Here we present the crystal structures of the SECRET domain of vTNFR CrmD encoded by ectromelia virus and its complex with chemokine CX3CL1. The SECRET domain adopts a β-sandwich fold and utilizes its β-sheet I surface to interact with CX3CL1, representing a new chemokine-binding manner of viral CKBPs. Structure-based mutagenesis and biochemical analysis identified important basic residues in the 40s loop of CX3CL1 for the interaction. Mutation of corresponding acidic residues in the SECRET domain also affected the binding for other chemokines, indicating that the SECRET domain binds different chemokines in a similar manner. We further showed that heparin inhibited the binding of CX3CL1 by the SECRET domain and the SECRET domain inhibited RAW264.7 cell migration induced by CX3CL1. These results together shed light on the structural basis for the SECRET domain to inhibit chemokine activities by interfering with both chemokine-GAG and chemokine-receptor interactions.Pathogens have evolved sophisticated mechanisms to evade detection and destruction by the host immune system. Large DNA viruses encode homologues of chemokines and their receptors, as well as chemokine-binding proteins (CKBPs) to modulate the chemokine network in host response. The SECRET domain  to sequester chemokines during the infection. The SECRET domain represents a new family of viral CKBPs that was originally identified as a C-terminal extension of the viral tumor necrosis factor receptors (vTNFRs). We determined the three-dimensional structures of the SECRET domain and its complex with chemokine CX3CL1, revealing a new chemokine-binding manner of viral CKBPs. We also showed that other chemokines from different classes may be bound by the SECRET domain in a way similar to that observed in the SECRET/CX3CL1 complex structure. Our biochemical and chemotaxis assays also suggest that the SECRET domain is able to interfere with both chemokine-GAG and chemokine-receptor interactions, both of which are essential for chemokine activities  Chemokines orchestrate leukocyte migration during immune surveillance, inflammation, and development The chemokine network is an important component of host immune response to viral infection smallpox virus-encoded chemokine receptor) de novo model Four different genes encoding viral tumor necrosis factor receptors (vTNFRs) have been identified in poxviruses, consisting of cytokine response modifier B (CrmB), CrmC, CrmD, and CrmE 2, the size exclusion chromatography revealed that it is monomeric in solution  was determined at a resolution of 1.57 Å by using single-wavelength anomalous dispersion (SAD) method with a Br-soaked derivative . There aThe SECRET domain monomer adopts a β-sandwich fold, consisting of two parallel β-sheets and the connecting loops . The β-sThe overall β-sandwich topology of the SECRET domain is similar to that of vCCI and A41 The electrostatic complementarity plays a critical role in the binding of chemokines by vCCI and A41 workR and freeR factors of 19.6% and 25.0%, respectively , a 3-stranded anti-parallel β-sheet , a C-terminal helix (Q56−A69) packing against the β-sheet, and the 30s loop (N30−A38) and 40s loop (R44−H46) connecting the strands in the β-sheet  method. We performed two independent measurements for each protein sample and the results are listed in 2, can be divided into two patches. The patch 1 between the N-loop of CCL4 and vCCI is primarily composed of hydrophobic interactions around CCL4 residue F13 and salt-bridge interactions around CCL4 residue R18  is composed one contact patch with a small hydrophobic core and surrounding hydrophilic interactions as described above  and C-terminal domain (CTD)  to test if the SECRET domain can interfere with the interaction of CX3CL1 with GAG It has also been shown that the SECRET domain can inhibit the CCL25-mediated Molt4 cell migration, indicating its ability to interfere with binding of CCL25 with its cellular receptors The measured binding affinity (∼0.68 µM) between the SECRET domain and CX3CL1 in our experiment is lower than previous reported binding affinities between the SECRET domain and CCL28, CCL25, CXCL12, CXCL13, CXCL14, XCL1, and CCL20 that are in nM range in vivo function of chemokines, including helping immobilize chemokines to form a concentration gradient along which cells can migrate directionally, protecting chemokines from proteolysis, and inducing chemokine oligomerization GAG binding plays important roles in the The inhibitory ability of the SECRET domain for CCL25 and CX3CL1 induced cell migration indicates that it is able to interfere with the chemokine-receptor interaction. It is generally accepted that the N-termini of chemokines is the key signaling domain, and other residues in the N-loop and core domain can also be critical for the binding with chemokine receptors. For example, the residues 12–17 in the N-loop of CXCL12 were shown to be important for receptor binding The previous study reporting the discovery of the SECRET domain has shown that it is capable of binding CCL28, CCL25, CCL20, CXCL12, CXCL13, CXCL14 and XCL1 that are from the CC, CXC, and C classes. We have shown here that it is also able to bind CX3CL1, the only member in the CX3C class. The ability of previous reported M3 to bind a subset of chemokines from all four classes is attributed to its structural plasticity  and the use of flexible loops as primary contact sites for chemokines from different classes Besides CrmB and CrmD, genome analysis also identified other genes encoding SECRET domain containing proteins (SCPs) E. coli BL21 (DE3) competent cells. Three liters of LB media containing 100 µg/ml ampicillin were inoculated and grown to A600 of 0.8 and then induced with 0.6 mM IPTG. Induced cultures were grown for an additional 4 h at 37°C and harvested by centrifugation for 10 min at 5,000 rpm. Cells were resuspended in 25 mM Tris-HCl, 50 mM NaCl, pH 8.0, lysed with sonication and centrifuged for 50 min at 15,000 rpm. The SECRET domain was found exclusively in the inclusion bodies. The inclusion bodies were washed three times in wash buffer A  and once in wash buffer B . Washed inclusion bodies were solubilized in 8 M Urea, 50mM DTT and diluted into a refolding buffer  and stirred at 4°C overnight, and then dialyzed against 25 mM Tris-HCl, 50 mM NaCl, pH 8.0. The refolded SECRET domain was bound to HisTrap column, then washed with 25mM Tris-HCl, 50 mM NaCl, 20 mM Imidazole, pH 8.0 and eluted with 25 mM Tris-HCl, 50 mM NaCl, 500 mM imidazole, pH 8.0. Fractions containing the SECRET domain were examined by SDS-PAGE gel, pooled and further purified with size exclusion column. The SECRET mutant (D167A/E169A/D316A) was expressed and purified by the same method as wild type SECRET domain. To check the SECRET domain is a dimer or monomer in solution, molecular weight standards and the SECRET domain  were loaded onto Superdex 75 size exclusion column with a flow rate of 0.5 ml/min.The gene encoding the SECRET domain of CrmD (residues 162−320) was cloned into EcoRI and NcoI restriction sites of the pProEx HTb expression vector. The resulting plasmid was transformed into E. coli BL21 (DE3) competent cells. Three liters LB media containing 100 µg/ml ampicillin were inoculated and grown to A600 of 0.8 and then induced with 1.0 mM IPTG. Induced cultures were grown for an additional 4 h at 37°C and harvested by centrifugation for 10 min at 5,000 rpm. Cells were resuspended in PBS buffer (pH 7.2), lysed by sonication and centrifuged for 50 min at 15,000 rpm. CX3CL1 was found exclusively in the inclusion bodies. The inclusion bodies were washed three times in wash buffer A  and once in wash buffer B . Washed inclusion bodies were solubilized in 8 M Urea, 50 mM DTT and diluted into a refolding buffer  and stirred at 4°C overnight. Precipitated material was removed by filtration. Refolded protein was bound to a HisTrap column and washed with PBS buffer, 20 mM Imidazole then eluted with PBS buffer, 500 mM Imidazole. Fractions containing CX3CL1 were examined by SDS-PAGE gel, pooled and further purified with size exclusion column. All CX3CL1 mutants were expressed and purified by the same method as wild type CX3CL1. Purified SECRET domain and wild type CX3CL1 were mixed, left on ice for 1 h, and subjected to size exclusion column purification to obtain the SECRET/CX3CL1 complex.The gene encoding the chemokine domain of human CX3CL1 (residues 1–76) was cloned into the EcoRI and NcoI restriction sites of the pProEX HTb expression vector. The resulting plasmid was transformed into The SECRET domain and the SECRET/CX3CL1 complex were concentrated by ultrafiltration to ∼15 mg/ml. Crystals of the SECRET domain were grown from a mother liquor of 0.4 M Magnesium formate dehydrate, 0.1 M Bis-Tris propane, pH 7.0 with hanging-drop vapor diffusion method at room temperature. Crystals of the SECRET/CX3CL1 complex were grown from 0.2 M Lithium sulfate monohydrate, 0.1 M Tris·HC, pH 8.5, 20% PEG4000 with hanging-drop vapor diffusion method at room temperature.Crystals of the SECRET domain were cryoprotected in well solution plus 20% (v/v) glycerol and cooled to 100 K before data collection. For the SAD data collection, crystals were soaked in well solution with 0.2 M NaBr for 30 s before data collection. Crystals of the SECRET/CX3CL1 complex were cryoprotected in well solution plus 20% (v/v) glycerol and cooled to 100 K before data collection. All diffraction data were collected at Shanghai Synchrotron Research Facility (SSRF) beamline BL17U. All data were indexed and integrated and scaled with program HKL2000 workR is 16.4%, and the freeR is 19.9%. The structure of the SECRET/CX3CL1 complex was solved using the molecular replacement method with the SECRET domain and the CX3CL1 structures as search models in the program PHASER workR of 19.6% and freeR of 25.0%. All structural figures were made by using PYMOL (http://www.pymol.org).The structure of the SECRET domain was solved using the Br-SAD method. The positions of the Br were determined using the program SHELXD −1. The binary complexes were allowed to associate for 90 s and dissociate for 90 s. The surfaces were regenerated with 5 mM NaOH between each cycle if needed. Data were analyzed with BIAcore 3000 evaluation software BIAevaluation 4.1.The binding affinity between the SECRET domain and CX3CL1 was determined by surface plasmon resonance (SPR) using BIAcore 3000 at 25°C. The SECRET domain was immobilized to about 350 Response Unit (RU) on a research-grade CM5 sensor chip in 10 mM sodium acetate, pH 4.1 by standard amine coupling method. The flow cell 1 was left blank as a reference. To measure binding affinity of CX3CL1 wild type and mutants by the SECRET domain, CX3CL1 in 10 mM HEPES, pH 7.2, 150 mM NaCl, and 0.005% Tween-20 were injected over the flow cells at different concentrations at a flow rate of 30 µl minTo investigate the interference of heparin in the binding of CX3CL1 by the SECRET domain, 1 µM wild type CX3CL1 was pre-incubated with increasing concentrations  of heparin sodium salt  at 4°C for 1 h. SPR analysis was performed as above.To compare the binding ability of the SECRET domain wild type and mutant by chemokines, CX3CL1 purified by ourselves, CCL28, CCL25, and CXCL12 purchased from PeproTech were immobilized on the CM5 chip to ∼200 RU. SPR analysis was performed as above.2 incubator. Serum-starved RAW 264.7 cells with a total number of 1×107 were suspended in PBS buffer with 1 µM CellTracker Green CMFDA (Invitrogen) and incubated at 37°C for 5 minutes. The labeled cells were collected, washed three times with PBS buffer to remove the excessive CMFDA, and then suspended in RPMI 1640 medium for cell migration assays.RAW 264.7 cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FBS at 37°C in CO4 CMFDA-labeled cells in each transwell insert and incubated at 37°C for 4 hours. Cell migration was quantified by counting the number of cells that remaining in the upper transwell by FACS.Cell chemotaxis assay was performed using 8 µm−pore Cell Culture Inserts (Millipore). The inserts were placed into 24-well plates containing RPMI1640 in the presence or the absence of CX3CL1 and SECRET domain. We seeded 8×10The coordinates of the SECRET domain and SECRET/CX3CL1 structures have been deposited into the Protein Data Bank with accession numbers 3ON9 and 3ONA, respectively.Text S1Supporting data including eight supplemental figures.(PDF)Click here for additional data file.


