Co-translational protein targeting is an essential, evolutionarily conserved pathway for delivering nascent proteins to the proper cellular membrane. In this pathway, the signal recognition particle (SRP) first recognizes the N-terminal signal sequence of nascent proteins and subsequently interacts with the SRP receptor. For this, signal sequence binding in the SRP54 M domain must be effectively communicated to the SRP54 NG domain that interacts with the receptor. Here we present the 2.9 Å crystal structure of unbound- and signal sequence bound SRP forms, both present in the asymmetric unit. The structures provide evidence for a coupled binding and folding mechanism in which signal sequence binding induces the concerted folding of the GM linker helix, the finger loop, and the C-terminal alpha helix αM6. This mechanism allows for a high degree of structural adaptability of the binding site and suggests how signal sequence binding in the M domain is coupled to repositioning of the NG domain. The signal recognition particle plays a key role in the co-translational protein targeting of membrane and secretory proteins. Here the authors report a crystal structure of the ternary SRP complex in signal sequence bound and unbound forms, providing insight into how signal sequence binding is coupled to SRP receptor interaction. The signal recognition particle (SRP) co-translationally targets proteins to the endoplasmic reticulum in eurkaryotes or to the plasma membrane in prokaryotes. As the initiating step, SRP binds to the N-terminal signal sequence of nascent secretory or membrane proteins as they emerge from the ribosome. The SRP–ribosome nascent chain complex (RNC) is then targeted to the membrane through a GTP-dependent interaction with the SRP receptor (SR). The signal sequence is released from SRP and inserted into the translocon channel. Finally, GTP hydrolysis triggers the dissociation of SRP from SR, and SRP can start another cycle of protein targeting  bound to the coaxial stacked helices 5 and 8 of SRP RNA. It is this SRP core that carries out the key functions of signal sequence recognition and SR interaction.SRP54 comprises an N-terminal NG domain and a C-terminal methionine-rich M domain. The NG domain contains the GTPase activity and interacts with SR356769101112131511161711SRP-dependent N-terminal signal sequences are highly diverse in amino-acid composition and length, but they all contain a core of at least eight consecutive hydrophobic amino acids that acts as the major determinant for recognition by SRP19Sulfolobus solfataricus SRP54 complexed with the signal sequence of yeast dipeptidyl aminopeptidase B (DPAP-B)Methanococcus jannaschii SRP54 and a signal sequence mimic5Two crystal structures of archaeal SRP54-signal sequence fusion proteins have provided the first detailed views of the M domain-signal sequence interaction. The M domain contains five amphipathic α-helices (αM1-5) and includes a ‘finger loop' between αM1 and αM2 ref. . DiffereM. jannaschii SRP and its interaction with signal sequences. Here we report the crystal structure of a monomeric SRP, including the full-length (fl) M domain in complex with a signal sequence. This structure shows how signal sequence binding in the M domain is directly linked to NG domain repositioning via an induced helix formation of the GM linker.Communication between the M and NG domains is critical for SRP function. During protein targeting, the binding of a signal sequence in the M domain must be effectively transmitted to the NG domain to accelerate its interaction with SR. The molecular mechanism by which the signal sequence stimulates NG domain–SR complex assembly is currently not understood. To address this, we performed structural studies on Pyrococcus furiosus SRP54 (ref. 55M. jannaschii (aa1-451) fused to a strongly hydrophobic, idealized signal sequence comprising 14 leucine and alanine residues6M. jannaschii SRP54. Size-exclusion chromatography demonstrated that fl-SRP54-ss, but not SRP54-ssThe amino-acid sequence C-terminal to αM5 in the M domain varies between species in length and composition, but is found in all SRP54 proteinsP54 ref. . The SRPM. jannaschii SRP core complexes comprising SRP54, SRP19 and SRP RNA. As expected, we observed that SRP complexes containing fl-SRP54-ss stimulated GTP hydrolysis at a rate ∼20 times faster than SRP complexes containing SRP54-ss  positioned between the NG domain and GM linker, which allows for some degree of NG domain movements10Crystals of the M. jannaschii SRP RNA) diffracted to 2.9 Å resolution. These crystals belonged to space group P22121 with two complexes in the asymmetric unit. The structure was solved by molecular replacement , finger loop K346-H364) and C-terminal sequence (K431-G451) are structurally undefined . In this-H364 and2 of surface area  and bound (complex B) forms of the M domain. This crystal packing is best explained by crystal packing interactions unique to complex A that selectively pack only unbound particles into the crystal lattice at this site. This provided us with a unique opportunity to directly compare the structures of unbound and signal sequence bound forms of the fl-SRP54 M domain.In this study, we report the crystal structure of the complex of SRP RNA, SRP19, and a fl-SRP54 M domain-signal sequence fusion from M. jannaschii SRP structures67810In its unbound form (complex A), the M domain is structurally defined only by its core elements, that is, αM1-αM5, excluding the finger loop. The M domain core is rigid and unchanged from the ones present in complex B and in previously determined M. jannaschii assumes the shape of an α-helix (αM6) thereby shielding the signal sequence from solvent. M. jannaschii SRP54-ss, which is lacking the C-terminus, forms monomers and dimers in a roughly 1:1 ratio in solutionOnly limited structural data are available for the SRP54 C-terminus and its role in signal sequence binding has not been elucidated. Here we show that the C-terminus in -helix αM thereby -helix αM thereby M. jannaschii, however, differs from those seen in the crystal structure of the S. solfataricus SRP54-ssE. coli SRP–RNC complex12M. jannaschii and S. solfataricus binding sites shows that some hydrophobic contacts are conserved, for example, I338 (L339 in S. solfataricus) in αM1, and T420 (M424) and I424 (L428) in αM5. The different binding sites could represent discrete binding modes that occur during the recognition event. This interpretation agrees well with recent data demonstrating that SRP binds to RNCs with exposed signal sequences in multiple interconverting conformationsE. coli SRP–RNC complex12Superimposition of the SRP54-ss and SRPM-ss structures shows a virtually identical orientation of the bound signal sequence relative to αGM and the M domain core. Moreover, the same contacts are made by the signal sequence with the hydrophobic residues in αGM and αM1. The importance of some of these residues for SRP function is confirmed by mutational analysisSuccessive events during the SRP cycle require a rearrangement of the relative position of the NG and M domains1112141718The coupling of ligand binding and folding of the recognition region in the M domain  provides a simple and efficient mechanism to communicate signal sequence recognition in the M domain to the NG domain. Moreover, we propose that signal sequence recognition via a disorder-to-order transition of multiple structural elements facilitates specific recognition of widely diverse signal sequences. Future structural studies with signal sequences of different composition and length will be necessary to gain a more comprehensive view of how promiscuous binding by SRP is achieved.srp19 gene from M. jannaschii was cloned into the pET-3a vector (Novagen) and expressed in JM109(DE3) cells (Promega). The SRP19 protein was purified by heat treatment followed by sequential column chromatography on Heparin Sepharose, Mono S, and Superdex 75 . The DNA sequence for M. jannaschii fl-SRP54, fl-SRP54-ss, SRP54-ss and SRP54M-ss was cloned into the pNZ8048 vector and expressed in Lactococcus lactis. The SRP54 proteins were purified by heat treatment followed by sequential column chromatography on Heparin Sepharose, Mono S and Superdex 200 . The M. jannaschii SRP RNA was transcribed in vitro using T7 RNA polymerase and purified by denaturing gel electrophoresis. Before complex formation, the RNA was annealed in water by denaturation at 75 °C followed by snap cooling on ice. The annealed RNA was purified on Mono Q  and dialyzed against 10 mM Tris-HCl (pH 7.5), 5 mM MgCl2. The protein–RNA complex was reconstituted in a buffer containing 10 mM Tris-HCl (pH 7.5), 250 mM KCl, 5 mM MgCl2, 1% (v/v) ß-mercaptoethanol. Binding reactions were incubated for 15 min at room temperature after addition of SRP19, and for 1 h at 37 °C after addition of SRP54. After this, the complex was purified on a Mono Q column and dialyzed against 10 mM Tris-HCl (pH 7.5), 250 mM KCl, 5 mM MgCl2. The complex (3 mg/ml) was crystallized by the hanging-drop vapor-diffusion technique at 18 °C. Crystals (0.15 × 0.15 × 0.05 mm3) grew in 7 days when the complex solution was mixed with an equal volume of mother liquor containing 35% 2-Methyl-2,4-pentanediol, 200 mM NaCl, 80 mM MgCl2, 100 mM sodium acetate (pH 5.0).The λ=0.984 Å at 100 K. Data were processed and scaled using XDSM. jannaschii SRP54M-ss, SRP19 and SRP RNA complex belonged to space group P22121and contained two molecules in the asymmetric unit. The structure was solved by molecular replacement using the programme PHASER from the PHENIX suiteCrystals were cryocooled directly from mother liquor and diffraction data were collected at the European Synchrotron Radiation Facility beamline ID23-1 using X-ray radiation with M. jannaschii SRP RNA  ß-mercaptoethanol to form 0.5 μM of M. jannaschii SRP. M. jannaschii SR(aa93-408) was added to the SRP complex to final concentrations of 0, 1, 2, 5, 10 and 25 μM. GTP hydrolysis reactions, carried out at 25 °C, were started by adding 100 μM GTP with trace amounts of γ-32P-GTP (PerkinElmer). At different time points, 0.5 μl aliquots of the reactions were spotted on a polyethyleneimine–cellulose thin-layer chromatography (PEI-TLC) plate (Merck). Inorganic phosphate (Pi) and GTP were separated by chromatography in 0.75 M KH2PO4 and quantified with a Phosphorimager (Molecular Dynamics). Rates of GTP hydrolysis were averaged from three independent experiments and fitted to a single exponential model.One micromolar of M. jannaschii SR(aa93-408). Interaction experiments were performed in running buffer containing 10 mM Tris-Cl (pH 7.5), 150 mM KCl, 5 mM MgCl2 at 37 °C. Purified M. jannaschii SRP complexes in running buffer were injected at 0.5 and 1 μM concentrations in the absence of GTP and a flow rate of 5 μl min−1. Sensorgrams were corrected for non-specific interaction with the GST-only surface and by double referencing.Sensorgrams were recorded using a Biacore 3,000 instrument . An anti-gluthathion-S-transferase (GST) monoclonal antibody was immobilized on a CM-5 chip  by amine coupling according to the manufacturer's instructions. Binding surfaces were subsequently generated by the application of equimolar amounts of GST-only as reference, or the N-terminally GST-tagged Accession codes: Coordinates and structure factors have been deposited with the Protein Data Bank under the accession code 4XCO.How to cite this article: Hainzl, T. et al. Signal sequence induced conformational changes in the signal recognition particle. Nat. Commun. 6:7163 doi: 10.1038/ncomms8163 (2015).Supplementary Figures 1-7


