Escherichia coli and phototrophic Rhodopseudomonas palustris. In this coculture, E. coli excretes waste organic acids that provide a carbon source for R. palustris. In return, R. palustris cross-feeds E. coli ammonium (NH4+), a compound that both species value. To explore the potential for interpartner competition, we first used a kinetic model to simulate cocultures with varied affinities for NH4+ in each species. The model predicted that interpartner competition for NH4+ could profoundly impact population dynamics. We then experimentally tested the predictions by culturing mutants lacking NH4+ transporters in both NH4+ competition assays and mutualistic cocultures. Both theoretical and experimental results indicated that the recipient must have a competitive advantage in acquiring cross-fed NH4+ to sustain the mutualism. This recipient-biased competitive advantage is predicted to be crucial, particularly when the communally valuable nutrient is generated intracellularly. Thus, the very metabolites that form the basis for mutualistic cross-feeding can also be subject to competition between mutualistic partners.Many mutualistic microbial relationships are based on nutrient cross-feeding. Traditionally, cross-feeding is viewed as being unidirectional, from the producer to the recipient. This is likely true when a producer’s waste, such as a fermentation product, has value only for a recipient. However, in some cases the cross-fed nutrient holds value for both the producer and the recipient. In such cases, there is potential for nutrient reacquisition by producer cells in a population, leading to competition against recipients. Here, we investigated the consequences of interpartner competition for cross-fed nutrients on mutualism dynamics by using an anaerobic coculture pairing fermentative  Mutualistic relationships, particularly those based on nutrient cross-feeding, promote stability of diverse ecosystems and drive global biogeochemical cycles. Cross-fed nutrients within these systems can be either waste products valued by only one partner or nutrients valued by both partners. Here, we explored how interpartner competition for a communally valuable cross-fed nutrient impacts mutualism dynamics. We discovered that mutualism stability necessitates that the recipient have a competitive advantage against the producer in obtaining the cross-fed nutrient, provided that the nutrient is generated intracellularly. We propose that the requirement for recipient-biased competition is a general rule for mutualistic coexistence based on the transfer of intracellularly generated, communally valuable resources. Suc12 (4+) , wherein12 (4+) , 17–20.  (4+) 17–.4+ excretion by N2-fixing bacteria  from Rhodopseudomonas palustris Nx . We. WeEscheE. coli ferments sugars into waste organic acids, providing essential carbon and electrons to R. palustris Nx. R. palustris Nx converts N2 into NH4+ and is genetically engineered to excrete low micromolar amounts of NH4+, providing essential nitrogen for E. coli  and a higher R. palustris maximum growth rate (μMAX) when NH4+ is used , as E. coli can acquire enough excreted NH4+ to be able to grow. In contrast, when the R. palustris affinity for NH4+ is high relative to that of E. coli , E. coli growth is no longer supported, because E. coli cannot compete for excreted NH4+. These trends are minimally impacted by the increase in the R. palustris growth rate when reacquiring NH4+  affected the simulated cell density values but not the overall trends. Despite the lack of E. coli growth, high R. palustris cell densities were still predicted . Download Copyright © 2017 McCully et al.2017McCully et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01620-17.1TEXT S1 TEXT S1, DOCX file, 0.1 MB.SyFFoN v3 equations and parameter descriptions. Download Copyright © 2017 McCully et al.2017McCully et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01620-17.2FIG S1 4+ affinities, whereas SyFFoN v2 does not. RA values for R. palustris NH4+ excretion were set to 0.15 fmol/cell and 0.5 fmol/cell for R. palustris strains Nx and Nx ΔAmtB, respectively. For SyFFoN v3, two simulations were performed using either equivalent NH4+ affinities or a slightly higher E. coli NH4+ affinity, based on the relative competitive affinities from KA, 0.01 mM; KAR, 0.05 mM). Symbols represent simulated values. Bars represent standard deviations from empirical data (n = 4). Simulated starting cell densities in panel A are indicated by dotted lines. Download FIG S1, TIF file, 0.4 MB.SyFFoN v2 and v3 predict similar cell densities and product concentrations. Predictions from SyFFoN v2 and SyFFoN v3 and empirical data for final cell densities (A) and product yields (B to E) are shown. Batch cultures (300 h) were simulated. SyFFoN v3 includes the roles of relative NHCopyright © 2017 McCully et al.2017McCully et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01620-17.3FIG S2 R. palustris growth rate boost on NH4+ do not change overall model predictions. (A) Simulation of 2,000-h cocultures instead of 300 h, as in E. coli population and higher R. palustris final cell densities. (B) Omitting the boost in the R. palustris growth rate on NH4+ did not affect the model trends. To prevent the boosted growth rate, μRpMax2 was changed from 0.0152 h−1 (A) to 0 h−1 (B) for simulated 2,000-h batch cultures.  Initial cell densities are indicated by dotted lines. Relative NH4+ affinity values represent the relative E. coli Km for NH4+ (KA) divided by that of R. palustris (KAR). For a ratio of 1, each species had a default Km for NH4+ of 0.01 mM. To the left of 1, the R. palustris Km value was raised. To the right of 1, the E. coli Km value was raised. Both panels show final cell densities (solid lines) of R. palustris  and E. coli  after simulated 2,000-h batch cultures. Starting cell densities (dashed lines) were based on a 1% dilution of cocultures containing 10% E. coli. Download FIG S2, TIF file, 0.2 MB.Simulation of longer  batch cultures and omitting the Copyright © 2017 McCully et al.2017McCully et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01620-17.10TABLE S2 TABLE S2, DOCX file, 0.02 MB.Strains, plasmids, and primers used in this study. Download Copyright © 2017 McCully et al.2017McCully et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the E. coli have a higher affinity for NH4+ than does R. palustris might seem at odds with other models of resource competition, wherein an increased cost of cooperation and/or decreased resource capture by the cooperator  can result in extinction of the cooperator . We. We4+ thprovided , consiststris Nx  and D. I to NH4+ , 38. Howl strain . These o10.1128/mBio.01620-17.4FIG S3 E. coli ΔAmtB and R. palustris ΔAmtB monoculture growth and metabolic trends. (A and B) Growth rates (A) and fermentation product yields (B) from WT E. coli (filled) or E. coli ΔAmtB (open) monocultures grown in MDC with 25 mM glucose and 15 mM NH4Cl. Lower concentrations of NH4Cl were not tested because other studies have shown that growth is only affected below 20 μM (n = 3). (C and D) Growth curves (C) and relative NH4+ excretion (D) of R. palustris monocultures grown in MDC with 3 mM sodium acetate and a 100% N2 headspace. Error bars indicate SD (n = 4). Download FIG S3, TIF file, 0.3 MB.ow 20 μM . In batcCopyright © 2017 McCully et al.2017McCully et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01620-17.5FIG S4 R. palustris ΔAmtB responds to NH4+-induced switch off of nitrogenase. The effect of either NH4Cl or NaCl on growth (A and C) and H2 production  by R. palustris Nx, ΔAmtB, or parental monocultures. (A to D) R. palustris monocultures were grown in MDC with 20 mM sodium acetate and 100% N2 headspace until mid-exponential phase, and then cultures were supplemented with either 15 mM NH4Cl or 15 mM NaCl at the time indicated by the arrow. Nitrogenase activity was measured using H2 production as a convenient alternative to an acetylene reduction assay, since H2 is an obligate product of the nitrogenase reaction and, unlike most N2-fixing bacteria that oxidize any H2 produced, our R. palustris strain accumulates H2 due to a ΔhupS mutation that inactivates hydrogenase. (E) R. palustris monocultures were grown in MDC with 20 mM sodium acetate and 100% N2 headspace with (gray) or without (white) 15 mM NH4Cl. Samples for determining H2 yields were taken 1 week after inoculation, within 24 h of entry into stationary phase. Error bars indicate standard deviations (n = 3). Download FIG S4, TIF file, 0.3 MB.Copyright © 2017 McCully et al.2017McCully et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the amtB would lower cellular affinity for NH4+, we directly tested all possible E. coli and R. palustris strain combinations in competition assays in which ample carbon was available for each species but the NH4+ concentration was kept low. Specifically, a small amount of NH4+ was added every hour to bring the final NH4+ concentration to approximately 5 μM, although it is possible that the NH4+ concentration exceeded 5 μM at early time points when consumption rates could have been slow due to low cell densities  E. coli was more competitive for NH4+ than any R. palustris strain. However, each R. palustris strain was able to outcompete E. coli ΔAmtB , final cell densities (B), and product yields (C) from cocultures of E. coli ΔAmtB paired with R. palustris Nx supplied with either 15 mM NaCl (gray) or NH4Cl (black). Final cell densities were taken within 24 h into stationary phase. Initial cell densities were approximately 3 × 106 CFU/ml for E. coli ΔAmtB and 1 × 108 CFU/ml for R. palustris Nx, which represented a 1% inoculum from stationary starter cocultures from single colonies. Error bars indicate standard deviations (n = 3). Download FIG S5, TIF file, 0.2 MB.Copyright © 2017 McCully et al.2017McCully et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the R. palustris strains lacking AmtB, the effects on population trends