Identification of aggregation-prone regions other than polyglutamine could greatly help the development of SCA1 treatment more specific than that based on targeting the low complexity polyglutamine region.The neurodegenerative disease spinocerebellar ataxia type 1 (SCA1) is caused by aggregation and misfolding of the ataxin-1 protein. While the pathology correlates with mutations that lead to expansion of a polyglutamine tract in the protein, other regions contribute to the aggregation process as also non-expanded ataxin-1 is intrinsically aggregation-prone and forms nuclear foci in cell. Here, we have used a combined approach based on FRET analysis, confocal microscopy and  The inherited disease spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative pathology characterized by progressive loss of Purkinje cells in the cerebellar cortex and of neurons in the spinocerebellum . The patIn agreement with this view, ataxin-1 is an intrinsically aggregation-prone protein known to form, also in its non-expanded form, diffuse cellular aggregates sometimes named foci . The sizRealization that ataxin-1 aggregation may be triggered by more than one region has suggested that this behaviour could inspire the development of new drugs that could target regions other than or in addition to the polyQ tract. Such drugs would be potentially more specific than compounds preventing the aggregation of the low complexity polyQ. A recent report has, for instance, shown that stabilization of the monomeric form of the AXH domain by formation of a complex with a peptide from the natural partner protein Capicua (CIC) prevents the aggregation and misfolding of the isolated domain . If the in vitro investigations of isolated regions of ataxin-1 to map the regions necessary for foci formation and explore the relationship between dimerization and self-association of non-expanded ataxin-1. FRET based approaches have proven to be a powerful tool for the analysis of protein homo-dimerization in cells  analysis and in cells . Our resNon-expanded (Q30) ataxin-1 and truncated ataxin-1 fusion proteins were constructed in pEYFP or pECFP vectors using the standard PCR and mutagenesis methods previously established in our laboratory . COS celSize exclusion chromatography was performed using a prepacked Superdex-75™ 10/300 GL column (Pharmacia) equilibrated with a 20 mM Tris–HCl pH 7, 150 mM NaCl, 1 mM TCEP buffer solution. Aliquots of 200 µl of 150 µM AXH and TLND2AXH incubated for 24 h at 37 °C in 20 mM pH 7, 150 mM NaCl, 1 mM TCEP were injected separately and eluted using a 0.8 ml/min flow rate. Albumin (67 kDa), Ovalbumin (43 kDa), Carbonic anhydrase (29 kDa), and Ribonuclease A (13.7 kDa) were used as standards for the molecular mass, whereas the Blue Dextran 2000 was used for the determination of the void volume of the column.Ro is the Förster radius for CFP and YFP, which is 4.95 nm.Samples for FRET were imaged on a Zeiss LSM 510 confocal microscope using a 63× 1.4NA Plan NeoFluar oil immersion objective and FRET analysis was carried out as previously described in detail . Pre- anWe first explored the relationship between foci formation and polyQ in non-expanded ataxin-1. We used non-expanded ataxin-1 to be able to detect the intrinsic properties of the functional protein that could then be transferred to the expanded form. We created several deletion constructs for expression in mammalian cells in which the protein was N-terminally attached to the yellow fluorescent protein (YFP) . The firAs expected, wild-type ataxin-1 fused to YFP readily formed nuclear foci  and 2B. per se a major foci forming factor. These results thus indicate that the C-terminus of ataxin-1 alone is involved in foci formation which does not involve the polyQ tract.The observation that Atx1ΔNTQ, which lacks the polyQ tract, is able to form foci demonstrates that polyQ region is not In order to further map the region required for nuclear foci formation, we expressed deletion mutants from the C-terminal region of ataxin-1 fused with YFP. We first expressed the region starting at the AXH domain and ending at the last residue of ataxin-1 , which also contains the endogenous Nuclear Localisation Signal (NLS). As expected, this protein was mostly nuclear, but expressed itself in a diffused pattern  and 3B. These results thus suggest that the aggregation-prone AXH domain is insufficient for foci formation by its own.Since TLND2End formed foci while AXH2End did not, we next expressed the TLND2AXH region (residues 410–561) as YFP fusion to analyse if this is independently capable of foci formation. We observed that this construct formed foci that were, however, smaller in size as well as in number –4C. ThisThese results show that foci formation takes place independently from the AXH domain and that the nuclear localization signal has an influence on foci localisation.in vitro . Lower retention volumes are observed for TLND2AXH as compared to AXH despite their similar molecular weight. This would be justified by the elongated shape of the putatively unfolded TLND2AXH construct  as compaThese results provide solid evidence of the existence of a newly identified self-association region within ataxin-1 that is independent from the AXH domain.in vitro studies of dimerization, we next tested the self-association regions identified in cultured cells and explored if they are capable of direct interaction leading to self-association also in cell. To verify that the full-length ataxin-1 protein is indeed capable of self-association, we expressed full-length non-expanded ataxin-1 as CFP and YFP fusions and carried out FRET analysis. Self-association of ataxin-1 was evident from positive FRET signal  peptide (residues 34–48) of the protein that we have recently been shown to interact with the AXH domain with high affinity and to force it in a monomeric form . UnfortuWe resorted to a different strategy as a proof of principle. We have recently reported that mutation of a glycine at position 567 keeps the AXH domain in a predominantly monomeric form . We thusWe can thus conclude that both dimerization domains of ataxin-1 contribute to the self-interaction of the protein in cells.One aspect that distinguishes ataxin-1 from most other proteins is its ability to form small dense nuclear bodies . These bin situ investigations have now revealed that non-expanded ataxin-1 has two self-association motifs. Both AXH domain and the TLND2AXH motif are able to self-associate in transfected cells, as shown by FRET analysis. An earlier description of ataxin-1 self-association had identified this property almost exclusively with the AXH domain (independently capable of self-association while the former but not the latter is essential for foci formation. These observations are further supported by in vitro analysis of the dimerization properties of these motifs. Interestingly, gel filtration analysis demonstrates that the TLND2AXH motif is a dimer in equilibrium with the monomer, much like the AXH domain itself (Our H domain . AccordiH domain . We haven itself . Collectin vitro (In the attempt to define the relationship between dimerization and self-association, we also questioned if events that potentially reduce dimerization of individual motifs are able to influence the self-association tendency of ataxin-1 as measured by FRET analysis. In light of a recent report  and of oin vitro . The FREin vitro . Our resIn conclusion, our study contributes to clarify the self-association properties of ataxin-1 and the relationship between these and the dimerization observed at the level of individual domains. This information may be used in further studies to probe the effect of the CIC interactions on aggregation and be helpful for designing new approaches to SCA1 therapy.


Studying the effects of pathogenic mutations is more complex in multidomain proteins when compared with single domains: mutations occurring at domain boundaries may have a large effect on a neighbouring domain that will not be detected in a single-domain system. To demonstrate this, we present a study that utilizes well-characterized model protein domains from human spectrin to investigate the effect of disease-and non-disease-causing single point mutations occurring at the boundaries of human spectrin repeats. Our results show that mutations in the single domains have no clear correlation with stability and disease; however, when studied in a tandem model system, the disease-causing mutations are shown to disrupt stabilizing interactions that exist between domains. This results in a much larger decrease in stability than would otherwise have been predicted, and demonstrates the importance of studying such mutations in the correct protein context. Moreover, the severity of disease correlated with the extent of destabilization. Such a ‘cut-off’ has been observed in other studies A key area of interest in the post-genomic era is to relate changes in gene sequence to phenotypic variation. As more than 70% of eukaryotic proteins are composed of multiple domains, when studying the effects of pathogenic mutations in multidomain proteins, we must determine the effect that a mutation in one domain may have on neighbouring domains 2), there are significant interactions between adjacent domains To demonstrate this effect, we use the well-studied protein domains R15, R16 and R17 from chicken brain α-spectrin as model systems to study the effects of pathogenic mutations in human spectrin domains. These domains are a common component of proteins involved in cytoskeletal and membrane-associated structures, including spectrin, α-actinin and dystrophin Erythrocyte and brain spectrin most commonly exist as a tetramer: two antiparallel spectrin molecules, one α and one β, associate laterally to form heterodimers that further associate to form tetramers www.uniprot.org/uniprot) and the Human Genome Mutation Database (www.hgmd.cf.ac.uk) to compile a list of 12 disease-related and 20 non-disease-related nsSNPs in the spectrin domains of the human proteins α-and β-spectin (UniProt designations SPTA1_HUMAN and SPTB1_HUMAN), dystrophin (DMD_HUMAN) and α-actinin (ACTN3_HUMAN). These 32 SNPs were found in 24 spectrin domains spread between the four proteins. We ignored all mutations found at the tetramerization site in α-and β-spectrin. The sequences of these domains were compared with those of the previously well-characterized chicken brain α-spectrin R15, R16 and R17, using alignments compiled using ClustalW . These were subsequently verified by comparison with the Pfam alignment (www.pfam.sanger.ac.uk)  . As obser.ac.uk) . We idenr.ac.uk) . In somer.ac.uk) .The effect of mutation on the stability of the single-domain model proteins was determined using the equilibrium denaturation method . Intereskf and ku, respectively), both alone and in the two-domain system, to determine the effect of a mutation on the stability of a two-domain protein kf and ku, extrapolated to 0 m denaturant, for each domain in the two-domain protein constructs. The method of analysis is explained in detail in Most of the pathogenic mutations are clustered in the linker helix between domains. It has previously been demonstrated that spectrin domains are stabilized by their neighbours −1 (−1) (kf of ∼ 560 s−1 (column 8) compared with the mutant single-domain protein kf of ∼ 7.6 s−1 (column 6), and the stability of the R16 domain in R1617*I18V is the same as that in WT R1617 (column 11). Thus the stabilizing interactions are retained and the loss of stability of the two-domain protein is the same as the loss of stability of the single-domain protein (columns 12 and 14). We found essentially the same results for all the non-pathogenic mutations. Thus the overall loss of stability resulting from these non-pathogenic mutations, even in the multidomain context, was < 2.0 kcal·mol−1 in all cases. This is consistent with the threshold that has been observed previously for other proteins For the non-pathogenic mutations, we found that the stabilizing interactions between the domains were retained, such that the total loss of stability in the two-domain protein was essentially the same as the loss of stability in the single domain. As an example, the mutation I18V in R17   to 15 s−1 (mutant). This means that the mutation N105P destabilizes R15 in R1516 by ∼ 3.4 kcal·mol−1. Moreover, R16, which was originally stabilized by R15 (folding more rapidly and unfolding more