UBE3A gene non-functional. Duplication of the chromosomal region containing the UBE3A gene is associated with ASDs. Although the causative role for UBE3A gene mutations in AS is well established, a long-standing challenge in AS research has been to identify neural substrates of UBE3A, an E3 ubiquitin ligase. A prevailing hypothesis is that changes in UBE3A protein levels would alter the levels of a collection of protein substrates, giving rise to the unique phenotypic aspects of AS and possibly UBE3A associated ASDs. Interestingly, proteins altered in AS are linked to additional ASDs that are not previously associated with changes in UBE3A, indicating a possible molecular overlap underlying the broad-spectrum phenotypes of these neurogenetic disorders. This idea raises the possibility that there may exist a “one-size-fits-all” approach to the treatment of neurogenetic disorders with phenotypes overlapping AS. Furthermore, while a comprehensive list of UBE3A substrates and downstream affected pathways should be developed, this is only part of the story. The timing of when UBE3A protein functions, through either changes in UBE3A or possibly substrate expression patterns, appears to be critical for AS phenotype development. These data call for further investigation of UBE3A substrates and their timing of action relevant to AS phenotypes.Angelman syndrome (AS) is a debilitating neurodevelopmental disorder that is characterized by motor dysfunction, intellectual disability, speech impairment, seizures and common features of autism spectrum disorders (ASDs). Some of these AS related phenotypes can be seen in other neurodevelopmental disorders (Williams,  Angelman syndrome (AS) is a neurodevelopmental disorder that affects one in 15,000 individuals , although the contribution of AS phenotype has not been interrogated  that is highly expressed to prevent overexpression of UBE3A. When this transcript is silenced, paternal expression is reinstated up to 90% of control level. Utilizing antisense oligonucleotides to disrupt the UBE3A-ATS and induce paternal expression of UBE3A protein, some phenotypes, such as fear conditioning and body weight, were ameliorated even upon treatment as juveniles. However, many phenotypes, including the robust and reproducible motor phenotypes and marble burying, were not rescued . GLS is supported by NSF Graduate Research Fellowship grant no. 1232825.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Death-Associated Protein Kinase 1 (DAPK1) belongs to a family of five serine/threonine (Ser/Thr) kinases that possess tumor suppressive function and also mediate a wide range of cellular processes, including apoptosis and autophagy. The loss and gain-of–function of DAPK1 is associated with various cancer and neurodegenerative diseases respectively. In recent years, mechanistic studies have broadened our knowledge of the molecular mechanisms involved in DAPK1-mediated autophagy/apoptosis. In the present review, we have discussed the structural information and various cellular functions of DAPK1 in a comprehensive manner. Death-associated protein kinase 1 (DAPK1), a part of a family of Ser/Thr kinase, was originally isolated in an unbiased antisense based genetic screen for genes whose protein products were imperative for interferon Gamma (IFN-γ) induced death in HeLa cells . This direct DAPK1-NR2B interaction thereby potentiating NR2BR activity via triggering the DAPK1-mediated phosphorylation of NR2B subunit at Ser-1303 -dependent serine-threonine kinase  domains, which overlap with a cytoskeletal-binding region, a death domain and a serine-rich C-terminal tail  was determined at 1.5 Å resolution and has provided structural hints as to DAPK1’s mechanism of activation, interactions with substrates, and potential inhibitor design  motif that require activation loop phosphorylation, common to other kinases in which the conserved arginine in this motif creates a salt bridge with the phosphorylated residue in the activation loop. In DAPK1, a change at residue 138 where the arginine is replaced by a conserved phenylalanine, substitutes phosphorylation dependent active-site interactions with a local hydrophobic core that maintains an active kinase conformation. According to active site analysis of the X-ray crystallographic structure of human DAPK1 complex with respective inhibitor (PDB ID: 1IG1) using Chimera 1.11 version, the active sites of DAPK1 accommodates certain highly conserved amino acid residues such as Gln23, Phe24, Lys42, Glu94, Val96, Glu100, Glu146 and Asp161; involved in H-bond interaction with their respective ligands and hydrophobic interactions includes Leu19, Val27, Ile77, Met146 and Ile160. Here, the hydrogen bond interactions with Val96 and Glu94 are especially important since they reside in a hydrophobic enclosure. There are some additional hydrophobic interactions with Ile77 and Leu93. The amino terminal lobe of the kinase domain contains an interaction surface for the chaperon heat shock protein 90 (HSP90), which recognizes specific neutral and positively charged residues within the alphaC-beta4 loop. This interaction with Hsp90 facilitates kinase maturation, activity and stability. Inhibition of HSP90 leads to degradation of DAPK1 , which is a critical signaling protein that modulates the function, stability and/or localization of numerous phosphoproteins through isomerization of proline that follows a phosphorylation site , Fas-associated protein with death domain (FADD) and Tumor necrosis factor (TNF) receptor. In DAPK1, death domain is largely associated with protein-protein interactions, kinase activity and apoptotic functions. DAPK1’s death domain is located near its C-terminus between residues 1312–1396, followed by a 17-aa tail rich in Serine residues, a common feature in other death domain-containing proteins  and DAPK1 /Kelch protein, Kelch-like protein 20 ( KLHL20) that act as an adaptor for the Cullin3-ROC1 ubiquitin E3 ligase complex. IFN results in the sequestration of KLHL20 away from DAPK1 cause high protein level of DAPK1 upon IFN treatment  affinity regulating kinases (MARK1/2). MARK is a Ser/Thr kinase that destabilizes microtubules by phosphorylating tau  is an isoform of the glycolytic enzyme, binds the death domain, which is commonly expressed in cancer. DAPK1 leads to an enhanced glycolytic rate and lactate production by activating PKM2’s activity Another interaction is between DAPK1’s death-domain and death-domain containing protein Unc-5 homolog 2 (UNC5H2), a dependence receptor which triggers apoptosis in the absence of its ligand netrin-1. This interaction is probably mediated through domains of both proteins as well as through additional domains and was shown to be necessary for UNC5H5-induced apoptosis, as co-transfection of a dominant-negative DAPK1 mutant together with UNC5H2 decreased cell death , a negative regulator of the mechanistic target of energy, growth factors and nutrient that controls biosynthetic pathways and multiple cell growth  or intrinsic  damage) stimuli, resulting in death. Inappropriate apoptosis (either too little or too much) is a cause of many human conditions including autoimmune disorders, neurodegenerative diseases, ischemic damage and many types of cancer Elmore, . The abiIn a number of cell culture models, DAPK1 has been implicated in pathway leading to apoptosis and in some cases may function prior to the commitment steps of the signal transduction mechanism -β is related to DAPK1-regulated pathways and apoptosis, which can stimulate apoptosis in developing neurons and other tissues , overexpression of constitutively active DAPK1 increases levels of p53, p53-regulated proteins Mdm-2 as well as p21, causing apoptosis  by DAPK1, which further phosphorylates and activates Vps34. In the second mechanism, DAPK1 directly phosphorylates Beclin1, a necessary component of the Vps34 complex, thereby releasing it from its inhibitors, B-cell lymphoma 2 (Bcl-2). Bcl-2 binds and inhibits Beclin 1, preventing its association with the Vps34 complex. DAPK1 disrupts the Beclin 1/Bcl-2 interaction, in response to ionomycin, via phosphorylation of Beclin 1 within the Bcl-2 homology domain 3 (BH3) domain. In response to starvation, a Ser/Thr rho-associated, coiled-coil-containing protein kinase 1 (ROCK1) can also phosphorylate this residue. DAPK1 phosphorylates PKD, under oxidative stress, which in turn phosphorylates and activates Vps34 directly. Activated Vps34 proceeds to phosphatidyl inositol-3 phosphate (PI3P) formation, which recruits PI3P effector such as Double FYVE-domain containing protein 1 (DFCP1), leading to autophagy. The interaction of Beclin 1 with Bcl-2 interaction can be disrupted by c-Jun N-terminal kinase (JNK)-mediated phosphorylation of Bcl-2, binding of high mobility group box 1 (HMGB1) to Beclin 1 or uniquitination of Beclin 1 by TNF receptor-associated factor 6 (TRAF6). During oxidative stress, PKD can lead to JNK phosphorylation through the mitogen-activated protein kinase 3K (MAPK3K) Apoptosis signal-regulating kinase 1 (ASK1); it is not known if and when DAPK1 can modulate Bcl-2 through a PKD-JNK pathway Figure .The various scenarios, in which DAPK1 was shown to be necessary for autophagy, have another factor in common: they all involved cell death; but in some scenarios  other death programs were also evident. As a strong case for autophagic cell death was presented, i.e., death was blocked by inhibition of autophagy and occurred in the absence of another death program.Although there are many regulators of autophagy but DAPK1 is unique in its potential to impact several different stages within the autophagy flux pathway, including autophagosome induction, trafficking and fusion. Some of these regulatory events are more substantiated and proven than others but it is not known what triggers these regulatory mechanisms, and whether one or more are concurrently activated. Nevertheless, by activating and enhancing multiple steps in the process, DAPK1 has shown the potential for being an autophagy “super-activator”  in cortical neurons of stroke mice and either blocking DAPK1-tau interaction by systematic application of a membrane permeable peptide or genetic deletion of kinase domain (KD) in mice (DAPK1-KD−/−) protects spine damage and improves neurological functions against stroke insults. As the interaction of DAPK1-KD has been confirmed with the MT repeat binding domain 1 of tau (R1D) consisting of amino acids IGSTENLK, recently, a membrane-permeable peptide is generated by fusing the peptide IGSTENLK to the transduction domain of the HIV TAT protein, named TAT-R1D. Treatment with TAT-R1D (IGSTENLK) results in effective dissociation of DAPK1-tau complexes and significant decrease in the level of pS262 causes apparent alleviation of infraction area as well as the neurological deficits induced by cerebral ischemic stroke (Pei et al., Here we can target DAPK1 to prevent ischemic brain damage by disrupting DAPK1-NR2B interaction promotes neuronal death by potentiating functions of “calcium–activated death-signaling proteins” in the NDC. An interference peptide, Tat-NR2B-CTIn addition, MT dynamics can be affected by DAPK1’s activation of the MARK kinases, leading to taupathies (Wu et al., PS wrote the manuscript. PT and PR designed the study and contributed in manuscript preparation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer TD and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