varied. Consistent with our previous work, R. palustris NxΔAmtB supported higher WT E. coli percentages and cell densities , which acidify the medium and inhibit R. palustris growth . We therefore tested whether coculture conditions stimulated higher nitrogenase activity by using an acetylene reduction assay. In agreement with our hypothesis, R. palustris ΔAmtB had increased nitrogenase activity under coculture conditions compared to monocultures, whereas R. palustris Nx, which exhibits constitutive nitrogenase activity, showed similar levels under both conditions . Si. SiR. pa NxΔAmtB  resulteds growth  (28). Suumulated  and D. T NxΔAmtB  and D. UtB  or coculture (Co) was measured in an acetylene reduction assay. Ethylene levels were divided by total R. palustris CFU in the test tube and then normalized to the R. palustris Nx monoculture value. Error bars indicate standard deviations (n = 4). *, statistical difference between monoculture and coculture conditions ; ns, no significant difference. Download FIG S6, TIF file, 0.1 MB.Copyright © 2017 McCully et al.2017McCully et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the R. palustris ΔAmtB paired with E. coli ΔAmtB showed little growth when started from a single colony of each species  and empirical data (B and C) showing the effects of the initial CFU ratio of R. palustris (Rp) to E. coli (Ec) on population and coculture growth trends are shown for E. coli, with a lower affinity for NH4+ than R. palustris. (A) Batch cultures (300 h) were simulated with a relative R. palustris:E. coli affinity ratio (Rp:Ec) for NH4+ of 1000 . (B and C) Changes in cell densities after 1 week of growth (B) and growth curves of cocultures inoculated at different species ratios (C). In all three panels, a ratio of 1 represents 2.7 × 106 cells/ml (A) or CFU/ml (B and C). Thus, the listed ratios were achieved by diluting either species rather than concentrating them. Error bars indicate standard deviations (n = 3). Download FIG S7, TIF file, 0.2 MB.Higher initial cell densities of Copyright © 2017 McCully et al.2017McCully et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the R. palustris ΔAmtB over E. coli ΔAmtB for NH4+ called into question why cocultures pairing E. coli ΔAmtB with either R. palustris Nx or R. palustris NxΔAmtB did not collapse as well  and varied the R. palustris NH4+ excretion level  as a mechanism leading to coculture collapse within the time period of a single culturing. However, we wondered if a subtle discrepancy in NH4+ affinities could lead to coculture collapse if given more time. We therefore simulated serial transfers of cocultures with partners having different relative NH4+ affinities , cell densities of both species were predicted to decrease over serial transfers . These results indicate that the recipient population must have a competitive advantage for a cross-fed nutrient relative to the producer population to avoid mutualism collapse.The above prediction prompted us to investigate if cocultures pairing partners . This de4+. This competition can impact partner frequencies and mutualism stability. We demonstrated that efficient nutrient reacquisition by the producer can render nutrient excretion levels insufficient for mutualistic growth, starving the recipient and leading to tragedy of the commons . Co. Co4+. T commons  and C. I commons . Intrace commons , our resIt is expected that for mutualistic relationships based on the extracellular generation of nutrients, such as the release of sugar from a polymer, a high affinity for the nutrient by either partner can collapse the mutualism . It has 35–12  agar, and R. palustris was cultivated on defined mineral (PM) agar (4)2SO4 was omitted from PM agar for determining R. palustris CFU. Monocultures and cocultures were grown in 10 ml of defined M9-derived coculture medium (MDC)  and glucose , unless indicated otherwise. E. coli monocultures were also supplemented with 15 mM NH4Cl. All cultures were grown at 30°C laying horizontally under a 60-W incandescent bulb with shaking at 150 rpm. Starter cocultures were inoculated with 200 μl MDC containing a suspension of a single colony of each species. Test cocultures were inoculated using a 1% inoculum from starter cocultures. Serial transfers were also inoculated with a 1% inoculum. Kanamycin and gentamicin were added to final concentrations of 100 μg/ml for cultures of R. palustris and 15 μg/ml for E. coli cultures when appropriate.Strains, plasmids, and primers are listed in regation  and to pPM) agar  with 10 um (MDC)  in 27-mlR. palustris mutants were derived from wild-type CGA009 , the WT nifA gene was amplified using primers JBM1 and JBM2, digested with XbaI and BamHI, and ligated into plasmid pJQ200SK to make pJQnifA16. This suicide vector was then introduced into CGA4021 by conjugation, and sequential selection and screening were performed as described (nifA* with WT nifA. Reintroduction of the WT nifA gene was confirmed by PCR and sequencing.e CGA009 . Generate CGA009 . To geneescribed  to replakm from the Keio Collection strain JW0441-1 (E. coli MG1655. The ΔamtB::km genotype of kanamycin-resistant colonies was confirmed by PCR and sequencing.P1 transduction  was usedJW0441-1  into E. 660) using a Genesys 20 visible spectrophotometer . Growth curve readings were obtained in culture tubes without sampling . Specific growth rates were determined using OD660 readings between 0.1 and 1.0, a range for which there is a linear correlation between cell density and OD660. Final OD660 measurements were taken in cuvettes, and samples were diluted into the linear range as necessary. H2 was quantified using a gas chromatograph  with a thermal conductivity detector as described  in MDC containing limiting nutrients , and 1 ml of each species culture was inoculated into the serum vials. These competition cocultures were incubated at 30°C under a 60-W incandescent bulb with stirring at 200 rpm for 96 h. Each serum vial was constantly flushed with Ar to maintain anaerobic conditions. NH4Cl was fed from a 500 μM NH4Cl stock via a peristaltic pump on an automatic timer at a rate of 0.33 ml/min once an hour for a final concentration of ~5 μM upon each addition. The NH4+ concentration was below the known concentration at which AmtB transporters become important for NH4+ uptake  cation solution, and 20 mM sodium acetate. Unlike acetic acid, which + uptake . SamplesE. coli and R. palustris for NH4+ as follows: (i) an equation for the R. palustris growth rate on NH4+ was added to boost the R. palustris growth rate when acquiring NH4+ and (ii) the ability for R. palustris to consume NH4+ was added along with an R. palustris Km for NH4+ (KAR). Default NH4+Km values were set to 0.01 mM for both species, to achieve a ratio of 1. To achieve higher R. palustris or E. coli relative NH4+ affinities, the E. coli or R. palustris Km value was raised, respectively. Simulated cultures were run for 300 h unless noted otherwise. Normally, full glucose consumption occurs by ~100 h under typical experimental conditions and in corresponding simulations, but 300 h was allowed to capture trends that would take longer to emerge in response to parameter changes while still approximating a reasonable experimental time frame. Equations and default parameter values derived from our experimental data can be found in https://github.com/McKinlab/Coculture-Mutualism.A Monod model describing bidirectional cross-feeding in batch cultures, called SyFFoN v3 , was modified from our previous model  to allow


Haemophilus surface fibril was determined at a resolution of 3.3 Å, revealing a novel domain arrangement.The crystal structure of PD1 from the  Haemophilus surface fibril (Hsf) is an unusually large trimeric autotransporter adhesin (TAA) expressed by the most virulent strains of H. influenzae. Hsf is known to mediate adhesion between pathogen and host, allowing the establishment of potentially deadly diseases such as epiglottitis, meningitis and pneumonia. While recent research has suggested that this TAA might adopt a novel ‘hairpin-like’ architecture, the characterization of Hsf has been limited to in silico modelling and electron micrographs, with no high-resolution structural data available. Here, the crystal structure of Hsf putative domain 1 (PD1) is reported at 3.3 Å resolution. The structure corrects the previous domain annotation by revealing the presence of an unexpected N-terminal TrpRing domain. PD1 represents the first Hsf domain to be solved, and thus paves the way for further research on the ‘hairpin-like’ hypothesis.The  One such virulence factor utilized by Hib is the Haemophilus surface fibril (Hsf), a trimeric autotransporter adhesin (TAA) protein that shares significant homology with another, better-characterized H. influenzae TAA known as Hia  domains : the interaction has been mapped to HsfBD2 and the C-terminal Vn residues 352–374  cloning. The PD1 gene was amplified by polymerase chain reaction from a pET-16b-hsf1–2414 plasmid. The primers were designed to generate a ‘megaprimer’ containing the PD1 gene with complementary overhangs to the destination vector, pIBA-GCN4tri-His6 (Supplementary Table S1). pIBA-GCN4tri-His6 was linearized by restriction digestion with XhoI (New England Biolabs) and used as a template in a secondary round of PCR to insert the PD1 gene (contained within the ‘megaprimer’) into the plasmid. Expression of PD1-GCN4 was induced at an OD600 of 0.6 by the addition of anhydrotetracycline hydrochloride to a final concentration of 8.6 µM for 4 h. The cells were collected by centrifugation (2000g for 10 min at 277 K), stored at 193 K overnight and resuspended in buffer A consisting of 50 mM NaH2PO4, 500 mM NaCl pH 8.0. The cells were lysed by sonication and supernatants were collected by centrifugation (16 000g for 10 min at 277 K). The protein was purified by immobilized metal ion-affinity chromatography (IMAC). The cleared supernatant containing PD1-GCN4 was applied onto a Ni–NTA