slowly), loses this stability  . Howevertability . R16 in −1. However, there is also a mutual stabilization between neighbouring domains when both domains are folded . The folding pathways for R1516 and R1617 are essentially the same (G) may be calculated from the folding and unfolding rate constants ΔG = −RT ln(ku/kf). The stability of the entire two-domain spectrin construct is thus:GN and ΔGC are the free energies of unfolding of the N-and C-terminal domains, respectively, ΔGextension is the gain in free energy of the N-terminal domain from simple extension , and ΔGinterface is the stabilization of one domain by its folded neighbour.Early equilibrium studies showed that spectrin domains were stabilized by their neighbours the same  32,35. FAs a full analysis of the folding of individual domains and the sometimes very complex two-domain constructs is necessary to fully characterize spectrin repeats, a systematic analysis of the thermodynamic and kinetic effects of pathogenic (and non-pathogenic) variants in their natural environment is simply prohibitive. Here, for instance, we investigate nine mutations in seven different α-spectrin domains, as well as two mutations in different domains of β-spectrin and one mutation of dystrophin. We had previously characterized three different wild-type single repeat proteins plus two tandems Here we find that the point mutations in the single domains result in proteins that are somewhat destabilized relative to the WT domain . This isFor the non-pathogenic mutations, the effect of a mutation in the two-domain system is approximately the same as the effect in the isolated domain. All stabilizing interactions between the domains are intact. This is shown in −1 or more, an increase of ∼ 3.5–4 kcal·mol−1 over and above the effect of the mutation in the single domain. The one exception was the mutation E106D in both R15*16 and R16*17. This mutation was created to model the mutation D791E in human α-spectrin, which has been shown to cause hereditary elliptocytosis However, the case for the pathogenic mutations is entirely different. With one exception, all pathogenic mutations caused a loss of the stabilizing interactions between the domains. The result is quite remarkable . Thus, t−1 per interface) is very significant when compared to the stability of spectrin domains that we have studied, which ranges from 3.5–6.5 kcal·mol−1. In a previous study, Johnson et al. Undoubtedly, spectrin repeats gain significant stability through nearest-neighbour effects, mediated through the linker helix. The stabilization conferred by these effects . Mutagenesis was performed using a QuikChange site-directed mutagenesis kit , and the identity of the mutants was confirmed by DNA sequencing. Protein expression and purification were performed as described previously m. Dithiothreitol was added to a final concentration of 5 mm for R17 and R1617 proteins. All experiments were performed at 25 ± 0.1 °C in 50 mm sodium phosphate buffer (pH 7.0). The data were fitted to a two-state transition curve as described previously The stability of the mutant single-domain proteins was determined by equilibrium denaturation using urea as the denaturant. Folding was monitored on the basis of intrinsic tryptophan fluorescence, measured using a Perkin Elmer  fluorescence spectrometer with a final protein concentration of 1 μm sodium phosphate buffer (pH 7.0). The final protein concentration was 1 μm, with 5 mm dithiothreitol added for R17 and R1617. Samples were excited at a wavelength of 280 nm, and the emission was monitored above 320 nm. At least six overlying traces were obtained at all concentrations of urea. Single-jump experiments on all proteins were performed using 10: 1 mixing (buffer:protein). Double-jump experiments were performed on tandem repeats R1516 and R1617. For R1617, interrupted unfolding experiments allowed the folding rate of R17 to be observed: proteins were initially unfolded in urea at a 1:5 ratio (protein:urea) for a delay time of 500 ms, and then jumped into refolding solutions at 1:10 ratio (protein:buffer). For R1516, interrupted refolding experiments allowed the unfolding rate of R15 to be observed: unfolded protein in 8 m urea was allowed to refold to a final concentration of 4 m with a delay time of 100 ms, and then jumped into unfolding solutions. Data for all experiments were fitted using Kaleidagraph .Kinetic experiments on the mutant proteins were performed using a stopped-flow fluorimeter  at 25 ± 0.1 °C in 50 m


Plasmodium falciparum PfEMP1 adhesin is a virulence factor and a prime target of humoral immunity. It is encoded by a repertoire of functionally differentiated var genes, which display architectural diversity and allelic polymorphism. Their serological relationship is key to understanding the evolutionary constraints on this gene family and rational vaccine design. Here, we investigated the Palo Alto/VarO and IT4/R29 and 3D7/PF13_003 parasites lines. VarO and R29 form rosettes with uninfected erythrocytes, a phenotype associated with severe malaria. They express an allelic Cys2/group A NTS-DBL1α1 PfEMP1 domain implicated in rosetting, whose 3D7 ortholog is encoded by PF13_0003. Using these three recombinant NTS-DBL1α1 domains, we elicited antibodies in mice that were used to develop monovariant cultures by panning selection. The 3D7/PF13_0003 parasites formed rosettes, revealing a correlation between sequence identity and virulence phenotype. The antibodies cross-reacted with the allelic domains in ELISA but only minimally with the Cys4/group B/C PFL1955w NTS-DBL1α. By contrast, they were variant-specific in surface seroreactivity of the monovariant-infected red cells by FACS analysis and in rosette-disruption assays. Thus, while ELISA can differentiate serogroups, surface reactivity assays define the more restrictive serotypes. Irrespective of cumulated exposure to infection, antibodies acquired by humans living in a malaria-endemic area also displayed a variant-specific surface reactivity. Although seroprevalence exceeded 90% for each rosetting line, the kinetics of acquistion of surface-reactive antibodies differed in the younger age groups. These data indicate that humans acquire an antibody repertoire to non-overlapping serotypes within a serogroup, consistent with an antibody-driven diversification pressure at the population level. In addition, the data provide important information for vaccine design, as production of a vaccine targeting rosetting PfEMP1 adhesins will require engineering to induce variant-transcending responses or combining multiple serotypes to elicit a broad spectrum of immunity.The clonally variant  Plasmodium falciparum malarial disease affects children and adolescents, while older subjects usually experience asymptomatic infections. This is thought to reflect the gradual acquisition of immunity by cumulated exposure to successive episodes of malaria caused by diverse parasite strains Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) variant adhesin that the parasite inserts into the membrane of the erythrocyte in which it develops. Surface expression of PfEMP1 bestows on the infected red blood cell (iRBC) the capacity to cytoadhere to host cells Plasmodium species and considered as an important contributor to falciparum pathology. PfEMP1 is encoded by a family of ∼60 var genes, each of which codes for a protein displaying specific binding and serologic characteristics. Successive expression of distinct var genes by clonal antigenic variation is a strategy used by the parasite to escape the host immune response and to establish a persistent infection, thereby optimising transmission.In sub-Saharan Africa, the main burden of var paralogs (with the exception of var3) differ in sequence, number of modules and flanking regulatory sequences, and are classified into groups A, B, and C, and B/A and B/C hybrid groups. Most var genes are located in the unstable sub-telomeric regions of the chromosomes and gene recombination during both meiosis and mitosis between these paralogs is an important mechanism of repertoire diversification var repertoires differ in their mosaic arrangement within individual modules, contributing to extensive sequence  diversity of the domain orthologs. Chimerism is particularly marked in var genes from group A , whose expression tends to be associated with severe malaria and with infection in young children with a poorly developed antibody response to the erythrocyte surface antigens PfEMP1 is a multi-modular adhesin, with an extracellular binding region consisting of a variable number of different (five types) Duffy-binding-like (DBL) and (three types) cysteine-rich interdomain region (CIDR) adhesion domains, and a more conserved cytoplasmic tail Factors regulating variant antigen diversification remain unclear but likely include a trade-off between diversifying antibody-driven selection and purifying function-constrained selection. Longitudinal follow-up studies indicate that malaria episodes tend to be caused by parasites expressing iRBC surface variants corresponding to gaps in the acquired antibody repertoire, suggesting negative selection by pre-existing immune responses 1 adhesion domains mediating rosetting , a cytoadherence phenotype associated with severe malaria in African children 1 domain, homology between the downstream domains of the three PfEMP1 variants is modest, allowing to explore the consequence of allelic polymorphism of the adhesion domain associated with non-orthologous domains.To gain insight into the immunological consequences of chimerism and allelic polymorphism, we compared the antibody response to a set of monovariant parasite lines that display an allelic form of a PfEMP1 adhesion domain. We chose to study the Palo Alto 89F5/VarO 1 adhesion domains, on one hand, and between the iRBC surfaces of the three lines, on the other, to study how allelic diversity impacts on antigenicity. We produced a soluble recombinant NTS-DBL1α1 adhesion domain from each variant. A prerequisite for the analysis of the iRBC surface serotype is the use of monovariant cultures in which all parasites express the same PfEMP1 variant. This had been achieved for VarO by positive selection using variant-specific antibodies raised to the recombinant NTS-DBL1α11 domain cross-reacted with their allelic forms by ELISA but displayed strictly variant-specific iRBC surface reactivity and variant-specific rosette disruption. Analysis of naturally acquired antibodies from humans exposed to P. falciparum living in Dielmo, Senegal, showed positively correlated ELISA reactivities to the recombinant NTS-DBL1α1 domains. Here also, however, reactivity with the iRBC surface was variant-specific and the age-acquisition pattern differed for the three variants. Absorption of sera with the recombinant domain showed that the anti-NTS-DBL1α1 antibodies represented a large fraction of the iRBC surface reactivity. Taken together, these data demonstrate that the related rosette-forming parasites display variant-specific iRBC-surface epitopes. Thus, PfEMP1 allelic variation causes antigen diversification and participates in immune evasion at the population level. In addition, these data provide important information for vaccine development since a strategy targeting rosetting will need either to combine multiple serotypes or to engineer the antigen so as to broaden specificity.We set out to analyse the serological relationships between the three NTS-DBL1αvar genes with a similar gene architecture and a high sequence identity score across the entire gene length, although the best identity scores for individual domains were found in the subset of group A genes 1 domain mediates rosetting, we focussed further work on genes harbouring the orthologous domain, namely PF13_0003  and R29 (IT4var9)  from in the 3D7 and IT4 genomes, respectively. Both R29 and PF13 belong to the group A/UpsA var genes with a typical PoLV Group 1 (2 cysteines/MFK*) sequence signature 1 protein sequence. The downstream protein architecture of VarO, R29 and PF13 differs in the number and type of DBL domains, and the presence of a second CIDR domain in R29 and PF13 that is absent in VarO (1 have limited homology with the downstream domains of R29 and PF13 (<30%), apart from the DBL2β and DBL5β domains of PF13 (38% identity)  . Thus, V1 domain was produced in E.coli for R29 and PF13 with a C-terminal His-tag. Protein purity was checked by SDS-PAGE