Xenopus, Wnt-regulated convergent extension (CE) is readily affected by Dvl mutants unable to form the closed conformation than by wild-type Dvl. We also demonstrate that while Dvl cooperates with other Wnt pathway elements to activate canonical Wnt signaling, the open conformation of Dvl more effectively activates Jun N-terminal kinase (JNK). These results suggest that together with other players in the Wnt signaling pathway, the conformational change of Dvl regulates Wnt stimulated JNK activity in the non-canonical Wnt signaling.The intracellular signaling molecule Dishevelled (Dvl) mediates canonical and non-canonical Wnt signaling via its PDZ domain. Different pathways diverge at this point by a mechanism that remains unclear. Here we show that the peptide-binding pocket of the Dvl PDZ domain can be occupied by Dvl's own highly conserved C-terminus, inducing a closed conformation. In DOI:http://dx.doi.org/10.7554/eLife.08142.001 The development of an animal embryo depends on a number of signaling pathways that pass information from the outside of the cell to the inside. These pathways include Wnt signaling, which also regulates cell growth. The pathways must be precisely controlled; abnormal Wnt activity has been implicated in several human diseases, ranging from heart disease to cancer.Wnt signaling is complex, and actually comprises two major pathways: the canonical pathway  and the PCP pathway (which doesn't depend on β-catenin). Both pathways are triggered when Wnt molecules bind to receptors on the outside of the cell. These receptors pass the signal into the cell and to a protein called ‘Dishevelled’ (or ‘Dvl’ for short). This protein then passes the signal on through either the canonical or PCP pathway. Nevertheless it is not clear how the Dishevelled protein can direct the signal specifically down either one of these pathways.Xenopus, which are commonly used in research) reveal that mutant versions of Dishevelled that were unable to take on the closed form strongly affected an aspect of the frog's development that involves the PCP pathway.Lee et al. now show that the Dishevelled protein can take on at least two different shapes. When it is ‘closed’, one end of the protein is tucked inside a pocket elsewhere on the protein's surface. But when Dishevelled is ‘open’, this end of the protein moves out of this pocket. Further experiments using frogs (called Lee et al. then demonstrate that while Dishevelled cooperates with several other Wnt pathway components to activate the canonical pathway, the open form of Dishevelled activates the PCP pathway. The next challenge following on from this work is to find out how Wnt molecules binding to the receptor trigger the shape change in Dishevelled.DOI:http://dx.doi.org/10.7554/eLife.08142.002 Drosophila), a key component of both Wnt signaling pathways, relays Wnt signals downstream from the membrane-bound Wnt receptor Frizzled (Fz)  . While Dled (Fz) . HoweverDvl contains highly conserved DIX, PDZ, and DEP domains and a highly conserved extreme C-terminus  Wharton. The PDZXenopus model, that disruption of this intramolecular interaction activates Jun N-terminal kinase (JNK) and enhances the CE phenotype associated with activation of non-canonical Wnt signaling. Further, we demonstrate that a Dvl PDZ-binding peptide or small molecule that inhibits canonical β-catenin signaling enhances JNK activity by releasing the Dvl C-terminus from its autoinhibitory closed conformation.Here we use biophysical methods to investigate the interaction of the Dvl extreme C-terminus with the Dvl PDZ domain and demonstrate that Dvl adopts a closed conformation. We also show, in a Drosophila Dsh (‘Dsh-C peptide’). We first used a competitive binding assay. Binding of the PDZ domain to a fluorescently-labeled peptide (Rox-DprC) derived from the C-terminus of Dapper (Dpr), a known binding partner of the Dvl PDZ domain (I) calculated from two independent experiments (12.3 ± 7.8 μM for the Dvl-C peptide and 26.8 ± 8.4 μM for the Dsh-C peptide) were similar to the binding affinity of Rox-DprC to the PDZ domain (KD ∼ 7.9 ± 0.9 μM)  , 2009b.  0.9 μM)  and obta15N-labeled Dvl-1 PDZ domain. During these titrations, the NMR resonance of the residues in the PDZ domain's peptide binding site , containing the PDZ and DEP domains (with an intact PDZ-binding motif) and mC1-CΔ7 (residues 251–688), lacking the PDZ-binding motif. We then examined the binding of Rox-DprC to the two constructs. Little polarization change was observed when mC1 proteins were added to Rox-DprC solution, whereas large polarization changes were induced by adding mC1-CΔ7 , indicatXenopus model  (XDsh (residues 1–732) . To avoid interfering the function of Dvl C-terminus, we placed the myc-tag at the N-terminus. However, the tag may affect the N-terminal DIX domain or potentially have some nonspecific effects. Therefore, in the Xenopus studies, we always paired the two constructs in the experiments to minimalize the potential of unexpected effects.After establishing that Dvl can adopt a ‘closed’ conformation in which its C-terminus binds to its PDZ domain, we used a 2, XDsh)  to invess 1–732)  and mutasiamois promoter reporter  of XDsh and XDsh-CΔ8 mRNAs were used. At low and intermediate doses, neither XDsh nor XDsh-CΔ8 substantially affected canonical Wnt signaling (not shown). At the high dose, both constructs enhanced canonical Wnt signaling, but wild-type XDsh was a stronger activator , XDsh-CΔ8 mRNA caused greater body axis shortening and dorsal tail flexion than did wild-type XDsh , we injected three different doses of wild-type ype XDsh , suggestype XDsh . Like XD of Xdd1 , suggestXenopus embryos, JNK is known to regulate CE through non-canonical Wnt-PCP signaling  of XDsh-CΔ8 or wild-type XDsh mRNA were injected into the dorsal region of 4-cell Xenopus embryos and luciferase activity was assayed at the late gastrula stage, XDsh-CΔ8 clearly activated AP1-luciferase activity more strongly  defects and produce CE phenotype  as compared to controls, while wild-type XDsh had little effect , DEP (residues 377–503), mC1 (residues 251–695), and mD1-CΔ7 (residues 251–688) domains of mouse Dvl-1 were sub-cloned into the pET28a vector. The N-terminally 6xHis-tagged proteins were expressed in BL21(DE3) eviously  and descPeptides were synthesized by the Hartwell Center for Bioinformatics & Biotechnology at St. Jude Children's Research Hospital; they were purified by reverse-phase high-performance liquid chromatography and confirmed by MSI-MS as described in the supplementary information.I of both peptides was determined in two independent experiments by using the equation KDapp = KD (1 + [I]/KI), where KDapp is the apparent KD of Rox-Dpr-C with the Dvl-C or Dsh-C peptide and [I] is the concentration of both peptides  with a 10 × 4 mm quartz cell (Hellma Inc.) with magnetic stirring was used. To confirm that the binding site of the Dvl-1 PDZ domain was occupied by intrinsic C-terminus, we generated two proteins, mC1 and mC1-CΔ7, which were separately titrated into the fluorescence-labeled peptide Rox-DprC. The Kpeptides . For thepeptides . The KD All NMR experiments were performed at 15°C using Bruker Avance 800-MHz spectrometers equipped with triple-resonance, 5-mm triple axis–shielded gradient probes. For titration experiments we used the Varian Unity INOVA 600 MHz spectrometer equipped with a triple-resonance, 5-mm triple-axis shielded gradient probe at 25°C.Xenopus PDZ domain (1L6O:A), whose amino acid residues were modified to fit the mouse Dvl PDZ domain (13C-NOESY-HSQC experiments using the 13C/15N PDZ domain of Dvl with unlabeled Dvl-C peptide. NOE restraints were grouped into distance ranges according to their relative intensity: strong (1.8–2.5 and 1.8–3.0 Å), medium (1.8–4.0 Å), and weak (1.8–5.0 Å). A total of 45 unambiguous restraints  were used. Two types of restraints were combined to generate 2000 initial structures of the Dvl PDZ/Dvl-C peptide complex; 200 of these structures were selected by using NOE-derived restraints, and 100 structures were then obtained for final refinement. We ultimately selected the 15 lowest-energy conformers from the final 100 complex structures for further structural analysis.We used NMR-derived data and a simulated annealing protocol using the program CNS within the HADDOCK software (ver. 1.2) . CoordinZ domain . Two difXenopus eggs were obtained from females previously injected with 500 IU of human chorionic gonadotropin (Sigma) and artificially fertilized. Synthesis, microinjection of capped mRNAs, and treatment of ectodermal explants with activin were previously described  or the TOPFLASH reporter DNA construct (200 pg) was injected, alone or with mRNA (500 pg) encoding wild-type XDsh or a mutant XDsh-CΔ8 lacking the PDZ-binding motif, into the animal pole region of 2-cell Xenopus embryos. Ectodermal explants were dissected from injected embryos at the late blastula stage. To assess non-canonical Wnt signaling, we used the AP1-luciferase reporter (Xenopus whole embryos. The reporter DNA (200 pg) was injected alone or coinjected with equal quantities (500 pg) of XDsh-CΔ8 or wild-type XDsh mRNA into the dorsal region of 4-cell Xenopus embryos, which were allowed to develop to late gastrula stage. A Lumat LB9507 luminometer (Berthold Technologies GmbH & Co) was used to perform the luciferase assays (Promega). We used cell lysates from 10 explants or five whole embryos to measure luciferase activity. All experiments were performed at least in triplicate using different batches of embryos and the mean value was calculated using Student's t-test.To examine canonical Wnt signaling, both the reporter  and the reporter  to monitXenopus embryos at the 4-cell stage. At the early gastrula stage, 10 ventral mesoderm explants were dissected and analyzed by western blot using anti-phospho-JNK  antibody (Millipore) and anti-myc 9E10 antibody (Santa-Cruz Biotechnology).Synthetic mRNAs (500 pg) corresponding to myc-tagged wild-type XDsh and different mutants were injected into the ventral blastomeres of Xenopus embryos were injected with mRNA (500 pg) encoding XDsh, XDsh-CΔ8, or Xdd1, either alone or with mRNA (500 pg) encoding dnJNK. For the dose-depending studies, three different amounts of XDsh and XDsh-CΔ8 mRNAs  were used. The embryos were cultured to the larval stage, then grouped and counted according to normal, mild, or severe CE abnormal (JNK gain-of-function) phenotype. To investigate the effect of XDsh, XDsh-CΔ8, and Xdd1 on CE in vitro, the same mRNAs were injected, alone or with dnJNK mRNA, into the animal pole region of 2-cell Xenopus embryos. Ectodermal explants were dissected at the early blastula stage, incubated with activin for 1 hr, cultured to the early neurula-stage equivalent, and examined for the extent of explant elongation. To investigate how the C-terminal region of TMEM88 affects Dvl function, equal amounts of mRNAs (500 pg) encoding the C-terminal half of TMEM88 (TMEM88-C) and wild-type XDsh were coinjected into the two dorsal blastomeres at the 4-cell stage, and the embryos were again grouped and counted as having normal, mild, or severe CE phenotypes. The above experiments were performed at least twice using different batches of embryos.The two dorsal blastomeres of 4-cell  review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent , a Reviewing editor, and three reviewers, one of whom, Sergei Sokol, has agreed to share his identity.Thank you for submitting your work entitled “Conformational change of Dishevelled plays a key regulatory role in the Wnt signaling pathways” for peer review at The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission. The three full reviews (lightly edited) are included in this letter, as there are some specific and useful suggestions in them that will not be repeated in the summary here.All of the reviewers were impressed with the importance and novelty of your work. We look forward to receiving the revised version of your manuscript.Reviewer #1:Lee et al. present evidence that the Dvl PDZ domain binds to the Dvl C-terminus, and that disrupting this interaction leads to activation of Wnt/JNK signaling. Since Dvl critical for both Wnt /β-catenin and PCP signaling, this is a very intriguing set of findings. The work seems technically solid. There are a few points that the authors should address, however.1) The intramolecular interaction of the PDZ and C-terminus is described as a “closed conformation”. Do the authors see differences in hydrodynamic radius of the purified proteins  when the interaction is disrupted?2) The data in 3) Since deletion of either the PDZ or the C-terminus produces similar phenotypes, neither is implicated in directly activating JNK signaling. It is not clear from the Discussion how the authors think the activation may be occurring. Also, do the authors have an idea about a potential role of the PDZ-Fzd interaction?4) I originally thought that the “closed conformation” is the one used for beta-catenin activation, but this is not what the authors are claiming – they think ablating the PDZ-C-term interaction is important for both, although they muddle through this point in the penultimate paragraph. I think we agree that they need to clarify their discussion on beta-catenin signaling. I also agree that they need to discuss the earlier papers on Dvl/JNK signaling more carefully.5) Re Reviewer #2:This work relates to the mechanism of Dishevelled activation by the Wnt pathway. The authors provide significant evidence uncovering a physical