agarose column  previously equilibrated with buffer A  and allowed to bind for 1 h with agitation. Proteins were eluted in buffer B consisting of 50 mM NaH2PO4, 500 mM NaCl, 10% glycerol, 300 mM imidazole pH 8.0. The quality of the purified protein was assessed by size-exclusion chromatography coupled to a multi-angle laser light scattering (SEC-MALLS) apparatus . SEC-MALLS was carried out using a Superdex 200 5/150 column pre-equilibrated with buffer C consisting of 50 mM Tris, 500 mM NaCl, 10% glycerol pH 8.0 at a flow rate of 0.2 ml min−1 and was detected using a DAWN 8+ multi-angle light-scattering (LS) detector, an Optilab T-rEX differential refractive-index (dRI) detector and a UV-absorbance (UV) detector (Wyatt).The Hsf domain PD1 was cloned between two GCN4 anchor proteins. GCN4 is a well characterized yeast transcription factor that forms a coiled-coil dimer in its native state. However, mutagenesis of specific residues in its hydrophobic core allows GCN4 to adopt various oligomeric states. Owing to this, variations of GCN4 are often used as partners for fusion proteins to facilitate stable oligomerization. In this case, the idea was to add a well characterized trimer-forming variant of GCN4 to both the N- and C-terminus to facilitate the stable trimerization of HsfPD1  and then divided into five equal volumes for elution of protein in different buffers: buffer B containing 0, 0.5, 1, 2 and 4 M urea. The protein was eluted and fractions were collected for native PAGE analysis .To prevent the aggregation of PD1-GCN4 (demonstrated by SEC-MALLS), the purification was repeated in the presence of increasing concentrations of urea. The protein was expressed and the cells were lysed as above. Subsequently, the cleared supernatant was applied onto Ni–NTA agarose resin (2 ml) and allowed to bind for 1 h with agitation. Purification was performed in batch mode. The resin was washed with buffer is Fig. 1b.2.1.2.6 plasmid generated using restriction-free (RF) cloning in the same way as PD1-GCN4 (§PD1 gene was amplified by PCR from the pIBA-PD1-GCN4tri-His6 plasmid using primers capable of producing a ‘megaprimer’. The pET-28-Tcfa-His6 destination vector was linearized by restriction digestion with XhoI and NcoI (New England Biolabs) to remove the tcfA gene (while retaining the His6 tag). This plasmid backbone was the template for a secondary round of PCR, utilizing the ‘megaprimer’, to insert the PD1 gene into the plasmid  to a final concentration of 0.5 mM for 4 h. The cells were collected and stored as before . The protein was purified via IMAC on a Ni–NTA agarose column previously equilibrated with buffer C  and allowed to bind for 1 h with agitation. Proteins were eluted in buffer D consisting of 50 mM Tris, 150 mM NaCl, 300 mM imidazole pH 8.0, and the pooled fractions were concentrated to 500 µl. Further purification was carried out by size-exclusion chromatography (SEC) on a Superdex 200 10/300 column pre-equilibrated with buffer E consisting of 50 mM Tris, 600 mM NaCl pH 8.0 and eluting imidazole-free protein at a flow rate of 0.2 ml min−1. The purified fractions were then pooled and concentrated to 15 mg ml−1 for crystallization. The quality of the purified protein was assessed prior to crystallization by SDS–PAGE and SEC-MALLS, carried out as described above, using buffer C  polyethylene glycol (PEG) 6000. Crystallization was performed at 293 K using the sitting-drop vapour-diffusion method, in which 100 nl protein solution was mixed with an equal volume of reservoir solution. Drops were set up using a Formulatrix NT8 crystallization robot. Since the initial crystals diffracted poorly, further crystallization optimization of PD1 was performed. The best diffracting crystals grew from 0.75 M LiCl, 0.1 M sodium citrate pH 3.9, 17.6%(w/v) PEG 6000  using the following conditions: protein concentration 15 mg ml0 Table 2. Owing t2.3.XDS  of 39.95 and log-likelihood gain (LLG) of 2010 indicated a correct MR solution. Refinement was carried out with PHENIX . Although radiation damage restricted the data set to the first 1100 images, the crystals diffracted to 3.3 Å resolution Table 3. Indexin6 Table 4.3.3.1.et al., 2008et al., 2014a). This was unexpected, as 18 crystal structures of TAA domains have already been solved using this method. We thought that the aggregation might be owing to hydrophobic interactions between the head domains and/or owing to improper folding of these domains arising from their flanking by the GCN4. We therefore decided to purify the protein in the presence of increasing concentrations of urea (0.5–4 M) to prevent aggregation and to use native PAGE to assess the level of aggregation . However, this method was unsuccessful in reducing aggregation, as the protein still did not migrate as expected in the gel, suggesting that the GCN4 anchors cause extensive misfolding and not just a small amount of reversible aggregation. We therefore decided to remove the GCN4 anchors. The construct lacking GCN4 (§dASTRA software (Wyatt). This is within experimental error (±5%) of the expected molecular weight of 47.8 kDa. It was clear from the chromatogram that no aggregates were present, and this construct yielded diffracting crystals .Initial efforts to purify PD1 utilizing GCN4 anchors  of 2.83 Å3 Da−1. The number of residues identified in the density of each monomer varied between 129 and 133 residues. The missing loops in the monomers are owing to poor electron density. Typical density is presented in Supplementary Fig. S3.In our effort to characterize the full-length Hsf protein, we determined the crystal structure of trimeric PD1 at a resolution of 3.3 Å, thus providing the first insight into the molecular arrangement of Hsf to date. HsfPD1 crystallized in the monoclinic space group 2 Table 1, with nib). The N-terminus of HsfPD1 spans 29 amino acids participating in the unexpected formation of three β-sheets, βW11, βW12 and βW13 (where ‘W’ represents tryptophan), which share considerable structural homology with the C-terminal TrpRing domain. Although the sequence identity between these two regions is low , a structural alignment of the 29 N- and C-terminal residues from one HsfPD1 monomer  confirms that the N-terminal region is indeed a TrpRing domain. The KG domain is composed of two β-strands, βKG1 and βKG2, as well as three α-helices, αKG1, αKG2 and αKG3. The C-terminal TrpRing is composed of five β-strands: βW21, βW22, βW23, βW24 and βW25. All domains participate in extensive intertwining, where the C-terminal α-helices (αKG3) from each monomer come together to create the central core of the trimer interface. The KG and C-terminal TrpRing domains were easily identified by simple structural observation and comparison with other TAAs . In contrast, although a superposition of HsfPD1 with HiaBD1 , suggesting that HsfPD1 is indeed a non-adhesive domain.Hsf and Hia are remarkably similar in their domain arrangement, as both possess adhesive domains (BDs) and domains of unknown function (PDs). Whilst BD domains have contiguous Neck-TrpRing architecture, the PD domains have a KG domain instead of the Neck domain. The adhesive activity of the BD domains in Hia results from the formation of an acidic pocket created by residues Asp618 and Ala620 of αD1 Fig. 3b, sugge3.4.i.e. N-TrpRing:Neck:TrpRing-C, and Hsf is approximately double the length of Hia. Thus, PD domains may have evolved via the duplication of BD domains or vice versa. This duplication certainly contributes to the overall length of Hsf, and whilst it is consistently reported that the PD domains are of unknown function, one implication of this evolution is that the additional length created by these domains conveys a survival advantage on those strains of H. influenzae that express Hsf. This perhaps explains why Hsf is expressed by all typeable strains of H. influenzae  whilst Hia is not, i.e. it is long enough to extend beyond the bacterial lipopolysaccharide layer and thus bind to complement regulators and ECM molecules to evade attack by the host.Owing to the high sequence identity between the shared regions of Hia and Hsf , we predict that, had the N-terminus of the Hia307–422 construct been extended by ∼40 residues, the same N-TrpRing:KG:TrpRing-C arrangement would have been observed. Moreover, this triplicate arrangement indicates an evolutionary link between BD and PD domains, in that BD domains are indeed triplicates, 4.Although HsfPD1 is in many respects a typical TAA domain, the novel domain arrangement (N-TrpRing:KG:TrpRing-C), revealing the N-terminal TrpRing domain, demonstrates the necessity of structural characterization of such proteins, as opposed to sequence analysis alone. This arrangement yielded insights into the evolution of PD domains, supporting the divergent nature of TAAs, and supersedes the previous domain annotation. Furthermore, the structure of HsfPD1 will contribute to the understanding and determination of the hypothesized ‘hairpin-like’ structure of Hsf. Inclusion of the N-terminal TrpRing domain in computer models may help to refine them. This combination may reveal unique protein–protein interactions between antiparallel PD and BD domains, generating exciting insights into the structure of TAAs, should this novel hypothesis be true.Hsf 1608–1749 putative domain 1, 5lnlPDB reference: 10.1107/S2053230X17001406/no5112sup1.pdfSupplementary Tables and Figures.. DOI:


Sensing external signals and transducing these into intracellular responses requires a molecular signaling system that is crucial for every living organism. Two important eukaryotic signal transduction pathways that are often interlinked are G-protein signaling and phospholipid signaling. Heterotrimeric G-protein subunits activated by G-protein-coupled receptors (GPCRs) are typical stimulators of phospholipid signaling enzymes such as phosphatidylinositol phosphate kinases (PIPKs) or phospholipase C (PLC). However, a direct connection between the two pathways likely exists in oomycetes and slime molds, as they possess a unique class of GPCRs that have a PIPK as an accessory domain. In principle, these so-called GPCR-PIPKs have the capacity of perceiving an external signal (via the GPCR domain) that, via PIPK, directly activates downstream phospholipid signaling. Here we reveal the sporadic occurrence of GPCR-PIPKs in all eukaryotic supergroups, except for plants. Notably, all species having GPCR-PIPKs are unicellular microorganisms that favor aquatic environments. Phylogenetic analysis revealed that GPCR-PIPKs are likely ancestral to eukaryotes and significantly expanded in the last common ancestor of oomycetes. In addition to GPCR-PIPKs, we identified five hitherto-unknown classes of GPCRs with accessory domains, four of which are universal players in signal transduction. Similarly to GPCR-PIPKs, this enables a direct coupling between extracellular sensing and downstream signaling. Overall, our findings point to an ancestral signaling system in eukaryotes where GPCR-mediated sensing is directly linked to downstream responses. G-protein-coupled receptors (GPCRs) are central sensors that activate eukaryotic signaling and are the primary targets of human drugs. In this report, we provide evidence for the widespread though limited presence of a novel class of GPCRs in a variety of unicellular eukaryotes. These include free-living organisms and organisms that are pathogenic for plants, animals, and humans. The novel GPCRs have a C-terminal phospholipid kinase domain, pointing to a direct link between sensing external signals via GPCRs and downstream intracellular phospholipid signaling. Genes encoding these receptors were likely present in the last common eukaryotic ancestor and were lost during the evolution of higher eukaryotes. We further describe five other types of GPCRs with a catalytic accessory domain, the so-called GPCR-bigrams, four of which may potentially have a role in signaling. These findings shed new light onto signal transduction in microorganisms and provide evidence for alternative eukaryotic signaling pathways. Oomycetes occupy environmental niches similar to those occupied by fungi but do not belong to the fungal kingdom . Instead century .2, are both important membrane components and universal signaling components. For some time, GPCR-PIPKs were thought to have limited phyletic distribution, as only one homologue was identified outside the oomycetes, namely, RpkA . De. DePhyto species .10.1128/mBio.02119-17.4TABLE S1 TABLE S1, DOCX file, 0.1 MB.Overview of the number of GPCR-bigrams in all species included in this study and references to genome papers. Download Copyright © 2018 van den Hoogen et al.2018van den Hoogen et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02119-17.5TABLE S2 TABLE S2, XLSX file, 0.2 MB.Overview of PIPKs. (a) Details of oomycete GPCR-PIPKs. (b) Details of non-oomycete GPCR-PIPKs. (c) Details of oomycete type I, II, and III PIPKs. (d) Details of type I, II, and III PIPKs in species having one or more GPCR-PIPKs. Download Copyright © 2018 van den Hoogen et al.2018van den Hoogen et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Aureococcus anophagefferens   . In unikebozoans . GPCR-PI trahens . AdditioRhizaria . Naeglerears ago . In summears ago .10.1128/mBio.02119-17.1FIG S1 P. infestans. The numbers in bold represent the number of protein models present after each successive step. Download FIG S1, PDF file, 0.4 MB.(A) Search strategy used to identify GPCR-PIPKs. The total numbers of identified GPCR-PIPKs are highlighted in bold. (B) Search strategy used to inventory GPCRs and GPCR-bigrams in Copyright © 2018 van den Hoogen et al.2018van den Hoogen et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the To identify conserved amino acid motifs in GPCR-PIPKs, we made a multiple-sequence alignment of all identified GPCR-PIPKs. In most GPCR-PIPKs, conserved motifs, such as the catalytic DLGKS and MDYSL motifs  and the Reticulomyxa filosa, a species in the supergroup Rhizaria, targeted two truncated gene models, one containing a GPCR region N-terminal of the LRxGI motif and the other with a PIPK domain at the C terminus. The genome assembly of R. filosa is highly fragmented . Split-color bars indicate truncated gene models. The color coding of the species names corresponds to the coding of the supergroup colors described for Copyright © 2018 van den Hoogen et al.2018van den Hoogen et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02119-17.3FIG S3 FIG S3, PDF file, 0.1 MB.Simplified phylogeny of oomycetes and related organisms based on the supertree as presented by McCarthy et al. . DownloaCopyright © 2018 van den Hoogen et al.2018van den Hoogen et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the A. anophagefferens GPCR-PIPK forms a clade together with the oomycete GPCR-PIPK D9  Inventory of GPCRs and GPCR-bigrams in P. infestans. (b) Details of GPCR-TKLs. (c) Details of GPCR-INPPs. (d) Details of GPCR-AC. (e) Details of GPCR-PDEs. (f) Details of AP-GPCRs. Download TABLE S3, XLSX file, 0.3 MB.Overview of GPCRs in Copyright © 2018 van den Hoogen et al.2018van den Hoogen et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. infestans genome, 17 have a C-terminal tyrosine kinase-like (TKL) domain, making the GPCR-TKLs the largest group of GPCR-bigrams in P. infestans. The occurrence of bigrams consisting of a TKL domain and TM domains was already reported previously in a kinome inventory of P. infestans  domain located C-terminally of the kinase domain  domain as an accessory domain. omycetes . INPPs aCR-PIPKs .P. infestans gene models, has adenylyl cyclase (AC) as a C-terminal accessory domain, while the other type, encoded by three gene models, has phosphodiesterase (PDE) as a C-terminal accessory domain. Adenylyl cyclases catalyze the conversion of ATP into cyclic AMP, while PDE can hydrolyze cAMP or other cyclic nucleotides to ATP or NTP, respectively. Other oomycetes have comparable numbers of genes encoding GPCR-ACs and GPCR-PDEs, although genes encoding GPCR-PDEs could not be identified in some species  as an accessory domain. ng an AP . On the P  . This am38–Amoebozoa and GPCR-INPPs are found only in the stramenopile alga Nannochloropsis, while GPCR-ACs, GPCR-PDEs, and AP-GPCR are restricted to oomycetes. The large number of genes encoding GPCR-bigrams in oomycetes (around 44 in each species) strongly suggests that these organisms evolved toward G-protein signaling pathways. The catalytic domains found in GPCR-bigrams are also found in proteins with a canonical domain organization. For example, P. infestans has four GPCR-ACs in addition to eight ACs with a universal domain composition (data not shown).While GPCR-PIPKs are present throughout the eukaryotic kingdom, the distribution of other GPCR-bigrams is more limited. Apart from oomycetes, GPCR-TKLs are found only in sα) or Gβγ subunits and are deactivated by inhibitory Gα subunits (Giα). Higher metazoans have multiple Gα, Gβ, and Gγ subunits. Combinations of different subunits give rise to a large variety of heterotrimeric G-protein complexes, each with specific functions. In contrast, oomycetes have only one Gα, Gβ, and Gγ subunit and thus only one uniform heterotrimeric G-protein complex. Silencing of the Gα subunit gene or the Gβ subunit gene in P. infestans results in aberrant swimming behavior of zoospores or deficiencies in sporulation, respectively, but does not affect viability , Giardia (Excavates), and Dictyostelium (unikonts) , cAMP-dependent protein kinase A (PKA), or protein kinase C (PKC) . Howeverdetected . PKC is nikonts) , 44. It nikonts) . GPCR-TKnikonts) . In S. cesponses . The preP. infestans, only two proteins have similarity to Rho GTPases. Whether GPCR-PIPKs and GPCR-INPPs indeed bypass G-protein or other signaling intermediates by directly activating the catalytic domain upon ligand binding remains to be tested experimentally.With respect to phospholipid signaling, the presence of GPCR-PIPKs and GPCR-INPPs is indicative of alternative pathways in oomycetes. Among the typical activators of PIPKs are Rho GTPases , PKA 4848, and pP. infestans and P. sojae are differentially expressed during development  against the NCBI, UniProt, FungiDB  PIPKs, using the positions of SMART and InterProScan protein domain predictions as a guide (see the positions of PIPK domain regions listed in P. infestans proteins with a GPCR signature, the proteome was screened for proteins with 5 to 9 TM regions using methods described above. Next, the protein domain composition was predicted using methods described above. Protein models with sequence similarity to non-GPCR membrane proteins, such as ion channels, transporters, or non-GPCR receptor proteins, were used. All identified GPCRs and GPCR-bigrams are listed in For making an inventory of