analysis under reducing and non-reducing conditions and immunoblotting. The proteins migrated at the expected molecular mass . The endpoint ELISA titre to the immunogen was >6×10−7 , similarCross-reactivity of the various mouse sera was analysed on the recombinant VarO, R29 and PF13 domains by ELISA and compared to the reactivity with a domain from a paralog from a distinct PoLV of DBL1α. We chose NTS-DBL1α PFL1955w, encoded by an UpsB/C type paralog, with a Cys4, group 4 DBL1α domain and displaying 32–33% identity with the VarO, R29 and PF13 domains . The rec1-VarO sera cross-reacted with the R29 and PF13 alleles, with 30- and 16-fold reduction of the 95% titre, respectively; reaction with PFL1955w was quite modest as both signal and titre were low (E. coli NTS-DBL1α1-VarO proteins (data not shown). Cross-reactivity was not due to reaction with the His-tag present in the VarO, R29 and PF13 recombinant proteins, as the sera failed to react with e.g. the His-tagged CIDR-VarO construct . The anti-R29 sera cross-reacted with the VarO and PF13 alleles with a 30- and 80-fold reduced 95% titre, respectively, and reacted poorly with PFL1955w  were isolated from animals immunised with the R29 or PF13 recombinant domain and used bi-monthly for routine enrichment of R29-expressing IT4 cultures or PF13-expressing 3D7 parasites following the procedure described for VarO 1 antibodies was analysed using the VarO, R29 and PF13 monovariant cultures. Although each antiserum reacted with its cognate iRBC-surface, no cross-reactivity was observed with the heterologous cultures expressing an allelic form of the NTS-DBL1α1 domain . Serum samples of all individuals (0.5 to 85 year old) were analysed. The threshold for positive recognition was set from reactivity of non-immune French adults. Seroprevalence to the R29 and PF13 recombinant domains was very high, with 96.5% [95% confidence interval (CI) 94.07–98.9] and 99.1% [97.9–100] positive sera, respectively, compared to 87.7% [82.8–91.6] positive sera to VarO. As for VarO 1 seroconversion was observed in the 2–5 y age group    revealed1 domains in ELISA, with higher titres and values on the allelic forms than on the unrelated group C NTS-DBL1α. It appears that the group A/Cys2 DBL1α1 antigens studied here form a group of related serotypes, although more work is needed with additional domains from other subgroups to substantiate this conclusion. Accordingly, ELISA could potentially be used to define serogroups of related orthologs/alleles or paralogs. Similar ELISA cross-reactivity was reported with mouse or rabbit sera obtained by immunisation with recombinant domains from var2csa1 domains in the Senegalese community suggest that ELISA cross-reactivity occurs with human sera as well, although this was not formally tested by competition ELISA. Interestingly, limited intra-genome cross-reactivity of human antibodies with a panel of DBL1α and CIDR domains encoded by the 3D7 repertoire has been observed by competition ELISA but this was restricted to a few domains that shared more than 50% sequence identity Another important lesson of the present work is that the analysis of ELISA-reactive and surface-reactive antibodies reveals different aspects of the antibody response. Indeed, although no surface cross-reactivity could be observed, there was clear evidence of cross-reactivity between the recombinant NTS-DBL1αfalciparum pathology, although children from Dielmo are usually protected against severe malaria from a very young age - earlier than the mean age for acquisition of surface-reacting antibodies, including antibodies to VarO. While a different study design is needed to analyse the potential contribution of the three variants to severe malaria, we can conclude that acquisition of specific antibodies coincides with mounting protecting response against malaria disease. Although, the precise mechanism of this immunity is still unclear, the present data are consistent with immunity against clinical malaria being brought about by the acquisition of a large repertoire of antibodies preventing expansion of a large number of antigenic variants.The VarO, R29 and PF13 surface serotypes were quite frequent in the Senegalese community. They were acquired with different kinetics, the earliest response being apparently to VarO. Antibodies to R29 appear to be acquired later than antibodies to VarO or PF13. The low seroprevalence in young children is consistent with findings in Kenyan children 1 domains that are correctly folded, as they met not only different biophysical criteria 1-varO.indicates that the NTS region is an intrinsic part of the domain. The recombinant DBL1α domains used by other groups to assess the acquired antibody response lacked the NTS moiety and in some instances did not include a critical last pair of cysteine residues 1-R29 in Dielmo is in line with seroprevalence against DBL1α1-R29 in areas of high endemicity We studied the domain-specific response using recombinant NTS-DBL1α1-VarO in other parasite genomes are involved in rosetting as well. Our data also illustrate antigenic switching occurring in the IT4/R29 line, since after several months of culture including weekly enrichment for rosettes, surface reactivity to NTS-DBL1α1-R29 had dropped below 20%. This reflects switching expression from the dominant R29/IT4var9 gene to another rosette-mediating paralog. Rosetting is encoded by a subset of var paralogs whose exact number is unknown. To circumvent the consequences of antigenic switching within the rosetting population in each of the three lines, we developed specific mAbs to pan the culture at regular intervals.Of note, sequence identity of PF13 with VarO and R29 predicted the cytoadherence phenotype, as the three lines indeed formed dextran sulfate- and heparin-sensitive rosettes, suggesting that orthologs of NTS-DBL1αvar genes var repertoire var2csa, since it is single copy in most lines and the only gene associated with adhesion to CSA var2csa-expressing parasites, serology studies using monovariant lines are scarce. Analysis of seroreactivity of Kenyan sera to the monovariant A4u line panned using a specific mAb showed an age- and transmission-dependent acquisition of surface seroreactivity Surface seroreactivity to patient isolates has been extensively used to analyse the acquisition of responses falciparum malaria Both naturally acquired antibodies and mouse antibodies to the recombinant domains failed to show overlapping surface serotypes between the three related group A/Cys2 variants studied. This has major implications for vaccine development, as it indicates that specific strategies should be developed in order to target this particular subgroup considered serologically more conserved and cross-reactive than variants expressed in older children E. coli Rosetta-gami™ 2 (DE3) (Novagen). Protein expression was induced with 0.1 mM isopropyl-β-D-thio-galactopyranoside for 4 h at 30°C followed by 16 h at 20°C. Cells were resuspended in 20 mM Tris-HCl, 200 mM NaCl, pH 8 in presence of PMSF (Fluka) and Protease inhibitor cocktail complete EDTA-free (Roche) and lysed mechanically . Soluble proteins were purified by metal affinity  followed by size exclusion chromatography . The recodoned PFL1955w domain (without mutations or His tag) was cloned in pMAL-c2X (New England BioLabs) and expressed in E. coli SHuffle™ Express (New England BioLabs) as an MBP-fusion protein Production of the VarO soluble recombinant domain has been described Outbred mice were immunised as described P. falciparum parasites were cultured in human O+ erythrocytes −1, Abcys). For 3D7 (obtained from MR4), weekly rosette enrichment for 2.5 months yielded a rosette-forming parasite culture. After rosette dissociation with 10 µg.mL−1 dextran sulfate (Sigma) incubated for 30 min at 37°C, mature iRBC were magnetically selected on a CS column . For cell sorting, the iRBC pellet was resuspended in PBS, 2% foetal calf serum (FCS) and stained with anti-R29 or -PF13 sera , and a goat anti-mouse IgG Alexa fluor 488 conjugate (dilution 1/1000). Surface-positive iRBCs were sorted by flow cytometry  and sub-cultured in O+ erythrocytes. Rosette formation was then selected by weekly enrichment and bi-monthly panning with specific mAbs as described Serum samples from 235 villagers (.5–85 years old) living in Dielmo  were used. Experimental conditions for ELISA, surface immunofluorescence, flow cytometry and rosette-disruption assay using monovariant parasite cultures were as described −1 dextran sulfate. An equal volume of differentially stained iRBC was mixed and incubated 1 h at 37°C with human sera  and Hoechst dye (dilution 1/1000). Immune agglutinates were visualised using fluorescent microscopy (Leica). An iRBC agglutinate was defined as a clump containing more than three iRBC. Each serum was tested in duplicate in two independent experiments.After rosette disruption using dextran sulfate, iRBC were magnetically enriched. The iRBC membrane was labelled using PKH67 or PKH26 (Invitrogen) following the manufacturer's instructions, followed by three washes with PBS-2% FCS. Cells were resuspended in complete culture medium, 10 µg.mL10 µg of the VarO protein was absorbed on 10 µL of TALON resin (Clontech) for 1 h at 4°C. Unbound protein was removed by three washes with 20 mM Tris/HCl, pH 8, 200 mM NaCl. The resin was then incubated for 1 h at 4°C with human serum samples . The depleted sera were collected by centrifugation. Depletion was verified by ELISA.1-varO. The result was displayed using PyMOL The CONSURF web server ELISA OD and surface IFA values were log-transformed. The Student's t-test and Wilcoxon's nonparametric test were used to compare normal and non normal distributions of log-transformed data. Surface-reactive and domain-reactive antibodies were correlated using Spearman rank correlation test. Data were analyzed with STATA software  and P<0.05 was considered to be statistically significant.http://webcampus.pasteur.fr/jcms/c_98517/protocoles-usuels) and complied with the European Union guidelines for the handling of laboratory animals . The procedures were approved by the Institut Pasteur animal care and use committee. Animal care and handling was approved by the Ministère de l'Agriculture et de la Pêche  and the protocols and procedures used by the Direction départementale des services vétérinaires de Paris (Ref. RL- 07031395-30701147) isued on 29th August 2007 to OMP.This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Institut Pasteur  and by the National Council on Health Research of Senegal (reference n° 05 MSP/DS/CNRS in February 2008). Both review boards approved the verbal consent procedures. The project objectives were carefully explained to the assembled village population and the village head, the spokesperson for the women, for the younger generation, the religious authority of the village gave their informed consent. Individual verbal informed consent was then obtained, as most participants were illiterate, at each stage of the study. In formed consent was obtained individually from all participants (parents or ward of children) and was recorted in the individual villager's file. The possibility to withdraw from the project at any time was stated.Figure S1PfEMP1 protein sequence analysis. Pairwise amino acid sequence identity between domains of similar subtype from the Palo Alto/varO, IT4/R29, 3D7/PF13_0003 and 3D7/PFL1955w PfEMP1 proteins.(PDF)Click here for additional data file.Figure S2SDS-Page analysis of the recombinant domains. The recombinant NTS-DBL1α domains of VarO , R29 , PF13_0003  and PFL1955w  were migrated under reducing (even lanes) or non-reducing (odd lanes) conditions.(PDF)Click here for additional data file.Figure S3Cross-reactivity between allelic domains measured by ELISA. ELISA 50% and 95% titres of sera from mice immunised with individual recombinant NTS-DBL1α1 domains tested against the recombinant domains (top chart). Reduction factor of reactivity with the heterologous domains (bottom chart).(PDF)Click here for additional data file.Figure S4Rosette-forming, surface-positive sorted iRBC used to derive IT4/R29 and 3D7/PF13_monovariant lines. Microscopic visualisation (left) and immunostaining (right) of rosettes from the IT4/R29 (A) and 3D7/PF13_0003 (B) monovariant cultures obtained after cell sorting with mouse sera raised to the recombinant R29 and PF13_0003 domains respectively.(PDF)Click here for additional data file.