interaction between the PDZ domain of the protein and the C-terminal PDZ-binding motif. Since the two domains might be expected to interact, this result, on its own, is not a surprise, but the authors propose that this interaction is critical for the ability of Dishevelled to signal. According to their model, the intramolecular association is responsible for the closed conformation of Dishevelled, which prevents JNK activation and convergent extension defects . The comparison of Dishevelled constructs shows that the protein with a deleted C-terminus (C∆8) and the one with a nonfunctional PDZ domain (Xdd1) activate JNK and interfere with convergent extension stronger than the wild-type protein. Similar effects are observed for two Dvl antagonists that bind to the PDZ domain, consistent with the proposed model. Interestingly, the effects on beta-catenin reporters are opposite to those obtained for AP-1 luciferase, suggesting that the ‘open conformation’ of Dishevelled inhibits canonical Wnt signaling. This is another important point, although it is not emphasized in the manuscript.This study is interesting because of the critical role of Dishevelled in Wnt signaling, the experiments are overall solid, with some typos and controls missing. To verify their conclusions obtained for the C-terminally truncated construct (C∆8), the authors are recommended to make specific point mutations disrupting the PDZ binding motif and, possibly, the PDZ domain. They should also confirm that the inhibitors used (TMEM88-C and 8625) interfere with the PDZ-PBD binding in vitro. I feel that the paper would benefit from showing an effect of a Wnt signal on the proposed intramolecular interaction, although these experiments might be considered beyond the scope of the present study. The authors should discuss early papers on Dishevelled and JNK signaling, such as Reviewer #3:Xenopus that Dsh/Dvl is normally in a closed confirmation (with the C-term residues bound to the PDZ domain) and open Wnt-signaling activation it is “opened” up with the C-term residues being released from the PDZ and allowing the full length protein to bind to and activate downstream effectors, like for example JNK signaling in the Wnt/PCP signaling context or interaction with Axin in the canonical signaling pathway.The authors address the potential role of intramolecular interactions between the Dsh/Dvl C-terminal sequences and its PDZ domain. They establish through a series of biochemical/structural techniques and a follow up biology in eLife. I have however a few concerns that should be addressed prior to publication.The observations presented here are of significance and relevance and worth publishing in 1) Based on the presented data, both the canonical and the PCP  Wnt pathway are more “effective” in the open configuration. Yet, in the Abstract the authors state: “Wnt-regulated CE is more readily affected by Dvl mutants unable to form the closed conformation than wild-type Dvl...non-canonical signaling is mediated by the open conformation of Dvl...”. I assume the authors refer to CE and non-canonical signaling as the same  and yet they state that one is “more affected”. This part of the Abstract is very unclear and needs to be rewritten.2) The authors state (in the subsection “The binding pocket of Dvl PDZ is occupied by its intrinsic ligand”): “... suggesting not only that the Dvl PDZ domain binds intrinsically to the Dvl C-terminus but also that this binding interferes with interactions between the Dvl PDZ domain and its other binding partners in the Wnt signaling pathways”. How does this work with the presumed Fz C-tail Dvl PDZ interaction, which has been shown to be essential for Fz induced Dsh/Dvl membrane recruitment and thus activation; this was published by the same authors .3) Throughout the paper the biochemical and biological experiment quantifications lack statistical analyses, e.g. 4) The data presented in Drosophila) would have been helpful. The Xenopus experiments suffer from massive overexpression and pure gain-of-function approaches. Does the model hold up in a loss-of-function genetic context?5) Some biological assays in a real in vivo/genetic setting  The intramolecular interaction of the PDZ and C-terminus is described as a “closed conformation”. Do the authors see differences in hydrodynamic radius of the purified proteins  when the interaction is disrupted?We did not see definitive difference between “closed” and “open” forms of Dvl in the SEC studies. Our DLS studies were also unsuccessful partially because it was very difficult to concentrate the proteins. However, we did obtain a set of data from an analytical ultracentrifugation study shown in . The sha2) The data inare used to argue that the Dvl C-terminus has an important role in Wnt/β-catenin signaling, since deletion of this region doesn't produce a strong activation of luciferase activity. Yet high doses the full-length protein gives the well-known effect. Is this because some fraction of the overexpressed full-length protein is “open”? Also, what does deletion of the PDZ domain do in this assay?Indeed, some fraction of the full-length Dvl is in “open” form. This issue is discussed and showed in Because the Fzd-PDZ interaction plays a key role in Wnt/β‐catenin signaling, therefore, deletion of the PDZ domain will block the signaling. In fact, it was showed that deletion of the PDZ domain abolishes the activity of Dsh in canonical Wnt signaling (3) Since deletion of either the PDZ or the C-terminus produces similar phenotypes, neither is implicated in directly activating JNK signaling. It is not clear from the Discussion how the authors think the activation may be occurring. Also, do the authors have an idea about a potential role of the PDZ-Fzd interaction?As we discussed in the Discussion section, we clearly state that although we propose a few possibilities, we don’t know exactly how JNK is activated through Dvl. What we report in the manuscript is our observations that the “open” conformation of Dvl was more effective in triggering JNK activation.Regarding the potential role of the PDZ-Fzd interaction, as we discuss in the Discussion section, this interaction should open the “closed” Dvl to the “open” form.4) I originally thought that the “closed conformation” is the one used for beta-catenin activation, but this is not what the authors are claiming – they think ablating the PDZ-C-term interaction is important for both, although they muddle through this point in the penultimate paragraph. I think we agree that they need to clarify their discussion on beta-catenin signaling. I also agree that they need to discuss the earlier papers on Dvl/JNK signaling more carefully.Actually, at this moment, based on all the data we have, we believe that the “open” conformation of Dvl is more active in all the Dvl mediated Wnt signaling pathways. This issue is discussed in the Discussion section.5) Re, this also confused me until I realized that the protein alone lanes have no beads – these are just input protein markers. But, they don't do the control of beads alone nor (better) a scrambled peptide. In any case this needs to be corrected.This point is well taken; we removed the figure in the revised manuscript.Reviewer #2:[…] This study is interesting because of the critical role of Dishevelled in Wnt signaling, the experiments are overall solid, with some typos and controls missing. To verify their conclusions obtained for the C-terminally truncated construct (C∆8), the authors are recommended to make specific point mutations disrupting the PDZ binding motif and, possibly, the PDZ domain.Because the nature of PDZ domain, it is very difficult to identify such a mutant that has significant reduction in binding affinity to its binding partners yet maintain the integrity of the protein.They should also confirm that the inhibitors used (TMEM88-C and 8625) interfere with the PDZ-PBD binding in vitro.We did the experiment according to the reviewer’s suggestion and the data are incorporated in the new I feel that the paper would benefit from showing an effect of a Wnt signal on the proposed intramolecular interaction, although these experiments might be considered beyond the scope of the present study.We discuss this issue in the Discussion section.The authors should discuss early papers on Dishevelled and JNK signaling, such asin Cell, which are not fully consistent with the proposed model.The reference was mistakenly omitted during the editing of the original manuscript. We sincerely apologize for this mistake. The reference is added back.We think our proposed model is actually consistent with the Cell paper. This issue is discussed in the Discussion section.Reviewer #3:[…] The observations presented here are of significance and relevance and worth publishing in eLife. I have however a few concerns that should be addressed prior to publication.1) Based on the presented data, both the canonical and the PCP  Wnt pathway are more “effective” in the open configuration. Yet, in the Abstract the authors state: “Wnt-regulated CE is more readily affected by Dvl mutants unable to form the closed conformation than wild-type Dvl...non-canonical signaling is mediated by the open conformation of Dvl...”. I assume the authors refer to CE and non-canonical signaling as the same  and yet they state that one is “more affected”. This part of the Abstract is very unclear and needs to be rewritten.We reword the sentence in the Abstract according to the suggestion of the reviewer.2) The authors state (in the subsection “The binding pocket of Dvl PDZ is occupied by its intrinsic ligand”): “... suggesting not only that the Dvl PDZ domain binds intrinsically to the Dvl C-terminus but also that this binding interferes with interactions between the Dvl PDZ domain and its other binding partners in the Wnt signaling pathways”. How does this work with the presumed Fz C-tail Dvl PDZ interaction, which has been shown to be essential for Fz induced Dsh/Dvl membrane recruitment and thus activation; this was published by the same authors .Indeed, the Dvl PDZ domain can bind to Dvl C-terminus, Fz C-terminal tail, and many other binding partners. We propose that inside the cell, these interactions compete with each other, and the surrounding condition the Dvl inside the cell determines the binding partner(s) of the Dvl PDZ domain. We discussed this issue in the Discussion section. For example, in the current working model of the canonical Wnt signaling, the binding of Wnt to both its receptors, Fz and LRP5/6 triggers the formation of the signalosome. The network of interactions in the signalosome super-complexes stabilizes the signalosomes remarkably, although the individual interactions are relatively weak. Energetically, the supercomplex is more stable than the “closed” conformation of Dvl and therefore can capture the PDZ domain of Dvl. By ousting the Dvl C-terminus from its bond with the Dvl PDZ domain, the Fz binding motif also opens the conformation of Dvl as it is captured in the signalosome.3) Throughout the paper the biochemical and biological experiment quantifications lack statistical analyses, e.g.We now include statistical analyses in these figure legends. Particularly, for luciferase assays, Student t-test was used in our analysis and the P values are included. For whole embryo phenotypes, at least two independent experiments were performed, with total numbers of embryos being indicated.4) The data presented inlacks several controls. The authors need to include “uninjected” and “xDsh alone”.These injections are included in 5) Some biological assays in a real in vivo/genetic setting (mouse or Drosophila) would have been helpful. The Xenopus experiments suffer from massive overexpression and pure gain-of-function approaches. Does the model hold up in a loss-of-function genetic context?We truly believe that the model we proposed should hold up in different loss-of-function genetic contexts. However, we will not be able to perform these studies in a very limited time frame and we also think that the experiments are out of the scope of this manuscript. Nevertheless, we plan to carry out some of the studies along those lines in the future.


