gm18) methyltransferase) enzymes.

Transfer RNA (Gm18) methyltransferase (TrmH) catalyzes the methyl transfer from S-adenosyl-L-methionine (AdoMet) to the 2'-OH group of the G18 ribose in tRNA. To identify amino acid residues responsible for the tRNA recognition, we have carried out the alanine substitution mutagenesis of the basic amino acid residues that are conserved only in TrmH enzymes and not in the other SpoU proteins. We analyzed the mutant proteins by S-adenosyl-L-homocysteine affinity column chromatography, gel mobility shift assay, and kinetic assay of the methyl transfer reaction. Based on these biochemical studies and the crystal structure of TrmH, we found that the conserved residues can be categorized according to their role (i) in the catalytic center (Arg-41), (ii) in the initial site of tRNA binding (Lys-90, Arg-166, Arg-168, and Arg-176), (iii) in the tRNA binding site required for continuation the catalytic cycle (Arg-8, Arg-19, and Lys-32), (iv) in the structural element involved in release of S-adenosyl-L-homocysteine (Arg-11-His-71-Met-147 interaction), (v) in the assisted phosphate binding site (His-34), or (vi) in an unknown function (Arg-109). Furthermore, the difference between the Kd and Km values for tRNA suggests that the affinity for tRNA is enhanced in the presence of AdoMet. To confirm this idea, we carried out the kinetic studies, a gel mobility shift assay with a mutant protein disrupted in the catalytic center, and the analytical gel-filtration chromatography. Our experimental results clearly show that the enzyme has a semi-ordered sequential mechanism in which AdoMet both enhances the affinity for tRNA and induces formation of the tetramer structure.