Escherichia coli, comprise a superfamily of enzymes that synthesize and/or hydrolyze the alarmone ppGpp, activator of the “stringent” response and regulator of cellular metabolism. The classical “long” RSHs Rel, RelA and SpoT with the ppGpp hydrolase, synthetase, TGS and ACT domain architecture have been found across diverse bacteria and plant chloroplasts, while dedicated single domain ppGpp-synthesizing and -hydrolyzing RSHs have also been discovered in disparate bacteria and animals respectively. However, there is considerable confusion in terms of nomenclature and no comprehensive phylogenetic and sequence analyses have previously been carried out to classify RSHs on a genomic scale. We have performed high-throughput sensitive sequence searching of over 1000 genomes from across the tree of life, in combination with phylogenetic analyses to consolidate previous ad hoc identification of diverse RSHs in different organisms and provide a much-needed unifying terminology for the field. We classify RSHs into 30 subgroups comprising three groups: long RSHs, small alarmone synthetases (SASs), and small alarmone hydrolases (SAHs). Members of nineteen previously unidentified RSH subgroups can now be studied experimentally, including previously unknown RSHs in archaea, expanding the “stringent response” to this domain of life. We have analyzed possible combinations of RSH proteins and their domains in bacterial genomes and compared RSH content with available RSH knock-out data for various organisms to determine the rules of combining RSHs. Through comparative sequence analysis of long and small RSHs, we find exposed sites limited in conservation to the long RSHs that we propose are involved in transmitting regulatory signals. Such signals may be transmitted via NTD to CTD intra-molecular interactions, or inter-molecular interactions either among individual RSH molecules or among long RSHs and other binding partners such as the ribosome.RelA/SpoT Homologue (RSH) proteins, named for their sequence similarity to the RelA and SpoT enzymes of  Escherichia coli, regulate the concentration of the alarmone ppGpp  in response to various environmental cues such as temperature change Bacteria use several modified nucleotides as intracellular messengers, such as cAMP, c-di-GMP, c-di-AMP, cGMP, and ppGpp, with the latter being the first to be identified and one of the best studied E. coliin vivo to ppGpp by the enzyme pppGpp phosphohydrolase (GPP) in vitroE. coli has a second RSH protein, SpoT, which is bifunctional, with weak ppGpp synthetic activity in its SYNTH domain The first type of stringent response to be investigated was the RelA-mediated response to amino acid limitation in Vibrio cholera (RelV) Along with long RSHs, shorter and specialized RSHs (“short RSHs”) that contain either the SYNTH or the HD domain have been identified. SYNTH domain-only proteins have been found in firmicute bacteria  VibrioJust as there are many different RSH proteins, there are many targets of ppGpp in the cell. Thus, modulation of the intracellular ppGpp concentration acts as a hub, regulating transcription Streptococcus equisimilis (“RelSeq”) has been determined in silico analyses are valuable for predicting direct links between sequence and function, generating hypotheses that can be tested biochemically. By comparative analysis of patterns of sequence variation among RSHs, we have identified sites that we propose are involved in transmitting signals from the CTD region or from other interacting molecules. We suggest the Rel, RelA and SpoT system is an interesting case study for examining the fate of gene duplicates, as they show domain specific features of subfunctionalization and specialization.We have identified and classified RSHs and their domain architectures from available genomes across the tree of life to retrace the evolution of RSH form and function using the structural and functional information available for these proteins. The crystal structure of the Rel SYNTH and HD domains from http://www.phylo.org/sub_sections/portal/) with the PROTCATWAG model, with 100 bootstrap replicates. From this starting tree, 15 apparent paralogous subgroups (distinct clusters of sequences) were identified. Hidden Markov Models (HMMs) were created with HMMER 3.0b2  searching against completed genomes. For the first-pass survey, the full alignment of the synthetase domain was downloaded from Pfam , the DOE Joint Genome Institute , and the Cyanidioschyzon merolae Genome Project   domain from Pfam (PF01966) was also added at this stage in order to identify HD-containing proteins that may not necessarily be accompanied by a RelA_SpoT synthetic domain. An E value cut off of E−2 was also used for genome searches using the HD domain HMM. From the results of phylogenetic analysis as described above, we classified sequences into six HD-only subgroups, from which sequences were aligned and HMMs created. From the results of these searches, the HD and RelA_SpoT domain HMMs were remade and genomes rescanned with these and the 30 subgroup HMM models. In order to avoid confusion arising from the rather ambiguous name “RelA_SpoT” as used in Pfam for the ppGpp syntetase domain, the remade “RelA_SpoT” HMM is henceforth referred to as the SYNTH domain HMM.The predicted proteomes from 1072 organisms with complete genomes were downloaded from NCBI , additional NTD and CTD domains were used in this alignment to help resolve relationships in the long RSH part of the tree, with missing data coded for the SAS sequences in short poorly aligned regions upstream and downstream of the SYNTH domain. Final dataset dimensions were 670 amino acid positions from 1706 sequences for the SYNTH-containing data set and 168 amino acid positions from 1535 sequences for the HD-containing dataset. Phylogenetic analysis was also carried out separately on sequences that contain both the SYNTH and HD domains . Inspection of the final trees allowed classification of all the sequences into 30 subgroups.After examining the results of the HMM scan, the results were filtered in order to remove spurious SYNTH and HD domain hits to distant relatives. These spurious hits include the RNA binding domain of DEAD-box helicases in the case of the HD domain, and ribosomal protein L17 and the NADP-binding domain of glutamyl tRNA reductase in the case of the SYNTH domain. The new E value filtering thresholds were set at EIn order to examine branching patterns of subsets of the data more clearly, additional, separate phylogenetic analyses were conducted on the chloroplast RSHs (470 amino acid positions from 66 sequences) and the Mesh1/Mesh1-L subgroups (179 amino acid positions from 99 taxa). Phylogenetic analysis was carried out using RaxML as above, and with Bayesian inference, using MrBayes version 3.1.2 Consensus sequences from aligned sequences were generated using the Consensus Finder Python script to generate consensus alignments for analyzing differential conservation among subgroups http://bioinf.cs.ucl.ac.uk/disopred) http://www.cbs.dtu.dk/services/TargetP/) Secondary structure and regions of structural disorder were predicted with Psipred and Disopred respectively, using the Disopred server , suggesting it arose from gene duplication of Rel[HS] within this class. Its long branch length in the HD domain tree  for being a sister group of an RshD[HS] protein of Pelobacter carbinolocus. RshD[HS]s are second copies of Rel[HS] that are found in four δ-proteobacteria . Of these, only Desulfotalea psychrophilia and Syntrophobacter fumaroxidans form a significantly supported monophyletic group (100% MLBP). However, these sequences are classified as a subgroup by virtue of all being second copy RSHs from the same class of bacteria. As RshC[HS] and RshD[HS] have no support for their positions in the tree, they may be divergent paralogs, or could be xenologues, second copies of Rel[HS] that have originated via horizontal gene transfer (HGT).To better resolve relationships in the long RSH part of the tree using more homologous amino acid positions, maximum likelihood analysis was carried out on long RSHs (carrying at least the SYNTH and HD domains) alone. In this tree, branch support is 83% MLBP for RelA[hS] monophyly and 83% MLBP for SpoT[HS] monophyly  is available Seq and positions 49–59 in The consensus alignment of the RSHs allows the identification of differentially conserved sites indicative of a shift in functional constraints. As the crystal structure of the NTD (HD and SYNTH domains) of In the SYNTH domain, many sites are conserved across Rel/RelA/SpoT   and S3. uropaea, , suggesturopaea, .Mycobacterium, Aquifex and cyanobacteria) as an outgroup (Four subgroups of ppGpp synthetases were found in plants: Rsh1-4 . In the outgroup . In thisoutgroup . HoweverCyanidioschyzon merolae) that inherited their chloroplasts through vertical descent C. merolae RSH Rsh4[S] also groups with strong support  with the other Rsh4s, although it does not carry an EF-hand domain, and its HD domain is degrading. Thus, the EF-hand domain may have fused to an Rsh1-like protein in the archaeplastida lineage after the divergence of C. merolae. The boundary between the Rsh1 and Rsh4 groups is uncertain due to the presence of divergent algal Rsh1/4[S]s with degrading HD domains. The chromalveolates Aureococcus and Emiliania have some long branched HD-degrading RSHs that cluster with Rsh4[HS] with reasonable support , suggesting these may have also originated from the Rsh duplication that gave rise to Rsh4[HS], before the secondary endosymbiosis event  and there is strong support for the monophyly of these groups together  carry an N terminal domain of unknown function (“DUF429” in Pfam). DUF429 usually occurs on its own as a single domain protein in bacteria and archaea, but in a handful of bacteria, it is found in combination with other domains involved in nucleoside metabolism: the “Nudix” hydrolysis of nucleoside diphosphates domain, and the phosphomethylpyrimidine kinase domain, which is a phosphotransferase of the thiamine pyrophosphate (TPP) synthesis pathway.In comparison to the long RSHs, the taxonomic distributions of the SASs and SAHs are in general sporadic, with their presence and absence spread across the diversity of RSH-encoding bacteria . Among oMonosiga brevicollis and the fungus Cryptococcus neoformans. It was, however not identified in any other fungi. Scans of eukaryotic Mesh1[H]s on the TargetP server Methanococcoides burtonii, making it one of the few RSHs to be found in archaea.In eukaryotes, the only widely spread subgroup is Mesh1[H]. This is also the only previously reported SAH for any organism; surprisingly, no bacterial SAHs have been reported before the seven subgroups we describe here. Phylogenetic analysis of the Mesh1[H] and Mesh1-L[H] subgroups show eukaryotic Mesh1 groups tightly with a clade of α-, β- and δ-proteobacteria , in which 40 sequences matched the rickSpo[H] HMM , which in some lineages has experienced extensive proliferation, associated with transposable elements o[H] HMM  and S2 , and the algae encode Rsh1[HS] (plus a divergent Rel[HS] subgroup member in the case of T. pseudonana) as hydrolases, the fungi do not carry an RSH hydrolase. Similarly, one divRel[S] was found in the archaeon Methanosarcina acetivorans, also lacking an SAH. SAHs were found however in the archaeon Methanosarcina barkeri (divSpo[H]), Methanococcoides burtonii (Mesh1-L[H]), Natronomonas pharaonis (pbcSpo[H]) and uncultured methanogenic archaeon RC-1 (pbcSpo[H]), none of which carry an SAS  were found in the fungi y an SAS  and S2. Streptomyces), Y328, L331, G332, H335 and F345. These