Deinococcus radiodurans (DrRecQ) possesses an unusual domain architecture that is crucial for its remarkable ability to repair DNA. Here, we determined the crystal structures of the DrRecQ helicase catalytic core and its ADP-bound form, revealing interdomain flexibility in its first RecA-like and winged-helix (WH) domains. Additionally, the WH domain of DrRecQ is positioned in a different orientation from that of the E. coli RecQ (EcRecQ). These results suggest that the orientation of the protein during DNA-binding is significantly different when comparing DrRecQ and EcRecQ.RecQ DNA helicases are key enzymes in the maintenance of genome integrity, and they have functions in DNA replication, recombination, and repair. In contrast to most RecQs, RecQ from  RecQ helicases are a family of DNA strand-separating enzymes conserved from bacteria to humans that play a crucial role in the maintenance of genome stability . Bacteri E. coli RecQ (PDB 1OYW) and human RecQ1 (hRecQ1) (PDB 2V1X) [Most RecQ proteins share a helicase catalytic core and a C-terminal helicase-and-RNase-D-C-terminal (HRDC) domain. The minimal helicase catalytic core consists of two RecA-like domains, a Zn-binding domain (ZBD), and a winged-helix (WH) domain. Each individual domain has its own function and structure . The helDB 2V1X) , 6. ThesDeinococcus radiodurans is best known for extraordinary resistance to ionizing radiation, ultraviolet (UV) radiation, and chemical mutagens. This bacterium can survive under conditions of 7000 kGy of ionizing radiation with only 10% cell death; a radiation dose of greater than 50 Gy is lethal to most organisms [ D. radiodurans can become an excellent model system for studying the mechanisms of DNA repair. In this bacterium, RecD is the only member that is identified in the RecBCD pathway, whereas all of the members associated with the RecFOR pathway have been found in the genome of D. radiodurans, suggesting that the RecFOR pathway is the main repair pathway of DNA damage in this organism [rganisms , 8. Becaorganism .D. radiodurans has a unique RecQ homolog with three HRDC domains  at its C terminus. The function of DrRecQ has been characterized, confirming that the unusual domain arrangement of DrRecQ has the extraordinary ability to repair DNA damage in D. radiodurans [iodurans . HRDC1 aiodurans , 11. Howa = 85.7 Å, b = 98.5 Å, and c = 152.8 Å, with two molecules per asymmetric unit. All the DrRecQ crystals were cryoprotected with 25% glycerol added to the reservoir solution before flash cooling in a stream of nitrogen at 100 K. Crystals of DrRecQ diffracted at 2.8 Å at the National Synchrotron Radiation Research Center (NSRRC) BL13C1 in Taiwan. DrRecQ in complex with ADP was crystallized with a buffer containing 0.1 M imidazole (pH 7.2) and 16% PEG 20 K. The final crystals were orthorhombic with the following cell dimensions: a = 84.7 Å, b = 95.6 Å, and c = 183.8 Å, with two molecules per asymmetric unit. The crystal of the binary complex diffracted to a resolution of 2.9 Å at NSRRC BL13C1.The gene cloning, protein expression, and purification of DrRecQ have been previously reported . DrRecQ The crystal structure of the helicase catalytic core of DrRecQ was solved using the molecular-replacement method and the Phaser program with the coordinates of EcRecQ derived from a search model . The cry D. radiodurans was solved using the molecular-replacement method with the coordinates of EcRecQ from a search model and was refined at a resolution of 2.80 Å. The apoenzyme structure was subsequently used to solve the binary complex structure with ADP at resolution of 2.90 Å. In the binary complex structure, residues 5–517 of the polypeptide chain were well defined in the electron-density map; the exception was the 4 N-terminal end residues and residues 296–300, which were not visible. A ribbon representation of a monomer is shown in The crystal structure of the helicase catalytic core of RecQ fromβ-sheet surrounded by α-helices. Highly conserved signature sequence motifs are located in a cleft at the interface of D1 and D2. These motifs are typically involved in the hydrolysis of ATP and nucleic acid binding. The following Zn-binding domain (amino acids 342–407) possesses a Zn-binding motif and two antiparallel helices. The electron density maps of the Zn-binding motif reveal that the zinc ion is tetrahedrally coordinated by the side chains of four conserved cysteine residues   and D2 (amino acids 208–341), that are each composed of a central Cys 404) . The zinγ-phosphate cannot fit well, suggesting that the ATP has been hydrolyzed to ADP during crystallization despite not adding a magnesium ion (The nucleotide-binding pocket surrounded by conserved motifs is located at the D1 domain. Inspection of the electron density map revealed that electron density is strong in the pocket. Although we initially tried to model ATP into the experiment map, the sium ion . ADP is sium ion . The adesium ion . All of α, respectively. These data indicate that DrRecQ is more structurally similar to EcRecQ and more distant from hRecQ1. The domains of DrRecQ could individually superimpose well with both EcRecQ and hRecQ1, but the high rmsd value may be due to difference in the interdomain orientations between the structures. Comparison of the three structures demonstrates that there is conservation of domain architecture, specifically in the N-terminal portions of two RecA-like domains, the following Zn-binding domain and the C-terminal WH domain. A structural alignment was carried out for representing structures using the Pymol program with the resulting structural superimposition shown in β-hairpin structures in the WH domains of the human RecQ members are necessary for DNA unwinding, while mutational analysis shows that H491 in the β-hairpin of EcRecQ is not required for helicase activity, suggesting a different mode of action between the bacterial and human RecQs [β-hairpin of DrRecQ is shorter than hRecQ1 but resembles that of EcRecQ (β-hairpin of DrRecQ is similar to that of EcRecQ.Structural similarity searches with Dali revealed that DrRecQ is similar to other RecQ proteins . The besan RecQs . A strucf EcRecQ . This maA structural overlay of the free and ADP-bound forms of DrRecQ reveals that the two structures differ in the relative positions and orientation of D1 and WH domains . A strucIn this study, we described crystal structures of the DrRecQ helicase catalytic core and its ADP-bound form. Structural comparison of the two structures found that rigid-body movement occurs in the WH domain, indicating that the WH domain displays interdomain flexibility in DrRecQ. The motion may be important for the remarkable DNA repair mechanism performed by DrRecQ. Because DrRecQ is essential for maintaining genome stability, these structures provide important clues for understanding the molecular mechanisms of DNA replication, recombination, and repair.