Saccharomyces cerevisiae as an experimental model. We identified the yeast protein Pdp3 that contains a PWWP domain that closely resembles NSD3s PWWP. Our results indicate that the yeast protein Pdp3 and human NSD3s seem to play similar roles in energy metabolism, leading to a metabolic shift toward fermentation. The swapping domain experiments suggested that the PWWP domain of NSD3s functionally substitutes that of yeast Pdp3, whose W21 is essential for its metabolic function.WHSC1L1/NSD3, one of the most aggressive human oncogenes, has two isoforms derived from alternative splicing. Overexpression of long or short NSD3 is capable of transforming a healthy into a cancer cell. NSD3s, the short isoform, contains only a PWWP domain, a histone methyl-lysine reader involved in epigenetic regulation of gene expression. With the aim of understanding the NSD3s PWWP domain role in tumorigenesis, we used  Emerging evidence indicates that cancer is primarily a metabolic disease involving disturbances in energy production through respiration and fermentation . Even thAlthough compelling evidence shows that genomic instability is present to some degree in all tumor cells , it is uGene amplification is a major mechanism for oncogene activation in human cancers –14, resuNSD3 is the third member of the NSD (nuclear receptor SET domain- containing) family. All proteins from this family have been directly linked to multiple human diseases. A striking feature of the three NSD proteins is that they are highly similar within a region of about 700 amino acids spanning a catalytic SET domain together with a pre and post-SET (Enhancer of zeste) domain, two PWWP (proline-tryptophan-tryptophan-proline) domains, five PHD (plant homeodomain) fingers, and a NSD-specific Cys-His rich domain (C5HCH) . HoweverThe PWWP domain is exclusively found in eukaryotes, ranging from lower eukaryotes, such as protozoa and yeast, to human. The human genome encodes more than twenty PWWP-containing proteins, which are always located in the nucleus and play a major role in cell division, growth and differentiation. They are implicated in various chromatin functions, including DNA modification, repair, and transcriptional regulation , 24. TheSaccharomyces cerevisiae has been extensively used as a model for genetic analysis of various complex pathways and processes, including cell division, secretion, transcription and receptor-mediated signal transduction [S. cerevisiae and cancer cells, this microorganism has often been used as a tool for cancer research [The budding yeast sduction . Due to research . There aresearch . There aresearch . Yeast cresearch . Like maresearch –32. Yeasresearch . At leasresearch –32. A suresearch , 34. Howresearch .S. cerevisiae experimental model.In light of these findings, the aim of this study was to evaluate the human NSD3s PWWP domain functionality in the S. cerevisae energy metabolism, since overexpression of NSD3s was reported to transform a human healthy cell into a cell with tumorigenic characteristics [+ yeast strains displayed a specific growth rate (μ) higher than the control (WT). In addition, NSD3s+ cells exhibited a decrease in oxygen consumption when compared to WT cells  exposure  and results in oxidative damage –38. Downe Figure .To examine the heterologous expression of NSD3s in yeast, we used an anti-his tag antibody, since the construct contains an N-terminal six-histidine tag. We performed a western blotting analysis and found that the short isoform of NSD3 was successfully expressed in yeast .ab initio modelling. One can observe that both structures are composed of a N-terminal β-barrel responsible for recognizing and binding histone methylated lysines [Next, we screened protein databases for yeast proteins containing a PWWP domain. Among them, we selected Pdp3, which has a single PWWP domain with 25% sequence identity to that of NSD3s Figure . Pdp3 is lysines  and a C- lysines . TM-scor lysines , 47. The lysines . The TM- lysines . DespiteS. cerevisiae (Δpdp3) and transformed this mutant with a plasmid expressing Pdp3 harboring the tryptophan to alanine at position twenty one (W21A) mutation. Yeast strains were grown in glucose, a carbon source that favors fermentative metabolism, so the metabolic phenotype resembles that of a tumorigenic cell. We reasoned that both Δpdp3 and W21A+ would present similar characteristics when compared to the control. Both yeast strains showed lower specific growth rate compared to the control  exposure  methyltransferase highly selective for lysine 36 (H3K36) [set2(Pdp3)+ strains exhibited the same growth  . As showh Figure  and oxygh Figure . In addih Figure  and 8D.  and H2A . However and H2A . To verir Figure , suggestFinally, a chimeric form of Pdp3 carrying the NSD3 PWWP1 domain was constructed to analyze if the PWWP1 domain of NSD3s would be capable of functionally substituting that of Pdp3. When grown in glycerol, the chimera-expressing cells showed a higher specific growth rate Figure , a lowerS. cerevisiae. Using glycerol (respiratory metabolism fermentative) and glucose as the sole carbon sources we were able to mimic the metabolic profile of a cell with healthy and a tumorigenic characteristics, in order to evaluate their metabolic similarity.The work presented here explores the metabolic function of the PWWP domain of the short isoform of NSD3, a human oncogene, and Pdp3, a PWWP-containing protein from S. cerevisiae, but yeast also makes use of other carbon sources [HXK2 and MIG1, and genes required for derepression, as SNF1 [Glucose is the main carbon source consumed by  sources , 51. Ino sources . Gene ex sources –53. Gene as SNF1 .Replacing glucose by glycerol as the carbon source forces yeast to obtain energy through respiration. When respiratory metabolism is adopted by yeast, glycerol is phosphorylated by glycerol kinase (Gut1) generating glycerol 3-phosphate. Then, glycerol 3-phosphate is oxidized to dihydroxyacetone phosphate (DHAP) by glycerol phosphate ubiquinone oxidoreductase (Gut2) located on the surface of the inner mitochondrial membrane. Electrons from this oxidation are transferred to ubiquinone, entering the respiratory chain , 56. DHAin vivo, since it can adapt its metabolism to heterogeneous microenvironments for fast-growing conditions in malignant solid tumors [Healthy cells use mitochondrial oxidative phosphorylation for energy production , while td tumors . In thisd tumors , is capad tumors . Based od tumors . Taken tS. cerevisiae harbors one PWWP-containing protein, Pdp3, which does not contain any other domain [sas3. Therefore, our results suggest that, under respiratory conditions, the NuA3b complex is responsible for mediating the metabolic shift induced by Pdp3.NSD3s encodes a 645-amino acid protein containing one single PWWP domain , 62. Ther domain . The NuAr domain . Pdp3 depdp3. These results are in accordance with those of Gilbert and co-workers that demonstrated that all three mutations  independently abolish the interaction between Pdp3 and a H3K36me3 peptide, suggesting that Pdp3 requires a conserved aromatic cage to bind chromatin. Since the Pdp3 W21A+ mutant showed the same phenotype as the Δpdp3 strain, we concluded that the hydrophobic cage of Pdp3 PWWP is essential for its metabolic function. While overexpression of NSD3s was able to transform a healthy into a tumorigenic cell, knockdown of NSD3 in 8p11-12–amplified breast cancer cells resulted in loss of growth and survival [A conserved aromatic cage within the Pdp3 PWWP domain is required for H3K36me3 binding. The aromatic residues are conserved in the PWWP domain of Pdp3 at positions F18, W21, and F48 . We founsurvival . TherefoWe then concluded that overexpression of Pdp3 was capable of inducing a shift in metabolism toward fermentation, similar to overexpression of the human oncogene NSD3. The chimeric form of Pdp3, replacing its PWWP domain by that of NSD3s, functionally substitutes the yeast protein Pdp3. These data suggested that NSD3s and Pdp3 play similar roles in energy metabolism, shifting from aerobic to anaerobic metabolism. Cluntun and co-workers demonstrated that histone acetylation is sensed by glucose flux in a dose-dependent manner, which is a possible function of the Warburg effect . These rHuman NSD3s and yeast Pdp3 are both involved in regulation of gene expression and are reported in this work as capable of causing a metabolic shift from aerobic (respiration) to anaerobic (fermentation) metabolism. The phenotypical changes observed in the strains with different profiles of expression of NSD3s and Pdp3 in the yeast model are very similar and agree well with the metabolic shift from a healthy to a carcinogenic cell. These results indicate that the pattern of gene expression is important to the malignant transformation of cell metabolic phenotypes, in accordance with previous reports. In summary, NSD3s and Pdp3 seem to play similar roles in cell metabolism and cells overexpressing both proteins, under conditions that simulate respiration, appear to metabolically behave like a tumorigenic cell. The hydrophobic cage is essential for Pdp3 function. We suggest that the PWWP domain of NSD3s functionally substitutes that of the yeast protein Pdp3. Given that chromatin remodeling and metabolic dysfunction are gaining considerable attention in cancer studies, future researches on the PWWP domains of Pdp3 and NSD3s may contribute to the design of new anti-cancer drugs.MATa; his3; leu2; met15; ura3) and its isogenic mutants Δpdp3, Δset2 and Δsas3 harboring the PDP3, SET2 and SAS3 genes interrupted by KanMX4 were acquired from Euroscarf, Frankfurt, Germany. Stocks of both strains were maintained on solid 2% YPD  in appropriate conditions. For null mutants, the medium also contained 0.02% geneticine. pECUh6NSD3S  and pECUh6PDP3  2 μ plasmids, containing URA3 as selectable marker and harboring the NSD3s and PDP3 genes, respectively, under control of the CUP1 promoter, were used to transform WT strain using the lithium acetate protocol [pdp3 using the same vector. The Pdp3 (PWWP-NSD3s) chimera  (DNA sequence: ACAAAAGATATTAGAACAGGCGATTTAGTGTTATG CAAAGTTGGCTCGTTTCCACCTTGGCCAGCTGTA GTATTTCCACAGCGTTTGCTGCGAAACGATGTATA TAGAAAGAGAAAATCCAATTGTGTTGCTGTTTGTT TTTTCAACGATCCAACTTATTATTGGGAACAACCC AGTAGATTAAAGGAGCTAGATCAAGACAGCATTC ACAATTTCATATTAGAACATAGTAAAAATGCAAAC CAAAGGGAATTGGTCAATGCTTATAAGGAAGCAA AAAATTTTGATGATTTCAACGTATTTTTACAAGAA AAGTTTGAAGAAGAAAACAGGTTAAGTGATCTAA AAGCGTTTGAGAAAAGTGAAGGTTCTAAAATCGT TGCCGGAGAAGATCCCTTTGTAGGTCGAACAAAA GTAGTGAATAAAAGAAAAAAAAATTCAATATCC) by the PWWP1 domain of NSD3s  (DNA sequence: ATGACAACGGAAGTGTCCACTGGTGTTAAGTTTC AGGTTGGCGATCTTGTGTGGTCCAAGGTGGGAAC CTATCCTTGGTGGCCTTGTATGGTTTCAAGTGATCC CCAGCTTGAGGTTCATACTAAAATTAACACAAGA GGTGCCCGAGAATATCATGTCCAGTTTTTTAGCAA CCAGCCAGAGAGGGCGTGGGTTCATGAAAAACG GGTACGAGAGTATAAAGGTCATAAACAGTATGAA GAATTACTGGCTGAGGCAACCAAACAAGCCAGCA ATCACTCTGAGAAACAAAAGATTCGGAAACCCCG ACCTCAGAGAGAACGTGCTCAGTGGGATATTGGC ATTGCCCATGCAGAGAAAGCATTGAAAATGACTA GAGAAGAAAGAATAGAACAGTATACTTTTATTTAC ATTGATAAACAGCCTGAAGAGGCTTTATCCCAAG CAAAAAAGAGTGTTGCCTCCAAAACCGAAGTTA AAAAAACCCGACGACCAAGATCTGTGCT). WT and Δpdp3 strains were also transformed with the pECUh6 vector, as a control. Transformants were selected in drop out medium  supplemented with 2% glucose. For all experiments, cells were grown up to mid-exponential phase (0.8 mg dry weight/ml) in liquid drop out medium, with or without uracil, and with 2% glucose or 4% glycerol, at 28°C and 160 rpm, with the ratio/flask volume medium of 5:1. The CUP1-regulated expression was induced with 50 μM CuSO4 [WT strain BY4741 (protocol . Similarab initio modelling of the NSD3s and Pdp3 PWWP domains [The sequences of NSD3s (UniProt #Q9BZ95-3) and Pdp3 (UniProt #Q06188) PWWP domains were retrieved from the UniProt database . The seq domains . The TM- domains .The specific growth rate (μ) during the exponential phase was determined from a linear regression fit of the semilog plot of cell growth.