residues overlap with a differentially conserved motif of RelA[hS] and SpoT[HS]; RelA[hS] has a conserved acidic motif (EFDD) that is differentially conserved as basic RFKD in Rel[HS]/SpoT[HS]  that were identified in the previous evolutionary analysis of this protein superfamily Bifunctional Rel[HS] is by far the most widely distributed of the RSH proteins, being found in all phyla except members of the PVC superphylum, β- and γ-proteobacteria, the latter two of which encode the Rel[HS] duplicates RelA[hS] and SpoT[HS] . However, all these taxa have close relatives that are also pathogenic but carry RSHs. For example, Mycoplasma genitalium, which has one of the smallest genomes of free-living organisms, carries Rel[HS]. Thus, loss of the RSH system is possible in pathogenic bacteria, but rare, unless the bacterium is an obligate intracellular parasite.Other bacteria that lack RSHs are mostly obligate intracellular parasites or endosymbionts, as previously reported The co-distribution of RelA[hS] and SpoT[HS] in γ- and β-proteobacteria that lack Rel[HS] suggest they originate from a duplication of Rel[HS] in the proteobacterial lineage after the divergence of α-proteobacteria, the most closely related class to β- and γ-proteobacteria. The advantage of having a rapid ppGpp on/off switch may have driven the subfunctionalization of RelA[hS] and SpoT[HS] into mostly synthetic (RelA[hS]) or hydrolytic (SpoT[HS]) proteins following duplication of the ancestral Rel[HS]. In bacteria that encode RelA[hS] and SpoT[HS], concentrations of the synthetase and hydrolase domains are not kept equal as in the case of a single bifunctional Rel[HS] protein.Mycobacteria, where noise in Rel[HS] expression is further amplified via a positive loop acting on the mprAB operon The ppGpp-mediated regulatory circuits just as any sensory network, are challenged by noise originating from the stochastic nature of the chemical reactions constituting life E. coli is known to have interaction partners distinct from RelA[hS], such as the acyl carrier protein Apart from the potential kinetic advantages of encoding synthetic and hydrolytic activities in different polypeptides, the subfunctionalization of RelA[hS] and SpoT[HS] also allows bacteria to sense different environmental cues through different intermolecular interactions. SpoT[HS] in E. coli, intracellular ppGpp concentration is the primary factor controlling growth rate The expansion of SASs and SAHs possibly fine-tunes sensitivity and speed of reactions to the “classical” stringent response cues: different SASs and SAHs can be transcribed from different promoters, adding a transcriptional level of regulation to stringent response machinery. The SASs and SAHs may be expressed in response to different environmental triggers, so acting synergistically. Independent expression of long and small RSHs from multiple genes may also be beneficial to bacteria on a population level. In The core RSH set can accommodate addition of various combinations of SASs and SAHs. While 92% of the bacterial genomes sampled here carry Rel[HS] or SpoT[HS], just 44% of bacteria have Rel[HS] alone, SpoT[HS] alone, or RelA[hS] plus SpoT[HS] without any other RSHs. The SASs and SAHs have very scattered distributions across bacteria, with multiple subgroups often present in the same organism, and with the RSH complement often differing widely within families of bacteria, and sometimes within genera . This suAcidithiobacillus ferrooxidans, Candidatus Ruthia magnifica, Candidatus Vesicomyosocius okutanii, Methylovorus SIP3, and Nitrosomonas europaea) having only SpoT[HS] and no RelA[hS]. SpoT[HS] has some synthesis ability, explaining its presence without RelA[hS], however, it is also possible  for an SAH to be present alone in bacteria (Rickettsia conorii). Thus, although we have identified more SAS than SAH subgroups, the distribution of the HD domain is wider than the SYNTH domain. Knock-out experiments in E. coli corroborate well with our observations: strains with knock-outs of RelA[hS] or simultaneous knock-out of RelA[hS] and SpoT[HS] are viable, whereas knock-out of SpoT[HS] is lethal ΔspoT strains Treponema denticola is the only identified case of a bacterium that encodes no Rel[HS], but has SAS and SAH proteins that seem to have originated from HGT.The analysis of naturally occurring RSH systems in bacteria suggests certain organizational rules. Firstly, no bacteria with RelA[hS] or an SAS alone are observed, indicating that loss of the hydrolytic component (SpoT[HS] or SAH) is prohibited, presumably because that would result in run-away responses leading to cell death. Secondly, loss of the major synthetic component, is rare but possible, with several organisms  Animals have been found to carry Mesh1[H], an SAH Dictyostelium, Aspergillus and Gibberella), suggesting SASs may be particularly useful to eukaryotes in this environment, possibly by producing ppGpp as an extracellular nucleotide signal, similar to the use of cAMP in DictyosteliumDictyostelium has previously been reported, although others have failed to repeat this finding Similarly, the most likely explanation for the sporadic distribution of divRel[S] SASs in eukaryotes and archaea is HGT from bacteria. Phylogenetic relationships that might support possible donors of these RSHs unfortunately lack statistical significance. However, it is interesting to note that all of the recipient eukaryotes spend all or part of their life cycles in the soil  have SAHs apparently without SASs. These organisms may encode non-homologous ppGpp synthetases, as previously suggested Streptomyces morookaensis−7 for the presence of the SYNTH domain.SAHs can also occur alone in eukaryotes. Although the SAH Mesh1[H] is widespread in animals, our search did not identify any SASs in this kingdom. Similarly, five euryarchaea  in vitro system. When long RSHs are in close proximity, in cis cross talk may occur in trans through the interaction of the NTD of one protein molecule with the CTD of another. Thus, it is unclear whether, or just how significantly, oligomerization occurs in vivo.RSHs have been shown to regulate their catalytic activity by interacting with themselves in trans and in cis interactions in RSHs. Both types of interactions seem to be mediated by the same conserved Cys residues E. coli RelA[hS], C612, D637, and C638 of the CC domain are involved in dimerization, with mutations of D637, and C638 residues negatively affecting ppGpp synthetic activity Our hypothesis that oligomerization is a consequence of inter-domain cross talk is supported by the overlap in residues involved in both the Mycobacterium tuberculosis (“RelMtb”) and RelA[hS] from E. coli, this motif was shown to confer G nucleotide phosphorylation state specificity, with EXDD (in RelA) and RXKD (in Rel[HS] and SpoT[HS]) conferring GDP and GTP preference, respectively Single-domain RSHs are not capable of inter-domain cross-talk, thus their sequences are expected to lack the conserved residues of long RSHs that mediate these interactions in the NTD region. On the basis of differential conservation patterns, we propose 23 residues that are potentially involved in mediating inter-domain cross-talk in the long RSHs. These are 11 residues of the SYNTH domain and 12 of the HD domain . Of the Gene duplication results in a redundant copy of the original gene that is free from selective pressure and able to diverge in sequence In the Rel[HS], RelA[hS] and SpoT[HS] system, we see three main fates of the genes and their synthesis and hydrolysis functions following duplication . The firSuch refinement of RelA[hS] may involve its ppGpp synthesis function. The presence of sites that are strongly conserved in the RelA[hS] SYNTH domain and differentially conserved for a different amino acid or weakly conserved in Rel[HS]/SpoT[HS] suggests there has been some evolutionary fine-tuning of the synthetase domain in RelA[hS]. Specialization of RelA[hS] and SpoT[HS] probably also extends into the CTD: there are differentially conserved and rate shifting sites in all domains of the CTD , and difThe loss of hydrolysis function in RelA[hS] raises the question of why the HD domain has not been lost in its entirety in this protein. Although poorly conserved, the supposedly non-functional HD domain of RelA[hS] has been maintained at least structurally throughout the evolution of β- and γ-proteobacteria. As the sites that are conserved in RelA[hS] in the HD domain appear to be important for structural integrity , this inThe current analysis classifies RSHs into 30 RSH subgroups across the tree of life, 19 of which were previously unreported. These include previously unknown RSHs in archaea, fungi and Dictyostelid amoebae in addition to new bacterial RSHs with unusual domain structures. All these proteins now can be subjected to follow-up experimental analysis. The classification introduced in this study provides a unifying nomenclature for the RSH superfamily, resolving terminological confusion within the field . We suggM. tuberculosis Rel[HS] (“RelMtb”) The wide variety of SAS and SAH combinations that are present in bacterial genomes leads us to hypothesize that the small RSHs are accessory genes that are easily gained and lost during evolution, allowing bacteria to dynamically rewire and refine their stress responses in a lineage-specific manner. The long RSH component on the other hand represents a core ribosome-interacting hub that has evolved conservatively since its origin early in bacterial evolution, maintaining its six-domain structure and specializing some aspects of its function on a conserved structural frame. Systems biology investigations of different RSH systems similar to that undertaken for in vivo experiments will allow the wealth of the evolutionary and sequence data we have collated to be used to link sequence, structure and function.The prolonged conservation of the long RSHs RelA[hS] SpoT[HS] and Rel[HS] indicates their importance in the cell, however there is still much that remains unknown about their function. In order to understand how the long RSHs interact with their binding partners, we need more structural data such as crosslinking, cryoEM and X-ray structures of RSH complexes, as well as full length RSH proteins themselves. This information in combination with biochemical and Figure S1Phylogeny generated by DIVERGE for the analysis of site-specific rate shifts. RelA and SpoT are shown in pink and blue, respectively.(PDF)Click here for additional data file.Figure S2Maximum Likelihood phylogeny of the long RSHs using all alignable domains. The tree is generated from 699 amino acid positions. Bootstrap support values greater than 70% are shown on branches. All subgroups are labeled except for the paraphyletic Rel, which is shown in purple. Major taxonomic groups are indicated on the right.(PDF)Click here for additional data file.Figure S3Sequence alignment of RelA[hS] and SpoT[HS]/[Hs] from Escherichia coli, Psychrobacter arcticum and Acinetobacter baumannii. Domains are indicated below the alignment with colored lines, as per the coloring of domains in (PDF)Click here for additional data file.Figure S4Consensus sequence alignment of plant RSHs, RelA and SpoT. Domains are shown below the alignment with colored lines, as per the coloring of domains in (PDF)Click here for additional data file.Table S1Excel file showing all RSHs, the organisms that encode them, their ID numbers and database source.(XLSX)Click here for additional data file.Table S2Excel file showing the RSH complements of all sampled organisms, organized by taxonomy.(XLS)Click here for additional data file.