We also present here, for the first time, the phylogenetic tree for archaeal LOV domains that have reached a statistically significant number but have not at all been investigated thus far.Flavin-based photoreceptor proteins of the LOV  and BLUF (Blue Light sensing Using Flavins) superfamilies are ubiquitous among the three life domains and are essential blue-light sensing systems, not only in plants and algae, but also in prokaryotes. Here we review their biological roles in the prokaryotic world and their evolution pathways. An unexpected large number of bacterial species possess flavin-based photosensors, amongst which are important human and plant pathogens. Still, few cases are reported where the activity of blue-light sensors could be correlated to infectivity and/or has been shown to be involved in the activation of specific genes, resulting in selective growth patterns. Metagenomics and bio-informatic analysis have only recently been initiated, but signatures are beginning to emerge that allow definition of a  Listeria monocytogenes  that regulates growth patterns and biofilm formation in ytogenes a 1], a , a Listeaeroides ,3 , binding a single flavin chromophore . Their photosensing and photoresponding properties, characterized by defined photochemical reactions and spectral features (vide infra) are linked to diverse effector/regulator functions in a large array of different proteins -x(4)-[GEQR]-x-C, where x is any amino acid and the terminal C is the cysteine forming the covalent bond during the photocycle. For BLUF domains we used: Y-x-[NG]-x-[LMVIFTK]-x-[FALIYCV]-x(1)-Q, where the two residues at the N- and C-terminal ends are the Tyr-Gln reactive pair. To avoid exceedingly stringent criteria, which would miss some hits, we extended in some positions the choice among amino acids similar to those super-conserved. The length of variable intervals is obtained from alignment and from structural considerations of secondary structure elements.Surveys in databases yield so-called sequence logos ,86, obta domains . It is eE. coli, Acinetobacter and K. pneumoniae). The results are summarized in a histogram , 167 archaeal LOV proteins in 86 strains (82 species), and 1705 bacterial BLUF proteins in 1282 strains  and by the large occurrence of EAL domains (34%), putatively involved in c-di-GMP metabolism, sometimes these domains are degenerated and enzymatically inactive EAL domains, as illustrated above for YcgF in E. coli   of the halobacterial protagonists carry fused HTH effector domains , whereas this motif is found in only 0.6% of all bacteria. The general architecture of these putative photo-regulated DNA-binding proteins is depicted in nPAS+kGAF+HTH, with n = 0–8, and k = 0–2, where: RR = response regulator, receiver domain; PAS = Per Arnt Sim domain (the same fold as LOV domains); GAF = domain found in cGMP-specific phosphodiesterases, cyanobacterial adenylate cyclases, and formate hydrogen lyase transcription activator FhlA -x(4)-[GEQR]-x-C for LOV domains; Y-x-[NG]-x-[LMVIFTK]-x-[FALIYCV]-x(1)-Q for BLUF domains, where x is any amino acid. With these pattern we performed a search for LOV and BLUF domains over all annotated prokaryotic genome sequences (completed or in progress), within the non-redundant protein sequence database and the SwissProt/UniProt Knowledgebase [To build the sequence logos of escribed . From thescribed  and Shanescribed   . DetailePhotophysiological studies of BL-sensing in prokaryots, as mediated by non-membrane LOV and BLUF proteins, are just now starting to accumulate. Only the demonstration of light-regulated changes in the growth behavior and the infectivity of selected animal and plant pathogens that can cause deleterious social and economic impact slowly convinces (micro)biologists of the important role of BL-sensing photoreceptors. As far as can be deduced thus far, BL seems to operate both as a stress factor and as a source of environmental information able to elicit adaptative responses in metabolism, growth patterns, and interactions with other organisms. It is still a fully open and fascinating question, if and how these Fl-Blues exert an effect in the large number of radiotolerant, extremophilic, and xenobiotic-metabolizing bacteria that are prominently represented in our lists of LOV- and BLUF-carrying prokaryots. The ever-growing information of genomic information now calls for more elaborate tools to screen new genomes (and metagenomes) for the presence of BL-photoreceptor genes and the identification of their genomic neighborhood. Though, still in its infancy, search criteria are now being developed that allow a more sophisticated search for potential photoreceptors, and also allow suggesting scenarios by which BL-photoreceptors have an impact on the gene regulation. The usefulness of such search criteria has been demonstrated here for the first time for archaea in which a manifold of BL-sensing photoreceptors could be identified. None of these archaeal species have been probed so far for their response to and lifestyle changes caused by blue light. It has now to be accepted that the generic stress response mechanisms, well studied in several bacterial species, have now to be extended by the impact elicited by blue light.


E-values have been the dominant statistic for protein sequence analysis for the past two decades: from identifying statistically significant local sequence alignments to evaluating matches to hidden Markov models describing protein domain families. Here we formally show that for “stratified” multiple hypothesis testing problems—that is, those in which statistical tests can be partitioned naturally—controlling the local False Discovery Rate (lFDR) per stratum, or partition, yields the most predictions across the data at any given threshold on the FDR or E-value over all strata combined. For the important problem of protein domain prediction, a key step in characterizing protein structure, function and evolution, we show that stratifying statistical tests by domain family yields excellent results. We develop the first FDR-estimating algorithms for domain prediction, and evaluate how well thresholds based on q-values, E-values and lFDRs perform in domain prediction using five complementary approaches for estimating empirical FDRs in this context. We show that stratified q-value thresholds substantially outperform E-values. Contradicting our theoretical results, q-values also outperform lFDRs; however, our tests reveal a small but coherent subset of domain families, biased towards models for specific repetitive patterns, for which weaknesses in random sequence models yield notably inaccurate statistical significance measures. Usage of lFDR thresholds outperform q-values for the remaining families, which have as-expected noise, suggesting that further improvements in domain predictions can be achieved with improved modeling of random sequences. Overall, our theoretical and empirical findings suggest that the use of stratified q-values and lFDRs could result in improvements in a host of structured multiple hypothesis testing problems arising in bioinformatics, including genome-wide association studies, orthology prediction, and motif scanning. Despite decades of research, it remains a challenge to distinguish homologous relationships between proteins from sequence similarities arising due to chance alone. This is an increasingly important problem as sequence database sizes continue to grow, and even today many computational analyses require that the statistics of billions of sequence comparisons be assessed automatically. Here we explore statistical significance evaluation on data that is stratified—that is, naturally partitioned into subsets that may differ in their amount of signal—and find a theoretically optimal criterion for automatically setting thresholds of significance for each stratum. For the task of domain prediction, an important component of efforts to annotate protein sequences and identify remote sequence homologs, we empirically show that our stratified analysis of statistical significance greatly improves upon a combined analysis. Further, we identify weaknesses in the prevailing random sequence model for assessing statistical significance for a small subset of domain families with repetitive sequence patterns and known biological, structural, and evolutionary properties. Our theoretical findings in statistics are relevant not only for identifying protein domains, but for arbitrary stratified problems in genomics and beyond. PLOS Computational Biology Methods paper.This is a E-values two decades ago  gives the E-value.We briefly review the relevant FDR definitions; for a comprehensive overview, see . Given apotheses , V and Rp-values ,24,43. NThere are two closely-related versions of the FDR used in our work: the positive FDR (pFDR) and marginal FDR (mFDR) ,43, defip-values are drawn independently from the two-component distribution of H = 0 denotes that the null hypothesis holds. This quantity is sometimes called the “Bayesian FDR” ..lFDR(t)=q-value of a statistic t is the minimum pFDR incurred by declaring t significant [q-values are efficiently constructed from p-values, and conservatively estimate the pFDR [q-value and lFDR estimation are based on the above formulas, where π0,F(t) and f(t) are replaced by estimates. See the Supp. Methods in q-values and lFDRs.The nificant . Estimatthe pFDR . Specifip-value thresholds ti per stratum i that maximize the number of predictions across strata while constraining the maximum FDR of the strata combined. Optimality of the lFDR here is consistent with the related Bayesian classification problem, where posterior error probabilities are also optimal [Here we prove that the lFDR gives optimal thresholds for stratified problems. For domain prediction, each domain family defines a stratum. We wish to find  optimal .Ni, π0,i, Fi(ti) and fi(ti) be given per stratum i. We desire to maximize the expected number of predictions across strataQ, orLet the FDR model quantities Λ, with the constraint set to strict equality, in a formulation that avoids quotients:This problem is solved using the Lagrangian multiplier function Λ with respect to tj, we obtain a necessary condition for optimality,j. Optimality of the lFDR also holds when constraining the combined E-value instead of the combined FDR , and then estimate q-values and lFDRs with methods adapted for censored p-values (Methods). For comparison, we also use E-value thresholds and the “Standard Pfam” curated bitscore thresholds  is no different from a combined E-value approach in that the ranking of predictions is preserved, since the number of proteins, or tests, is the same per stratum; the stratified E-value threshold equals the combined E-value threshold divided by the number of strata. Similarly, a combined q-value or lFDR approach  also preserves the E-value rankings.Each of the 12,273 Pfam domain families was used to scan for domains in each of 3.8 million proteins of UniRef50  produce or “GA” ). Note tq-value estimates. We created or adapted five tests, each of which labels domain predictions as either true or false positives  using different statistical and biological criteria. The proportion of predictions labeled FP estimates the FDR.We estimate the true FDR via “empirical” FDR tests, to compare all methods on an equal footing, but also to test the accuracy of p-value, highest ranking domains are considered as TPs, domains that overlap a higher-ranking domain of the same clan are removed (since they would not be counted as separate predictions), and domains that overlap a higher-ranking domain of a different clan are considered FPs  test is based on the expectation that overlapping domain predictions should be evolutionarily related . Pfam anMethods, . All FPsMethods, .The principles behind the other three tests are described here briefly: OrthoC (“Ortholog Set Coherence”) is based on the expectation that orthologous proteins contain similar domains , RevSeq Methods are compared at the same empirical FDR based on the number of domain predictions , unique q-value thresholds outperform E-values in all tests  and perform similarly in RevSeq  and tends to restrict new predictions to proteins that already had Standard Pfam predictions. In contrast, q-values increase domains at the same rate as they increase protein coverage, which increases information the most. Thus, while stratified q-values