°C in cell suspensions at 10 mg dry weight/mL incubated in 100 mM glucose and 50 mM Tris–HCl (pH 4.5) using a computer-interfaced Clark electrode operating in an air-tight chamber with continuous stirring [- completely abolished the oxygen consumption of WT cells (both when CN- is added in the middle of the curve or when cells are previously incubated for 20 min), confirming that oxygen consumption was solely due to mitochondrial activity  equipped with a refractive index detector. An Aminex HPX-87H column  was used for separation. The HPLC apparatus operated with a mobile phase of 0.004 mM sulfuric acid at a flow rate of 0.6 mL/min , 72.2O2 [Cells at the first exponential phase growing on drop out medium glucose 2% or glycerol 4% were directly stressed (3 mM or 20 mM H2O2 , respect2O2 , 74, 75.2O2 –78.t -test, which denotes homogeneity between experimental groups at p < 0.05.Data were expressed as mean values ± SD of at least three independent experiments. Values were compared by Student's


Hence, the three dimensional structure of MLL5 SET domain unveils the structural basis for its lack of methyltransferase activity and suggests a new regulatory mechanism.Mixed Lineage Leukemia 5 (MLL5) plays a key role in hematopoiesis, spermatogenesis and cell cycle progression. Chromatin binding is ensured by its plant homeodomain (PHD) through a direct interaction with the N-terminus of histone H3 (H3). In addition, MLL5 contains a Su(var)3-9, Enhancer of zeste, Trithorax (SET) domain, a protein module that usually displays histone lysine methyltransferase activity. We report here the crystal structure of the unliganded SET domain of human MLL5 at 2.1 Å resolution. Although it shows most of the canonical features of other SET domains, both the lack of key residues and the presence in the SET-I subdomain of an unusually large loop preclude the interaction of MLL5 SET with its cofactor and substrate. Accordingly, we show that MLL5 is devoid of any  MLL5 was initially identified as a candidate tumor suppressor gene located in the commonly deleted 2.5 Mb segment of human chromosome band 7q22 in myeloid malignancies [gnancies . Knock-ognancies  and normgnancies . A recenDrosophila counterpart UpSET, respectively. Interestingly, both the Drosophila UpSET and the Yeast ortholog SET3, which are found in histone deacetylase complexes, were shown to contain inactive SET domains [in vitro and does not exhibit HMT activity on full-length histones. Having provided compelling evidence that MLL5 is not a functioning enzyme, the biological role of this protein remains to be established.Full-length MLL5 is a 205 kDa protein (1858 residues), although a smaller isoform (around 75 kDa) including a single Plant HomeoDomain (PHD) and a Su(var)3-9, Enhancer of zeste, Trithorax (SET) domain has been described [ domains ,11. To bThe MLL5 protein contains a SET domain (residues 323 to 433) followed by a POSTSET domain (residues 434 to 473). The prominent feature of the POSTSET domain is a zinc-binding cage formed from three cysteine residues of the C-terminal region with a fourth cysteine provided by the SET-C subdomain . A commoSequence analysis of the MLL5 SET domain suggests that it does not contain all the conserved sequence elements required for methyltransferase activity. Contrary to other SET-domain containing methyltransferases, MLL5 does not have the residues usually involved in cofactor binding . InsteadDrosophila, yeast and amphioxus sequences shows that MLL5 and SETD5 form a family that is clearly distinct from the MLL1-4, SETD1A, B group  family of SET domain-containing methyltransferases includes at least six members , all of which are involved in gene activation . All mem B group , Fig 1B.Drosophila UpSET also lack the XGXG, Y and NH catalytic sequence signatures present in MLL1-4  as well as a longer fragment containing an additional N-terminal helix , which was previously shown to be important for the structural integrity of the SET domain ,16. IsotSimilarly, we ran fluorescence anisotropy experiments to measure the affinity of the presumed substrate H3K4 peptide (ARTKQTARKST), with un-methylated or mono-methylated lysine 4 , for the-4–10-7 M range) did not reveal any resonances of atoms from the peptide in interaction with the protein. Identical results were obtained with all forms of the H3K4 peptide.To ensure that the MLL5/H3K4 peptide interaction was not inhibited by the fluorescein moiety attached to the N-terminus of the peptides, we conducted NMR titration and saturation transfer difference (STD) experiments to monitor the interaction between the SET-POSTSET fragment and the unlabeled non-, mono- and di-methylated H3K4 peptides. Titration and STD analyses were performed by increasing the protein/peptide ratio from 1/20 to 1/5 in a stepwise manner. As shown in As we did not observe any interaction of the MLL5 SET domain with non-, mono- or di- methylated H3K4 peptides, we sought to identify other histone substrates for the MLL5 SET domain using GST-pull down and a mixture of histones from calf thymus. As shown in Taken together, our data indicate that the isolated MLL5 SET domain in combination with the POSTSET region fails to bind either the cofactor SAM or any histone substrate. In addition, the presence of the N-terminal flanking region is not helpful for the SAM binding.in vitro histone methyltransferase assay (HMT) to address whether the MLL5 SET domain is enzymatically functional. We followed the same protocol as the one used in the study of UpSET, the Drosophila ortholog of MLL5, showing that this SET domain does not exhibit histone methyltransferase activity [Next, we performed an activity . We founactivity , the MLLIn order to better understand the molecular basis for the absence of HMT activity in the MLL5 SET domain, we solved its crystal structure. Attempts to crystallize the Nh-SET-POSTSET and SET-POSTSET fragments were unsuccessful. NMR studies  showed t121, and diffracted up to 2.1 Å resolution , due to the presence of a pseudo-translation along the c axis. A pool of 16 homologous SET models with less than 30% sequence identity was selected for initial molecular replacement trials. The best template models were then edited based on the local fit to the density maps to produce an ensemble of viable search models. Finally, by combining molecular replacement with this ensemble model in the large cell using Phaser-MR [The SET domain alone (residues 323 to 458) crystallized in the space group P3solution , Fig 4A.haser-MR  and cyclhaser-MR  and Parrhaser-MR  were equThe asymmetric unit contained two unliganded MLL5 molecules which superposed with a root mean square deviation (RMSD) of 0.64 Å (110 Cα atoms). Molecule A, which includes most of the SET polypeptide chain  was used for the structural analysis. Molecule B had well defined electron density for SET core, however residues 327 to 339 at the N-terminus and 449 to the end at the C-terminus were disordered and could not be modeled from X-ray data. However, the MLL5 POSTSET zinc binding site could be easily modeled using the MODELLER v9.2 program , and the10 helix (α2) is packed against the core on the opposite side by one two-strand anti-parallel β-sheet  and one parallel β-sheet . Finally, a typical pseudo-knot is formed by the C-terminal segment of the SET domain passing through a loop formed by a preceding sequence stretch.The overall tertiary structure of the MLL5 SET domain is similar to that of previously determined SET domain structures . The RMSA careful analysis of the crystal structure of the SET domain reveals several features that most likely account for the lack of catalytic activity of MLL5. A unique structural feature of the SET domain of MLL5 is a very short helix α1  where other SET domains usually display a four-turn α-helix (e.g. DIM-5 and MLL3) . None ofAccordingly, the shortening of this helix results in the formation of a long loop (from Gly 372 to Phe 381) that is very uncommon in SET domains. This large loop between α1 and β5 occupies a groove which, in other SET domains, serves as a substrate docking site . This grOf note, the NH motif that is usually highly conserved in SET domains and necessary for the binding of the SAM cofactor is replaced by a RR (Arg 408-Arg 409) motif in MLL5 Figs  and 6. TRegarding the catalytic center itself, the conserved Tyr 358 which favors the methyl transfer from the cofactor SAM to the histone peptide in other HMTs such as the MLL1-4 proteins ,16 appeaThe data presented here indicate that the isolated and bacterially expressed SET domain of MLL5, either alone or in combination with a complete POSTSET sub-domain, is not capable of binding either the cofactor SAM or the histone substrate and thus completely lacks histone methyltransferase activity. Even if we could not exclude a contribution of post-translational modifications in eukaryotic organisms, this absence of catalytic activity can be explained by several factors. First, the MLL5 protein lacks essential residues for cofactor and substrate binding. Interestingly, comparison of the human MLL5 SET sequence with those of orthologs from various mammalian species indicates that