The programmed cell death 4 (PDCD4) protein is induced in animals during apoptosis and functions to inhibit translation and tumor promoter-induced neoplastic transformation. PDCD4 is composed of two MA3 domains that share similarity with the single MA3 domain present in the eukaryotic translation initiation factor (eIF) 4G, which serves as a scaffold protein to assemble several initiation factors needed for the recruitment of the 40S ribosomal subunit to an mRNA. Although eIF4A is an ATP-dependent RNA helicase that binds the MA3 domain of eIF4G to promote translation initiation, binding of eIF4A to the MA3 domains of PDCD4 inhibits protein synthesis. Genes encoding PDCD4 are present in many lower eukaryotes and in plants, but PDCD4 in higher plants is unique in that it contains four MA3 domains and has been implicated in ethylene signaling and abiotic stress responses. Here, we examine the evolution of PDCD4 in plants.In older algal lineages, PDCD4 contains two MA3 domains similar to the homolog in animals. By the appearance of early land plants, however, PDCD4 is composed of four MA3 domains which likely is the result of a duplication of the two MA3 domain form of the protein. Evidence from fresh water algae, from which land plants evolved, suggests that the duplication event occurred prior to the colonization of land. PDCD4 in more recently evolved chlorophytes also contains four MA3 domains but this may have resulted from an independent duplication event. Expansion and divergence of the PDCD4 gene family occurred during land plant evolution with the appearance of a distinct gene member following the evolution of basal angiosperms.The appearance of a unique form of PDCD4 in plants correlates with the appearance of components of the ethylene signaling pathway, suggesting that it may represent the adaptation of an existing protein involved in programmed cell death to one that functions in abiotic stress responses through hormone signaling. Following transcription and processing of an mRNA, the ribosome is responsible for performing protein synthesis. Although the bacterial 30 S ribosomal subunit can identify the initiation codon through base-pairing between the 3′-end of its16 S ribosomal RNA subunit and the Shine-Dalgarno sequence upstream of the initiation codon, the 40 S ribosomal subunit of the eukaryotic 80 S ribosome requires several translation initiation factors (eIFs) for its binding to an mRNA and to identify the initiation codon . The heleIF4A binds to two regions of eIF4G that fold into HEAT  domains, characterized by the presence of antiparallel α-helical hairpins known as HEAT repeats ,13. One in vivo[In addition to eIF4G, other proteins containing an MA3 domain have been described. The programmed cell death 4 (PDCD4) protein is characterized by the presence of two tandem MA3 domains that fold into a subtype of HEAT domains. The N- and C-terminal MA3 domains of human PDCD4 contain four and three helical hairpins, respectively, while eIF4G-MA3 contains five helical hairpins . From a in vivo,26,28. Pin vivo,30. The in vivo. Althougin vivo. A singlArabidopsis thaliana ethylene receptors, ETR2 and EIN4, as well as EIN2, a downstream component of the ethylene signaling pathway required for the induction of ethylene responses [PDCD4 homologs are present in lower eukaryotes and plants although no PDCD4 homolog has been reported in yeast. PDCD4 proteins in higher plants are unique in that they contain four tandem MA3 domains. Higher plants also appear to lack a two MA3 domain PDCD4 homolog. One such four MA3 domain PDCD4 protein (ECIP1) was reported to bind esponses . Loss ofesponses . Thus, leIF4G contains a single MA3 domain which in plants, is present at the C-terminus as plant eIF4G lacks the HEAT-3/Mnk-binding domain found in animal eIF4G Figure . Plants Arabidopsis thaliana[A. thaliana genome but the A. thaliana gene family actually contains four members: MAT5 (At4g24800), MAT6 (At5g63190), MAT7 (At3g48390), and MAT8 (At1g22730). Comparison of the MA3 domains from MAT5-8 with eIF4G and eIFiso4G revealed conservation of several residues among all MA3 domains but only four residues were invariant in all MA3 domain proteins  suggesting that this gymnosperm species also expresses a four MA3 domain PDCD4 homolog. In contrast, only PDCD4 homologs containing two MA3 domains were found in extensive database searches of animal and lower, non-plant eukaryotic species, indicating that the four MA3 domain PDCD4 homolog is unique to plants.PDCD4 homologs are present throughout angiosperm species and the proteins exhibit considerable conservation both in domain organization and primary sequence Figure . All higi Figure , demonsts Figure . A partiChlamydomonas reinhardtii and Volvox carteri representing the Chlamydomonadales but only two MA3 domain PDCD4 homologs in algal species of the Mamiellales such as Micromonas and Ostreococcus  are thought to have derived from a common ancestor that likely contained a two MA3 domain PDCD4 form of the protein that was maintained in older algal lineages  but underwent duplication during later chlorophyte evolution.As land plants did not evolve from Chlamydomonadales but rather from charophytes , the appearance of a four MA3 domain PDCD4 homolog in Chaetosphaeridium globosum, representing the Coleochaetophyceae. Although the 5′-end of the cDNA is truncated, the predicted protein contains MA3 domains 2–4 and, unlike PDCD4 present in C. reinhardtii and V. carteri, the C. globosum homolog shares similarity to the regions between the MA3 domains of land plant PDCD4 homologs, particularly between the second and third MA3 domains  is not full-length but was included along with the second P. abies homolog (MA_2413g0010), which is full-length, to represent the two PDCD4 gene members of a more recent gymnosperm species.Gene divergence often follows gene duplication. To determine how the plant PDCD4 gene family members may have diverged during the evolution of land plants, phylogenetic analysis of the gene family was performed. Because of the poor sequence conservation of the N-terminus, only the region containing the four MA3 domains was used for the analysis. Although the C. reinhardtii and V. carteri clustered together but not with the C. globosum sequence  which is more closely related to MAT8-like gene members in other dicots such as C. rubella, T. halophila, B. rapa, G. raimondii, T. cacao, G. max, M. truncatula, R. communis, P. trichocarpa, V. vinifera, and S. lycopersicum, as well as in monocots such as O. sativa, S. bicolor, Z. mays, H. vulgare, B. distachyon than it is to the other three members of the A. thaliana PDCD4 gene family , suggesting that genes in the MAT8 cluster are true orthologs. Regardless of the size of the MAT gene family, only one MAT8 gene is observed in those species in which a MAT8 homolog is present. In many species examined, the PDCD4 gene family is composed of just two members so that there is a single member in each subgroup. In some species, however, there are more than two PDCD4 genes and those that are not similar to MAT8 clustered together. An example is the A. thaliana gene family which, in addition to the single MAT8 gene, contains three additional members , that are more closely related to one another than to any MAT8 homolog. As four members are present in C. rubella , which cluster similarly to the A. thaliana gene family members, the MAT5/6/7 subgroup likely underwent gene expansion to three members from the two members of the MAT5/6/7 subgroup present in T. halophila, and B. rapa , C. rubella, T. halophila, and B. rapa . Moreover, examination of the PDCD4 homologs of the basal angiosperms A. trichopoda, A. scandens, and I. floridanum revealed that they too contain deletions in each MA3 domain that are identical to those observed in C. micholitz and S. amarus  the evolution of A. thaliana was sufficient to alter ethylene sensitivity and tolerance to salt [Arabidopsis species contain five distinct ethylene receptors and MAT5 (ECIP1) interacts with just two of these, i.e., ETR2 and EIN4 (as well as EIN2). Whether other members of the PDCD4 gene family exhibit different specificities in their interactions with ethylene receptors, EIN2, or other proteins remains to be determined but the divergence of the MAT8 subgroup and the MAT5/6/7 subgroup might provide the basis for any functional specificity that exists within this family.These observations collectively raise the question of what advantage does a four MA3 domain PDCD4 protein confer to recently evolved algal species and land plants and what advantage does expansion of the PDCD4 gene family confer to plants as they transitioned from an aquatic to a terrestrial environment? Loss of expression of just one of the four PDCD4 proteins in  to salt . The abi to salt . Whether to salt ,34,35. T to salt . The expArabidopsis thaliana were used to query genome-wide analysis and comparative studies. MA3-containing sequences were obtained by BLAST searches of the NCBI [http://www.onekp.com. Predicted protein sequences from genomic and EST sequences were obtained using the ExPASy Translate tool [Arabidopsis relatives , legumes (Glycine max and Medicago truncatula), the castor oil plant (Ricinus communis), cereals and grasses , fruits and vegetables (Vitis vinifera and Solanum lycopersicum), cotton (Gossypium raimondii), trees (Populus trichocarpa and Theobroma cacao), basal angiosperms , gymnosperms , bryophytes and lycophytes (Physcomitrella patens and Selaginella moellendorffii), green marine and fresh water algae , and stramenopiles or algal relatives . A list of protein sequences and cognate genes used for the comparative analysis is provided in Table MA3-containing sequences obtained from the NCBI  Phytozomthe NCBI , and thethe NCBI  protein,ate tool . Proteinate tool  with manECIP1: EIN2 C-terminal interacting protein 1; eIF: Translation initiation factor; HEAT: Huntington, Elongation Factor 3, PR65/A, TOR; MAT: MA three; PABP: Poly(A) binding protein; PDCD4: Programmed cell death 4.The authors declared that they have no competing interest.DG conceived of the study and directed its design and coordination. DG, RL, and SC contributed to the sequence alignment, phylogenetic analysis, and preparation of the manuscript. All authors read and approved the final manuscript.Sequence comparison of the MA3 domains in A. thaliana eIF4G and PDCD4 proteins. Comparison of the amino acid sequence of the MA3 domains of eIF4G and PDCD4 proteins is shown with amino acid identity highlighted in yellow amino acid and similarity highlighted in green. Conserved residues for MAT5-8 proteins (MAT consensus) are indicated below the sequence alignment as are the conserved residues for eIF4G and eIFiso4G (4G/iso4G consensus). The eIFiso4G-like sequence is MAT4 (At4g30680). Those residues absolutely conserved among all MA3 domain proteins are indicated as bold residues in both consensus sequences.Click here for fileDomain organization of PDCD4 homologs in algae and algal relatives. The domain organization of PDCD4 homologs is shown for green algae and algal relatives with the MA3 domains indicated in blue. PDCD4 proteins for green marine algae , fresh water alga (Chaetosphaeridium globosum), and stramenopiles or algal relatives  are shown. The additional sequence interrupting the fourth MA3 domain of the C. reinhardtii and V. carteri homologs is indicated by a gap. The first MA3 domain is missing from the partial C. globosum cDNA but is proposed.Click here for fileSequence analysis of the MA3 domains of PDCD4 homologs in algae and algal relatives suggests domain duplication in at least some chlorophytes. A phylogenetic tree was generated using the sequences comprising MA3 domains 1–2 and MA3 domains 3–4 of the four MA3 domain PDCD4 homologs of chlorophytes Chlamydomonas reinhardtii and Volvox carteri, and MA3 domains 1–2 and MA3 domain 3 from the partial cDNA of the Chlorella variabilis PDCD4 homolog, MA3 domains 1–2 of the two MA3 domain PDCD4 homologs of prasinophyte Micromonas species and Ostreococcus species, and those of the stramenopiles Ectocarpus siliculosus, Aureococcus anophagefferens, Phaeodactylum tricornutum, Thalassiisira pseudoonana, Phytophthora species, and Albugo laibachii. The phylogenetic tree was constructed using the maximum-likelihood method. Numbers on each branch denote percentages of bootstrap support. Chlorophytes are shaded dark green, species of the Mamiellales are shaded light green, and the stramenopiles are shaded tan.Click here for fileAligned sequences used for the phylogenetic analysis of Figure2.Click here for fileAligned sequences used for the phylogenetic analysis of Additional file.Click here for fileAligned sequences used for the phylogenetic analysis of Figure6.Click here for file