predict fewer domains than dPUC, those domains tend to be more informative than the dPUC predictions at comparable FDRs.We also evaluated dPUC, a prediction method based on domain context ,52. dPUCq-values and our empirical FDRs tests . This threshold gives a greater FDR than the Standard Pfam . We find that most families  detect many families with significantly larger FDRs than expected  for increased-noise as compared to as-expected-noise families, but many increased-noise family thresholds remain too permissive  to assign families into mutually-exclusive classes by majority rule. The “increased-noise” families have significantly large positive deviations see  in at leq-values in as-expected-noise families than in all families combined, although some disagreement remains . The final FDR is approximately Qseq+Qdom|seq if both thresholds are small and under an independence assumption , as q-values do very efficiently. Our work is, to the best of our knowledge, the first attempt at applying q-values and lFDRs to domain identification, thus advancing the statistics of this field.In multiple hypothesis testing, the FDR and lFDR are straightforward approaches for controlling the proportion of false positives and the posterior error probability, respectively. The Hochberg . BenchmaHochberg ,48,52,53q-values are more robust than our lFDR estimates for domain predictions, where the underlying p-value estimates are imperfect [Our theoretical work revealed that the lFDR, which is the Bayesian posterior probability that a prediction is false, is the optimal quantity to control in stratified problems. Stratified lFDR control has previously been found to optimize stratified thresholds in the related problem of minimizing the combined false non-discovery rate while controlling the combined FDR . The lFDmperfect  Fig 3)..q-valuesq-value approach into what we call tiered stratified q-values, by setting q-value thresholds on both the sequence and domain statistics reported by HMMER. While accurate FDR estimation of this procedure remains a challenge, tiered q-values successfully leverage the additional signal of repeating domains to increase predictions  binding site identification, the position weight matrix of each TF may yield a p-value per match [p-values using taxonomy, since domain family abundances vary greatly across the kingdoms of life  [The basis of our work is a general theorem applicable to naturally stratified statistical tests. Whether the combined FDR or er match , and theviruses) ,59. In sp-value distribution is required to estimate q-values and lFDRs. HMMER reports two kinds of p-values. The “sequence” p-value combines every domain of the same family on a protein sequence, while the “domain” p-value is limited to each domain instance. The sequence p-value thus reports whether the protein sequence as a whole contains similarity to the HMM, whereas the domain p-value scores individual domain units within the sequence. We obtained domain predictions with p-values on UniRef50 [p-value thresholds of 0.1, 0.1, and 0.01, respectively. Moreover, we obtain p-values using “-Z 1--domZ 1”. Lastly, we remove domains with p>0.01 by adding “-E 1e-2--domE 1e-2”.A UniRef50  and OrthUniRef50  proteinsp-values over a protein database to estimate q-values and lFDRs. We use standard methods [p-values while standard methods require all p-values. Notably, HMMER3 does not provide complete p-values even if filters are removed [p-values are accurate [p-values is not useful. Moreover, the filters are desirable to reduce HMMER3's runtime. The Supp. Methods  [p-value. Overlaps between families in the “nesting” list are not removed  . For allp-values are computed. The five empirical tests we use instead label every prediction as either a TP or a FP, and these labels are used to compute empirical FDRs and E-values . Each test makes different assumptions, and together they provide independent and complementary evaluations. We describe our two primary tests in detail next; for the other three, see Supp. Methods  and domains ranked by p-value, a domain is labeled as a TP if it is the highest-ranking domain or a higher-ranking domain can be found such that their family pair is in L; otherwise it is labeled a FP. This test does not evaluate the Standard Pfam fairly, which gets an FDR of zero, since L is defined by the Standard Pfam observations  combining all domain families, whereas the “family-level” FDR evaluates the accuracy of q-values separately per family. These quantities are consistent estimators of the corresponding true pFDRs under weak dependence [t, let TPij(t) and FPij(t) be the observed number of true positives and false positives, respectively, for domain family j in protein sequence i.Given domain predictions labeled as either TPs or FPs as above, we compute empirical FDRs at two levels. Briefly, the “method-level” FDR evaluates an entire scoring method  over all proteins i with predictions, which corresponds to the expected FDR per protein. This per-protein FDR normalizes for domain counts, so proteins with hundreds of domain instances are weighted the same as proteins with fewer domains. Similarly, the empirical E-value is i(t) and E-values respectively.The method-level empirical FDR is the mean epFDRq-value threshold of 1e-2; ideally they agree. The empirical family-level FDR, or efFDR, of family j is defined asi, and the log-deviation is defined asThis procedure measures per-family deviations between the empirical FDRs and the j(q) so LDj is defined when there are no observed FPs. For families with few predictions, the LD may be artificially large or small. We compute a two-tailed p-value (pPoisson) of the empirical E-value pPoisson distribution across families is used to compute q-values , and a measurement is deemed significant if qPoisson≤ 1e-3. Families are also separated by effect size: positive deviations if LDj > 2, negative deviations if LDj < -2, and small deviations if |LDj| ≤ 2.A pseudocount of 1 is used in efFDRp-value threshold” of dPUC is a tunable parameter, which when set to p ≤ 1e-4 gives comparable empirical FDRs to q ≤ 4e-4 on the MarkovR and OrthoC tests. dPUC is not evaluated in ContextC because both are based on domain context , nor in ClanOv because dPUC requires overlap removal while ClanOv requires observing overlaps to compute its FDR. DPUC 2.0 is available at http://compbio.cs.princeton.edu/dpuc.DPUC improves domain prediction by taking into account the “context,” or presence of other domain predictions . A newerPairCoil2 , TMHMM , and SEGS1 TextContains the supplementary methods, results, tables and figures.(PDF)Click here for additional data file.S1 FileTable with family accessions and annotation, number of “votes” , HMM length, and “type” is the repetitive pattern category: T = transmembrane domain, L = low-complexity region, C = coiled coil, and N = other .(TXT)Click here for additional data file.S2 FileTable with family accessions and annotation, number of “votes” (how many tests declare it as having a significantly large positive effect size difference from the expected FDR), HMM length, and “type” is the repetitive pattern category: T = transmembrane domain, L = low-complexity region, C = coiled coil, and N = other .(TXT)Click here for additional data file.S3 FilesThis dataset is used for one of our empirical FDR tests see .(FA)Click here for additional data file.S4 FilesThis dataset is used for one of our empirical FDR tests see .(FA)Click here for additional data file.S5 FilesThis dataset is used for one of our empirical FDR tests see .(FA)Click here for additional data file.S6 FilesThis dataset is used for one of our empirical FDR tests see .(FA)Click here for additional data file.S7 FilesThis dataset is used for one of our empirical FDR tests see .(FA)Click here for additional data file.S8 FilesThis dataset is used for one of our empirical FDR tests see .(FA)Click here for additional data file.S9 FilesThis dataset is used for one of our empirical FDR tests see .(FA)Click here for additional data file.S10 FilesThis dataset is used for one of our empirical FDR tests see .(FA)Click here for additional data file.


Time-calibrated phylogenomic trees of protein domain structure produce powerful chronologies describing the evolution of biochemistry and life. These timetrees are built from a genomic census of millions of encoded proteins using models of nested accumulation of molecules in evolving proteomes. Here we show that a primordial stem line of descent, a propagating series of pluripotent cellular entities, populates the deeper branches of the timetrees. The stem line produced for the first time cellular grades ~2.9 billion years (Gy)-ago, which slowly turned into lineages of superkingdom Archaea. Prompted by the rise of planetary oxygen and aerobic metabolism, the stem line also produced bacterial and eukaryal lineages. Superkingdom-specific domain repertoires emerged ~2.1 Gy-ago delimiting fully diversified Bacteria. Repertoires specific to Eukarya and Archaea appeared 300 millions years later. Results reconcile reductive evolutionary processes leading to the early emergence of Archaea to superkingdom-specific innovations compatible with a tree of life rooted in Bacteria. A stem line of descent is a primordial propagating series of pluripotent cellular entities. It is believed that such a remarkable backbone of the cellular world resulted in sequential cellular lineage spin-offs  , which were derived directly from a ToD. The rooted ToL and ToD trees are shown to the left and below the heat map and were built using maximum parsimony from the data matrix and its transposed derivative, respectively.We have shown that the natural history of protein domain structures and their associated functions can be directly inferred from ToDs, comb-like trees that harbor domains as taxa , the relative number of internal nodes from the root to a leaf of the tree. (iii) Phylogenetic statements are linked to the geological record through time calibration points: A global molecular clock of domain structures establishes a significant linear relationship between the age of domains and the geological record  TheThe time of first appearance of a domain structure at FSF level in the chronology records the time of the origin of that FSF. Consequently, the chronology of FSFs should be viewed as a timeline of molecular innovation portraying the gradual rise of modern biochemistry  in archaeal organisms. These early diversification patterns suggest the emergence of archaeal grades by reductive evolution and the very early rise of Archaea as the first superkingdom of diversified life  that are preserved in 1.5–2.1 Gy old cherts throughout the world , revealing further weakening of the stem line in favor of grades Figure . This beTimetrees suggest that aerobic metabolism and planetary oxygenation appeared concurrently ~2.9 Gy ago (Wang et al., Phylogenomics makes explicit the differential evolutionary accumulation of domain structures in evolving proteomes. Evolutionary patterns suggest that the primordial stem line produced cellular grades 2.9 Gy ago, which slowly turned into archaeal and eukaryal lineages and into bacterial lineages at more accelerated pace. This differential behavior reconciles the early origin of Archaea inferred from molecular structures and functions with the canonical rooting of the ToL sometimes revealed by standard sequence analysis. The important realization that supports these findings is the continuous growth of the protein world and the fact that the complete loss of a domain structure in a proteome occurs more likely soon after its origin in the timeline, at a time when there are few domain copies encoded in the genome and recruitment is incipient.