the complete MLL5 SET domain is strictly conserved (not shown). Therefore, the selective pressure on residues within the putative SAM and histone binding sites is comparable to that on structural residues, which suggests that the results obtained here on the human MLL5 protein can likely be applied to all mammalian MLL5 sequences. Second, structural analysis and molecular dynamics simulations of the MLL5 SET domain reveal the presence of a large loop between α1 and β5 that stably folds back to the protein core and prevents histone peptide binding. Previous studies described the reduction or lack of catalytic activity for isolated SET domains due to the incorrect positioning of structural elements around the substrate binding groove , 26, 27.Drosophila, yeast and amphioxus sequences demonstrated that MLL5 and SETD5 belong to the same family, and that this family is clearly distinct from the MLL1-4 and SETD1A, B group. Contrary to MLL5, the SET domains of the MLL1-4 proteins have all the required catalytic residues and the methyltransferase activity of these proteins is well described [Phylogenetic analyses of human, escribed ,16. In tescribed . While cescribed . In addiescribed . CompariDrosophila and yeast) nonetheless suggests that they have important functions in the organism that are independent of the catalytic site. In addition, given the high level of sequence identity between SETD5 and MLL5, it is conceivable that they are functionally redundant, which might explain the relatively mild phenotypes observed in MLL5 knockout mice [Although MLL5 lacks HMT activity, the presence of members of the MLL5/SETD5 family in various species  to indirectly stimulate methylation . Furtherechanism ,33. Likeactivity ,9. Interactivity .In conclusion, we hypothesize that MLL5 functions as a mediator of gene regulation in humans, not through a HMT activity, but as a structural component ensuring multi-protein complex integrity. Thus, MLL5 could have been retained in the genomes of a wide variety of organisms because of its capacity to engage in protein-protein interactions.Homo sapiens), the cephalochordate amphioxus (Branchiostoma floridae), the fruit fly (Drosophila melanogaster) as well as the yeast (Saccharomyces cerevisiae). The first tree was calculated with 24 sequences and a total of 105 amino acid positions, while the second one was obtained based on 22 sequences and a total of 206 amino acid positions. The phylogenetic relationships were assessed using both the Maximum Likelihood (ML) and Bayesian inference (BI) methods. For the ML analyses, two different phylogenetic models were used: the human HMT tree was calculated with the Whelan And Goldman + Freq. model [Amino acid sequences included in the analysis are listed in q. model  and a diq. model  and a diq. model . All ML q. model . To valiq. model . BI phylq. model  in two r6-GST fusion protein (named SET). In order to limit dimerization of the protein, a mutation of cysteine 453 into an alanine was introduced using the QuikChange site-directed mutagenesis lightning kit (Stratagene) (named SETC453A). This mutant was used only for crystallization purpose. Two longer constructs including the POSTSET domain were prepared by sub-cloning a fragment encoding residues 323–473 (named SET-POSTSET) and residues 262–473 (named Nh-SET-POSTSET). These two latest constructs do not contain the C453A mutation. All the proteins were expressed in Rosetta (DE3) E. coli cells. Cultures were grown overnight at 20°C after induction with 0.5mM IPTG. The cell lysate was first applied to a nickel affinity column  washed in 50mM Tris, HCl pH 7.5, 150mM NaCl and 1mM DTT. After cleavage of the GST-tag with PreScission protease on the column, a final purification step was added consisting in a gel filtration on a Superdex 75 column  equilibrated in the same buffer. 20μM of ZnSO4 was added in all purification buffers for the constructs that include the POSTSET domain. Fractions containing the protein of interest were pooled and concentrated to 8 mg/ml. The intact mass of the purified protein was confirmed by ESI mass spectrometry.The human MLL5 cDNA was a gift from Shigeaki Kato . A fragment from this cDNA encoding the SET domain (residues 323–458) was sub-cloned into the pOPINJ vector from Ray Owens's lab , allowinThe protein SETC453A was crystallized by sitting drop vapor diffusion using a 1:1 drop ratio against a mother liquor containing 0.1 M MES pH 6.5 and 25% (w/v) PEG 3000. Crystals around 100 μm in size, were cryo-cooled by immersion in liquid nitrogen using 25% glycerol as cryoprotectant. Datasets were collected at the ID23-1 beam-line at the European Synchrotron Radiation Facility  and processed and scaled with XDS and XSCALE . For molD1) and 128 dummy scans were employed to reduce subtraction artifacts. All NMR Spectra were processed and analyzed using Gifa [The following protein buffer was used for NMR experiments: 50 mM Tris pH 7.5, 150 mM NaCl, 1 mM DTT (addition of 20 μM ZnSO4 for the SET-POSTSET protein). All spectra were recorded on a 500 MHz Bruker Advance spectrometer equipped with a cryogenic H/C/D/N probe with a Z-axis gradient at 300 K. For 1D titration experiments, series of 1D Saturation Transfer Difference (STD) NMR spectra  were recing Gifa  and TopsThe ITC experiment was carried out at 20°C on a MicroCal VP microcalorimeter. The Nh-SET-POSTSET protein sample was buffer exchanged by overnight dialysis into the ITC buffer . Titration of MLL5 Nh-SET-POSTSET domain (30 μM) in the cell (2 mL) was performed by sequential addition of SAM . Integrated raw ITC data were fitted to a one binding site model using nonlinear least squares regression analysis with the MicroCal Origin plugin after the control experiments (titration of the SAM from the syringe into the buffer) were subtracted.® Orange (Sigma) and different concentrations (ranging from 2 to 20 molar excess of protein) of SAM, S-adenosyl homocysteine (SAH) or histone peptide in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT were added to the wells of a 96-well PCR plate. The plates were sealed with an optical sealing tape (Bio-Rad) and heated in an Mx3005P Q-PCR system (Stratagene) from 25°C to 95°C at 1°C intervals. Fluorescence changes in the wells were monitored with a photomultiplier tube. The wavelengths for excitation and emission were 545 nm and 568 nm, respectively.Solutions of 15 μL containing 40 μM of MLL5 SET-POSTSET protein, 2X Sypro4, 2 mM β-mercaptoethanol, 10 mM MgCl2, 10% glycerol.The binding affinities of fluorescein-labeled H3 peptides  for MLL5 SET-POSTSET protein in the presence of SAM (at a concentration of 10 molar excess of the higher protein concentration) were measured using a Safire microplate reader (TECAN) with the excitation wavelength set at 470 nm and emission measured at 530 nm at room temperature. The following buffer solution was used for this experiment: 50 mM Tris HCl pH 7.5, 150 mM NaCl, 20 μM ZnSO9, Cilbiotech) at 4°C in buffer . 24 hours later, 5 μg of bulk histones from calf thymus (Roche) and 1 μl 3H-SAM  were added for 90 min at 30°C. Methylation assay with SET7 was used as a positive control by mixing 5 μg of histones with purified Set 7 (1 μg) in 20 mM Tris-HCl pH 8.0, 4 mM EDTA, 1 mM PMSF, 0.5 mM DTT, and 1 μl 3H-SAM  for 90 min at 30°C. Methylation was measured by liquid scintillation assay as described [3 (pH 9.0) and air dried for 2 hours. The amounts of methylated products were quantified by liquid scintillation on a Liquid Scintillation Analyzer Tri-Carb 2900 TR (Beckman Coulter).GST-MLL5 Nh-SET-POSTSET (1 μg) protein was incubated with or without HeLa nuclear extracts  that form hydrogen bonds with the cofactor SAM (yellow sticks) and the K4 histone peptide (yellow sticks). Corresponding residues of the MLL1 SET domain are superimposed .(TIF)Click here for additional data file.S2 Fig(A). Maximum Likelihood (ML) tree of human HMT proteins, with Bootstrap percentages (ranging from 0 to 100) as branch support indicated at each node. Branch lengths are representative of sequence substitution rates. (B). Bayesian Inference (BI) tree of human HMT proteins, with posterior probabilities (ranging from 0 to 1) as branch support indicated at each node. Branch lengths are representative of sequence substitution rates.(TIF)Click here for additional data file.S3 FigGST pull-down shows that MLL5 SET domain does not bind either histone H3 or a mixture of histones from calf thymus.(TIF)Click here for additional data file.S4 Fig(A). Model of the MLL5 SET-POSTSET fragment. The MLL5 POSTSET zinc binding site was modeled using the MODELLER v9.14 program (32), and the homologous POSTSET domain of SETD2 methyl transferase (pdb entry 4H12) (50) as template. (B). Superposition of the 1H-15N HSQC spectra of MLL5 SET domain (blue) and MLL5 SET-POSTSET fragment (green) recorded at 500 MHz. The 2D heteronuclear experiments were recorded at 300K, with the two proteins at a concentration of 400 and 100 μM concentration respectively, in 50 mM Tris pH7.5, 150 mM NaCl, 1mM DTT. Peaks labeled with a red asterisk are present only in the spectrum of the SET domain, whereas peaks framed in a box are present only in the spectrum of SET-POSTSET fragment.(TIF)Click here for additional data file.S5 Fig1H-15N HSQC spectra of MLL5 SET-POSTSET domain (green) and Nh-SET-POSTSET (purple) recorded at 500 MHz. The 2D heteronuclear experiments were recorded at 300K, with the two proteins at a concentration of 100 mM concentration, in 50 mM Tris pH7.5, 150 mM NaCl, 1 mM DTT.Superposition of the (PDF)Click here for additional data file.S6 FigSuperposition of the final MLL5 SET domain structure (same colors as in (TIF)Click here for additional data file.S7 FigPlot of the distance between Arg 365 and Glu 392 (in red) and, as comparison, of the distance between Lys 376 and Asp 449. Asp 449 is localized in the POSTSET flexible segment. These values correspond to the distance between the center of mass of the oxygens in the acidic side chain and center of mass of the nitrogens in the basic side chain. The cutoff distance between the oxygen atoms of acidic residues and the nitrogen atoms of basic residues is 3.2A.(TIF)Click here for additional data file.S1 File(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