Neurexins (Nrxs) are presynaptic membrane proteins with a single membrane-spanning domain that mediate asymmetric trans-synaptic cell adhesion by binding to their postsynaptic receptor neuroligins. α-Nrx has a large extracellular region comprised of multiple copies of laminin, neurexin, sex-hormone-binding globulin (LNS) domains and epidermal growth factor (EGF) modules, while that of β-Nrx has but a single LNS domain. It has long been known that the larger α-Nrx and the shorter β-Nrx show distinct binding behaviors toward different isoforms/variants of neuroligins, although the underlying mechanism has yet to be elucidated. Here, we describe the crystal structure of a fragment corresponding to the C-terminal one-third of the Nrx1α ectodomain, consisting of LNS5-EGF3-LNS6. The 2.3 Å-resolution structure revealed the presence of a domain configuration that was rigidified by inter-domain contacts, as opposed to the more common flexible “beads-on-a-string” arrangement. Although the neuroligin-binding site on the LNS6 domain was completely exposed, the location of the α-Nrx specific LNS5-EGF3 segment proved incompatible with the loop segment inserted in the B+ neuroligin variant, which explains the variant-specific neuroligin recognition capability observed in α-Nrx. This, combined with a low-resolution molecular envelope obtained by a single particle reconstruction performed on negatively stained full-length Nrx1α sample, allowed us to derive a structural model of the α-Nrx ectodomain. This model will help us understand not only how the large α-Nrx ectodomain is accommodated in the synaptic cleft, but also how the trans-synaptic adhesion mediated by α- and β-Nrxs could differentially affect synaptic structure and function. In the mammalian brain, precise synaptic connections between billions of neurons must first be established if normal brain functions such as perception, memory and cognition are to be successfully executed. This requires the involvement of molecular mechanisms that not only guide specific synaptic recognition processes, but also allocate specific roles to each synapse during development. Presynaptic neurexins (Nrxs) and postsynaptic neuroligins (NLs), both of which are type I membrane proteins containing a single-membrane spanning region, are two potential regulators of this process since both are physically capable not only of linking the two opposing membranes via their ectodomains, but also of recruiting specific sets of pre- and post-synaptic proteins near the junction 2+ channels The α-Nrx ectodomain is comprised of three repeats, each containing an epidermal growth factor (EGF)-like domain flanked by two LNS  domains  domains) 2+) β-Nrx ectodomain structure equivalent to LNS6 in α-Nrx has been extensively studied, yielding multiple crystal structures from different subtypes , carrying different splice insertions (SS4 + and −), or crystallized in different conditions ), encompassing the domains LNS5-EGF3-LNS6. The 2.3 Å resolution structure revealed the presence of unique molecular contacts that potentially limit inter-domain mobility, thus explaining why the B+ variant of NL1 is incompatible with α-Nrx binding. By combining this with single-particle image analysis using negatively stained α-Nrx1 ectodomain samples, we were able to successfully construct a three-dimensional structural model of the entire α-Nrx ectodomain.In order to elucidate the unique NL-binding mechanism found in α-Nrx, the structure of the LNS6 domain must be analyzed in the context of a larger fragment, one that contains the preceding domains at its N-terminal. As the extracellular domain of α-Nrx is comprised of three repeating units, each containing two LNS domains separated by an EGF-like module, we chose to use a subfragment NX1α(III) encompassing LNS5-EGF3-LNS6 for our structural analysis. This fragment was robustly expressed and secreted from the transfected mammalian cells, indicating that the truncation at the LNS4-LNS5 boundary did not have any adverse effects on either the folding or the stability of the protein. Furthermore, the fragment exhibited NL1-binding in a splice B-sensitive manner , which wThe NX1α(III) structure, refined at 2.3 Å resolution, contained one molecule in the asymmetric unit, including the LNS5 domain (residues 867–1045), the EGF domain (residues 1046–1084), and the LNS6 domain (residues 1085–1261). The LNS5 and LNS6 domains formed a globular structure with dimensions measuring approximately 35×40×75 Å, while the EGF domain assumed an elongated cylindrical structure with dimensions ∼15×20×30 Å. The central EGF domain was sandwiched between the two LNS domains and physically separated them, thereby making the entire molecule look like an off-centered dumbbell or “handset” . Further84TTYIF sequence, which curls back toward the top rim of the β-sandwich (1086TTYIF sequence of the LNS6 domain in NX1α(III) emanate away from the domain almost perpendicularly, due to a kink at Pro1085. Thus, the relative arrangement of EGF3 and LNS6 seems to be governed by a single Pro residue.The LNS5 domain, the structure of which had never been previously determined, adopted a 14-stranded β-sandwich fold typical of this domain class, and showed highest similarity with the laminin a2 LNS5 domain (1DYK) with root mean square deviations (RMSD) of 1.55 Å for the 163 matched residues. As for the LNS6 domain, six independent crystal structures of Nrx1β, determined either as a monomer or in complex with NL, were available sandwich . In cont2+ at the hyper-variable surface of the LNS6 domain, we found a clear electron density peak at this site. To examine the presence of Ca2+, we collected a diffraction data set at a wavelength of 1.7000 Å and calculated an anomalous difference Fourier map. A robust signal stemming from an anomalous dispersion (>4σ) was observed at this site, suggesting that the electron density entity was, in fact, Ca2+. This site seems to be only partially occupied, however, since the Ca2+ had a significantly higher B-factor value (41.6 Å2) compared with that of the coordinated protein and water ligands (19–23 Å2). The Ca2+ coordination assumed an octahedral geometry, as opposed to a more typical pentagonal bipyramid 2+ was found at the corresponding site in the LNS5 domain of NX1α(III)  requires the presence of Ca2+ in the crystallization buffer, suggesting a low affinity 2+ affinities for other LNS domains were in the range of several hundred µM 2+ in the extracellular milieu would enable full occupancy of the site, thus indicating that the α- and β-Nrx expressed on the cell surface are pre-loaded with Ca2+ at the LNS6 domain and are ready for subsequent interaction with NLs.Consistent with previous reports that Nrx1β has the potential to bind CaX1α(III) , left. A2 solvent accessible surface area (ASA) was buried by the interaction between residues from both domains, excluding those involved in the direct domain linkage . Strikingly, this interaction was almost exclusively mediated by Trp1065, which is located at the tip of the C3–C4 loop  of the EGF3. The side-chain of Trp1065 was deeply inserted into a cave at the top of the LNS5 created by the Cys1015–Cys1043 disulfide and the interdomain linker, resulting in numerous van der Waals contacts and hydrogen bonds with more than 10 residues in the LNS5 domain alone (Within the structure of NX1α(III), the central EGF3 domain made contact with the preceding LNS5 domain and the following LNS6 domain in unique and contrasting ways . At the in alone , left. Bin alone , suggest2 of ASA, although the nature of the interaction was drastically different from that seen in LNS5-EGF3  abolished binding to the B- variant of NL1 (lanes 2 and 3). Furthermore, introduction of bulky Arg residues at the core , but not the periphery (D1106), of the interface also resulted in the complete loss of binding capability (lanes 4–6). These results strongly suggest that the same protein surface is used by both α- and β-Nrx when docking with NL1. However, simple superposition of the NX1α(III) structure onto the Nrx1β/NL1 complex at the LNS6 domain was found to cause a steric clash between LNS5 and a part of NL1 distal to the homodimeric interface /NL1 complex also allowed us to understand the reason behind the deleterious effect of a splice site B insertion on the NL1's binding ability to α-Nrx. The insertion point for the B sequence  is located right at the foot of the bound LNS6, directly under the “roof” of the EGF3 domain . When thEC, residues 1–1263) and imaged it using negative staining electron microscopy. Each particle displayed 4 to 5 globular domains closely positioned together, which most likely represented an individual LNS domain (EC construct we used consisted of the splice site 1+ (SS1+) form containing a 19-residue insertion between the EGF1 and the LNS2 domains , which corresponded to the core-repeating unit of the entire ectodomain, prompted us to analyze a three-dimensional arrangement of this unit in the context of full-length Nrx1α. To this end, we expressed and purified an Nrx1α fragment comprising the entire ectodomain of bovine Nrx1α , the repeat III region ), or LNS6 . The segments described above were PCR amplified from pCMV-N1α-1 (a gift from T.C. Südhof) All neurexin residue numberings are based on full-length bovine Nrx1α without any splice site insertions . Expression constructs contain the entire ectodomain . Cell cultures were grown for 24 h, and then the culture media containing the secreted protein was collected. For stable expression, CHOlec 3.2.8.1 cells (provided by P. Stanley) 2, the beads were incubated with the culture supernatants containing tagged Nrx constructs -MycHis, NX1αEC-MycHis) and incubated at 4°C for an additional 2 h. Bound Nrx fragments were detected by Western blot using an anti-Myc antibody.The binding between the Myc-tagged fragments of Nrx1α and the hGH-fusion NL1 variants were evaluated using a solid-phase binding assay. Briefly, hGH fusion constructs  were transiently expressed in 293T cells and the culture supernatants were incubated with immobilized anti-hGH monoclonal antibody  at 4°C for 1 h to capture the fusion proteins on the beads. After washing with 20 mM Tris (pH 8.0), 150 mM NaCl, and 2 mM CaCl2+ was collected at 1.7000 Å. Diffraction data were processed with the HKL-2000 program package R-factor of 19.7% and a free R-factor of 24.7%. The final model contained 395 amino acid residues, a calcium ion, and 148 water molecules. NX1α(III) contains an N-linked glycosylation site at Asn-1186. The electron density map revealed the presence of an N-linked glycan at Asn-1186. The final model therefore included one N-acetylglucosamine residue at this position. The quality of the final model was validated with MolProbity Purified NX1α(III) was concentrated to 10 mg/ml and subjected to crystallization screening via the hanging-drop vapor diffusion method using a Wizard I & II screening kit . The NX1α(III) crystal was grown at 20°C in hanging drops with reservoir solution containing 0.3 M sodium malonate (pH 7.0), 0.1 M sodium acetate, and 18% PEG 3000. Prior to data collection, the crystals were soaked in cryoprotectant containing 0.3 M sodium malonate (pH 7.0), 0.1 M sodium acetate, 22% PEG 3000, and 25% glycerol, and were then flash-frozen in liquid nitrogen. Data collections were performed on SPring-8 BL-41XU, BL-44XU, PF BL-5A and PF-AR NW12A. A diffraction data set used for the structural determination was collected at a wavelength of 1.0000 Å using a SPring-8 BL41XU. The data set used for the identification of CaFor the structural analysis, the accessible surface area was calculated with AREAIMOL EC was subjected to size-exclusion chromatography on a Superdex 200 HR column immediately before sample preparation. The peak fraction corresponding to the monodispersed NX1αEC was negatively stained with an equal volume of 2% uranyl acetate for 30 s. The specimen was examined at 200 kv with a H-9500SD transmission electron microscope . The images were recorded on a 2K×2K CCD camera  using a homemade microscope control program for spot scan at a nominal magnification of ×80,000, leading to a final image resolution of 0.22 nm/pixel. Individual particles were boxed out from the original images and processed using the EMAN software suite EC 3-D reconstruction was performed using Chimera A solution of purified NX1αEC has been deposited in the EMDB (www.ebi.ac.uk/msd) with code EMD-5270.The atomic coordinates of NX1α (III) have been deposited in the Protein Data Bank with PDB ID 3ASI. The EM map of NX1α