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The domain architecture of a large RNA can help explain and/or predict folding, function, biogenesis and evolution. We offer a formal and general definition of an RNA domain and use that definition to experimentally characterize the rRNA of the ribosomal small subunit. Here the rRNA comprising a domain is compact, with a self-contained system of molecular interactions. A given rRNA helix or stem-loop must be allocated uniquely to a single domain. Local changes such as mutations can give domain-wide effects. Helices within a domain have interdependent orientations, stabilities and interactions. With these criteria we identify a core domain (domain A) of small subunit rRNA. Domain A acts as a hub, linking the four peripheral domains and imposing orientational and positional restraints on the other domains. Experimental characterization of isolated domain A, and mutations and truncations of it, by methods including selective 2′OH acylation analyzed by primer extension and circular dichroism spectroscopy are consistent with our architectural model. The results support the utility of the concept of an RNA domain. Domain A, which exhibits structural similarity to tRNA, appears to be an essential core of the small ribosomal subunit. The ribosome is a ribonucleoprotein complex that conducts one of life’s universal processes, which is the synthesis of proteins. The large ribosomal subunit (LSU) contains the peptidyl transferase center (PTC) and catalyzes transpeptidation. The small ribosomal subunit (SSU) contains the decoding center and reads messenger RNA (mRNA). Much of ribosomal function is performed by ribosomal RNAs (rRNAs)1245267In ancient stepwise evolutionary processes, the ribosome acquired capabilities for RNA folding, catalysis, subunit association, correlated subunit evolution, decoding, and energy transduction12To understand and explain the ribosome and its domain structure we use the following general definition. (i) The RNA comprising a domain is compact and modular, with a self-contained and integrated system of molecular interactions. (ii) Any given RNA helix or stem-loop is contained uniquely within a single domain. (iii) Local changes such as mutations or metal binding can have domain-wide effects. (iv) Molecular interactions between stem-loops within an RNA domain dictate their orientations, stabilities and interactions to be interdependent. (v) An RNA domain has the capacity to fold autonomously when excised from the surrounding RNA.The accepted canonical secondary structure of the SSU rRNA is based upon covariation16815This domain model has utility, and explains some dynamical properties of the SSU. The spokes are relatively flexible, allowing the domains to move relative to each other during initiation and translocation18202122Domain A imposes orientational and positional restraints on the other domains, which are depicted by arcs in . HelicesISO” , within helices 3, 19, and 28  and circular dichroism (CD) spectroscopy. In addition, we observe that the three-dimensional structure of domain A has analogy in other biological RNAs.One goal here is to test this domain model. Therefore we isolated domain A from the rest of the :SSU rRNA. We refer to isolated domain A as “domain AISO” . To form, and 28 . The steISO is characterized here by SHAPE reactivity and CD spectroscopy. We determine effects of mutations and of added Mg2+. We compare the SHAPE reactivity of domain AISO with that of the same rRNA elements within the intact SSU, previously published by Weeks and coworkersDomain A appears to satisfy the criteria of an RNA domain. Domain A2+ interactions change reactivity upon the addition of Mg2+ 262728+ alone, domain AISO forms helices 1, 2, 3, 19, 27 and 28  shows elevated SHAPE reactivity as expected of unstructured RNA. Similarly, the single-stranded nucleotides between stems 3 and 19  have higher reactivity than the flanking stems.Helices 2 and 28 are anomalously reactive in domain A2+ ions appear to stabilize domain AISO and facilitate folding to the native state. Monovalent cations generally allow RNAs to form secondary structures and a subset of tertiary interactions. Divalent cations are required for complete folding to the native state312+ to domain AISO increases the intensity of the diagnostic CD band at 265 nm. The intensity increases over the range of [Mg2+] from 0 to 700 μM after which it plateaus. These Mg2+ effects on domain AISO are similar to those of well-characterized globular RNAs such as tRNATetrahymena group I ribozymeMg2+ has subtle but widely distributed effects on the SHAPE reactivity of domain AISO. Mg2+ is expected to influence SHAPE reactivities of nucleotides that directly contact Mg2+ or are involved in Mg2+-dependent tertiary interactions. This pattern of Mg2+-dependent SHAPE reactivity has previously been observed for tRNA, RNase P, the P4-P6 domain of the Tetrahymena Group I intron and Domain III of the 23S rRNA272829352+, nucleotides in domain AISO show slight overall decreases in SHAPE reactivity while some loop regions and bulges show increases , helix 19 (C65), helix 27 (U90) and helix 28  (ISO (here) are consistent with those in the assembled SSU (McGinnis and Weeks)Pleij7, A108) . In addiISO. The C18A mutation, like helix 28 excision, lessens the effect on Mg2+ on the intensity of the 265 nm band by 50% (ISO). In sum, the data appear to support our domain model of the SSU rRNA.The C18A mutation affects the CD spectra of domain Ad by 50% . These rT. thermophilus, E. coli, S. cerevisiae, D. melanogaster, and H. sapiens In sum, we propose a predictive model of SSU architecture by defining domain A as a hub connecting to the peripheral domains. We show that the domain concept is applicable and useful for understanding the SSU. Domain A plays a crucial role in SSU structure and function, forming a scaffold that links to each of the other SSU domains and is an evolutionary ancestor to the SSU rRNA (9). Our results support and explain previous The chemical reagents used here are molecular biology grade or higher. DNA primers and oligonucleotides were purchased from Operon MWG. All aqueous solutions were prepared with deionized, distilled, nuclease free water . For the experiments in the absence of divalent cations, nuclease free water was treated with the Chelex 100 Resin (Biorad) chelating resin and recovered with 0.2 μm Ultrafree–MC–GV Centrifugal Filters (Milipore). All the experiments are reproducible and repeated at least 2 times unless otherwise stated.Thermus thermophilus HB8 strain SSU rRNA sequence was obtained from NCBI database. The domain AISO gene minus helix 28 was created by recursive PCR using the four oligonucleotides (5′ to 3′):The Forward 1: GGTGTGGGAATTCTAATACGACTCACTATAGGGTTGTTGGAGAGTTTGATCCTGGCTReverse 2: CAGTGAATCCGGGGCCTTACGGCCCCTGAGCCAGGATCAAACTCTCCAACForward 3: GTAAGGCCCCGGATTCACTGGGCGCCGTAAGGCGCCTGGGGAGTACGGCCReverse 4: CACCAAGCTTATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCThe flanking primers were;Forward: TGAGTCGTATTAGAATTCCCACACCReverse: GAAACTCAAAGGAATAAGCTTGGTG.ISO gene was cloned into the pUC19 vector using the EcoRI and HindIII restriction sites. The transformation used 5 μL of the ligation mix, which was added to 50 μL DH5α cells using the heat-shock method. Plasmids obtained by minipreps were sequenced bidirectionally by Operon MWG.The domain AACTGGCTCAGG and reverse CCAACAACCCTATAGTGAG primers. For SHAPE experiments, a primer binding tail was added to the 3′ end by PCR using the reverse primer CACCAAGCTTGAACCGGACCGAAGCCCGATTTGTGTACAAGGGCCTTACGGCCCCCCGTCAATTCCTTTGAGTTTCAGCCTTGC (5′ to 3′). The secondary structure of domain AISO with the SHAPE tail is shown in the Helix 28 was added with Q5 site-directed mutagenesis (NEB) using forward AAGCTTGGCGTAATCATGG and reverse TGTACAAGGGCCTTACGG primers. The C18A mutant was also made by Q5 site-directed mutagenesis, using forward AGAGTTTGATISO gene was digested with HindIII-HF (NEB) for 2 hours at 37 °C as described by manufacturer. The reaction mixture was incubated at 80 °C for 20 minutes to deactivate the enzyme. The reaction was purified with SmartSpin nucleic acid & purification columns (Denville Scientific Inc.) using DNA Clean & Concentrator Kit buffers (Zymo Research Corp.) Digested plasmid  was used as a template for T7 RNA polymerase (NEB) transcription. Run-off transcription reaction was prepared according to manufacturer’s description (NEB T7 High Yield RNA Synthesis Kit). The reaction mixture was incubated for 16 hours at 37 °C. After incubation, 1 μL Turbo DNAse (Ambion) was added to the reaction mixture, which was then incubated for 30 minutes at 37 °C. RNA was purified by ammonium acetate precipitation. Ultimately, 40 μL nuclease-free H2O was added to the dried pellet and the OD was measured with a Nanodrop (Thermo Scientific). RNA was further purified by G25 size exclusion chromatography .The pUC19 plasmid containing the domain AISO rRNA mixed with 10 μL (0.8 μM) 5′ 6-FAM labeled GAACCGGACCGAAGCCCG primer (Operon MWG). To anneal the primer to the RNA, the reaction was heated to 85 °C and slowly cooled to 30 °C at a rate of 1.5 °C per minute. For domain AISO rRNA lacking helix 28, 5′ 6-FAM labeled TATTCCTTTGAGTTTCAGCC primer was used. After primer annealing, 20 μL mixture of SuperScript® III Reverse Transcriptase (Invitrogen) reaction mixture prepared and added to domain AISO rRNA and primer mixture to give the final concentrations of 1X RT buffer, 2 mM DTT, 0.625 mM dNTPs, and 2.5 mM ddNTPs (TriLink BioTechnologies). The reverse transcription reaction was carried out by incubating 50 μL reaction mixture at 55 °C for 2 hours and quenched for 15 min by heating to 70 °C.Dideoxy sequencing reactions were carried out by heating a 20 μL solution of 50 ng/μL domain AISO rRNA was incubated for 4 min at 85 °C in the presence of 5 μM 1,2-diaminocyclohexanetetraacetic acid (Sigma) chelating agent and allowed to cool for 10 min into room temperature. This procedure depletes divalent cations from RNA. Divalent-free RNA was divided to two 32 μL samples and 4 μL of 10X folding buffer was added  for RNA folding with sodium. Four μL 10X folding buffer was added  for RNA folding with sodium and magnesium. The sample was folded by incubated at 20 min at 37 °C and was divided two 18 μL solutions. One of the solutions was added to 2 μL of 800 mM benzoyl cyanide in anhydrous DMSO. The other solution was added 2 μL of pure DMSO for a negative background control. The reaction mixture was incubated 2 min at room temperature. The modified RNA was purified using Zymo RNA Clean and Concentrator Kit and eluted in 25 μL modified TE buffer . Primer annealing and extension reactions were as described above.For the SHAPE reactions, a 70 μL solution of 150 ng/μL domain AFor the capillary electrophoresis, 1.5 μL of reverse transcription reaction mixture was mixed with 0.5 μL ROX-labeled DNA sizing ladder and 9 μL of HiDi Formamide (Applied Biosystems) in a 96-well plate. To denature the cDNA, the plate was incubated for 5 min at 95 °C. The mixture was resolved on a 3130 Genetic Analyzer (Applied Biosystems). Capillary electrophoresis data were processed using in-house MatLab scripts as described2+. The RNA was titrated first with the chelator, followed by back-titration with Mg2+, taking CD scans on a Jasco J-810 spectropolarimeter after each addition. Four CD spectra collected and averaged, from 350 to 220 nm with an integration time of 4 seconds, bandwidth of 4 nm, a scan speed of 50 nm/min. The temperature was kept at 20 °C. RNA concentrations were kept constant for mutant and intact RNAs. Since titration of 0.5–1 mM Mg2+ gives a plateau for all of the RNAs in the CD scans, titration was stopped after 1 mM divalent cations added. All RNAs are present in 74 μM of nucleotides, which corresponds to strand concentrations of 0.55 μM domain AISO, 0.55 μM domain AISO C18A mutant and 0.70 μM domain AISO w/o helix 28. CD experiments were repeated 3 times and results are reproducible.A solution of 25 ng/mL RNA, 5 mM sodium cacodylate, pH 6.8 was titrated with either a EDTA or Mget al.The domain A three-dimensional structure was modeled the ribosome structure of Ramakrishnan (PDB ID:4V51)Partial minimization of the re-ligated rRNAs was performed with Sybyl-X 1.2 software  with the AMBER FF99 force field using an implicit solvent model with the distance dependent dielectric function D(r) = 20r. The non-bonded cut-off distance was set to 12 Å. Each system was minimized by 1,000 steps of steepest decent followed by 5,000 steps of conjugate gradient minimization.Thermus thermophilus, Escherichia coli, Saccharomyces cerevisiae, Drosophila melanogaster, Homo sapiens were obtained from Protein Data Bank6737PDB IDs: 4V51, 4V9D, 4V88, 4V6W, 4V6X for SHAPE data are normalized and mapped on in-house RiboVisionShannon Entropies were calculated as previously describedFigures of three-dimensional structures are prepared with PyMol or Maxon Cinema 4D with the ePMV pluginHow to cite this article: Gulen, B. et al. Ribosomal small subunit domains radiate from a central core. Sci. Rep.6, 20885; doi: 10.1038/srep20885 (2016).