Immunoglobulins (IG) or antibodies and T cell receptors (TR) are managed and described in the IMGT® databases and tools at the level of receptor, chain and domain. The analysis of the IG and TR variable (V) domain rearranged nucleotide sequences is performed by IMGT/V-QUEST  and, for next generation sequencing (NGS), by IMGT/HighV-QUEST, the high throughput version of IMGT/V-QUEST . In vitro combinatorial libraries of engineered antibody single chain Fragment variable (scFv) which mimic the in vivo natural diversity of the immune adaptive responses are extensively screened for the discovery of novel antigen binding specificities. However the analysis of NGS full length scFv (~850 bp) represents a challenge as they contain two V domains connected by a linker and there is no tool for the analysis of two V domains in a single chain.IMGT®, the international ImMunoGeneTics information system® " has been implemented in IMGT/V-QUEST and, for NGS, in IMGT/HighV-QUEST for the analysis of the two V domains of IG and TR scFv. It proceeds in five steps: search for a first closest V-REGION, full characterization of the first V-(D)-J-REGION, then search for a second V-REGION and full characterization of the second V-(D)-J-REGION, and finally linker delimitation.For each sequence or NGS read, positions of the 5′V-DOMAIN, linker and 3′V-DOMAIN in the scFv are provided in the ‘V-orientated’ sense. Each V-DOMAIN is fully characterized . The functionality is generic and can analyse any IG or TR single chain nucleotide sequence containing two V domains, provided that the corresponding species IMGT reference directory is available.The “Analysis of single chain Fragment variable (scFv)” implemented in IMGT/V-QUEST and, for NGS, in IMGT/HighV-QUEST provides the identification and full characterization of the two V domains of full-length scFv (~850 bp) nucleotide sequences from combinatorial libraries. The analysis can also be performed on concatenated paired chains of expressed antigen receptor IG or TR repertoires. IMGT® comprises 7 databases, seventeen online tools and more than 20,000 pages of Web resources, available at the IMGT® Home page , 5. The IG and TR are managed and described in the IMGT® databases and tools at the level of receptor, chain and domain . A complThe analysis of the IG and TR V domain rearranged nucleotide sequences is performed by IMGT/V-QUEST  and, for NGS, by IMGT/HighV-QUEST, the high throughput version of IMGT/V-QUEST (online since October 2010), maximum of 500,000 sequences per batch, set comparison of 1 million results). IMGT/V-QUEST and HighV-QUEST use the same algorithm and the same IMGT reference directories .So far, the analysis has been performed on each V domain individually. The Sanger sequencing of single chain Fragment variable (scFv) was done on a case by case basis using IMGT/V-QUEST. Indeed scFv are single chains of approximate molecular weight of 26,000 Da, encoded by about 800–900 nucleotides with two V domains connected by a linker of about 45-60 nucleotides Fig. , and theIn this paper, we describe a new advanced IMGT/V-QUEST functionality “Analysis of single chain Fragment variable (scFv)” for the identification and characterization of the two variable domains of scFv, generic for IG and TR, and implemented, for NGS, in IMGT/HighV-QUEST.The algorithm proceeds in five steps Fig. : search For a selected species and receptor type (IG or TR), the IMGT/V-QUEST tool first searches the submitted sequence for the closest V-REGION by comparison with the IMGT reference directory of the V groups of the selected receptor type  . The IMGThe first closest V-REGION identified is the one with the highest score which would have been detected in a classical IMGT/V-QUEST analysis ”). There is no search priority for a given V group or for a respective order position (5′ or 3′) in the submitted sequence.If no V-REGION is identified, IMGT/V-QUEST complementary reverses the submitted (input) sequence automatically, and the search is performed again. If a closest V-REGION is identified, this defines the complementary reverse sequence as being in the ‘sense’ orientation for the V-REGION Fig. .The following steps of the algorithm are performed on scFv sequences in which the V-REGION has a ‘sense’ orientation, and therefore are designated as ‘V-orientated scFv’ (either from the direct input scFv sequence or as a result of a complementary reverse step).i.identifies the names of the closest germline V-GENE and allele and J-GENE and allele, with score and percent (%) of identity –26 by alii.adds gaps according to the IMGT unique numbering  and deteiii.delimits the V-(D)-J-REGION, i.e., the V-DOMAIN ,iv.provides a detailed analysis of the V-(D)-J junction and the identification of the D genes and alleles for IGH, TRB and TRD performed by the integrated IMGT/JunctionAnalysis tool , 28,v.provides an extensive analysis of the nucleotide (nt) mutations and amino acid (AA) changes, resulting for the IG from somatic hypermutations, by comparison with the closest V-REGION,vi.localizes the mutation hotspots in the closest germline V gene and allele,vii. and finally, annotates the V-(D)-J-REGION identified with IMGT labels using IMGT/Automat , 30.The full characterization of the first identified V-(D)-J-REGION -J-REGION , a similar search is performed between the 5′ end of the V-orientated sequence and the 5′ end of ‘D1’, provided that this part of the sequence has at least a length of 200 nt. If a V-REGION is identified, the full characterization of the second V-(D)-J-REGION -J-REGION (‘V-DOMAIN 1’ and ‘V-DOMAIN 2’) are characterized, the sequence between them is delimited and defined as ‘linker’ ” is freely available online for academics . The scFv sequences are submitted in FASTA format .The “Analysis of single chain Fragment variable (scFv)” is selected as an option in “Advanced functionalities” at the bottom of the IMGT/V-QUEST Search page Fig. .Fig. 3IMThe user can choose one of the three displays: A. Detailed view. B. Synthesis view. C. Excel file. Selecting the “Analysis of single chain Fragment variable (scFv)” adds automatically the file ‘12’ in the display C. Excel file.The results are displayed even if the V-(D)-J-REGION (V-DOMAIN) are partial and/or not fully characterized, the only requirement being that at least the V-REGION has been identified, which is the condition for IMGT/V-QUEST to give results.The top of the page IMGT/V-QUEST Detailed view results for scFv Fig.  recalls The Detailed results show the Number of analysed sequences  and the Number of analysed V-DOMAIN . The table “Identified scFv” comprises one line per identified scFv in the submitted sequence set. It includes the sequence ID (the one from the flat file header), the 5′V-DOMAIN ID, positions and length, the linker positions and length, the 3′V-DOMAIN ID, positions and length Fig. .The 5′V-DOMAIN ID and 3′V-DOMAIN ID consists of the sequence ID, followed by an underscore and a capital letter for the locus as identified by IMGT/V-QUEST , and preceded by a number which indicates the V domain analysis order in the submitted set. Clicking on the 5′V-DOMAIN ID or 3′V-DOMAIN ID link leads to the corresponding classical detailed view Fig. .The complete V-orientated sequence of the scFv is shown in the result of each domain, with the corresponding analysed V domain being highlighted in green.If the option ‘Search for insertions and deletions’ has been selected, insertions detected are reported in capital letters in the sequence of the corresponding V domain(s). The IMGT/V-QUEST results per domain are given after filling the deletions and removing the insertions .The Result summary table of each V domain Fig.  is folloThe top of the page IMGT/V-QUEST Synthesis view results for scFv Fig.  recalls,Each identified V-(D)-J-REGION (V-DOMAIN) appears individually on a different line of the Summary table. Pairs of V-DOMAIN belonging to the same scFv, are identified by having the same sequence ID Fig. . For eacClassically, the IMGT/V-QUEST Excel file or, for NGS, the IMGT/HighV-QUEST comma separated values (CSV) files results include eleven Excel spreadsheets or CSV files, respectively . TypicalAn additional file “12_scFv” Fig.  is speciThe IG scFv are usually made of a VH and of VL. The order of the V-DOMAIN in an scFv can be either VH-linker-VL or VL-linker-VH. Both V domains of an scFv are transcribed in a single chain and are necessarily in the same orientation of transcription. Different associations of V domains (the two domains being in the same orientation) are possible for the IG or for the TR. Analysis of scFv V-DOMAIN by IMGT/V-QUEST is done against a single species. For the TR, other V domain associations per permutation between any two V domains (not shown) are also analyzable.V-DOMAIN analysis order: order in the submitted scFv sequences set (as in the ten first spreadsheets or files),V-DOMAIN ID: V-DOMAIN identifier (SequenceName_LocusLetter),V-DOMAIN positions: begin and end position of the identified V-DOMAIN in the V-orientated scFv sequence,V-DOMAIN length: as determined by the begin and end positions,V-DOMAIN Functionality: productive or unproductive,V-GENE and allele: IMGT gene and allele name of the closest germline V-REGION,V-REGION score: alignment score with the closest germline V-REGION,V-REGION identity %: identity percentage with the closest germline V-REGION,V-REGION identity nt: number of identical nt with the closest germline V-REGION, J-GENE and allele: IMGT gene and allele name of the closest germline J-REGION, J-REGION score: alignment score with the closest germline J-REGION, J-REGION identity %: identity percentage with the closest germline J-REGION, J-REGION identity nt: number of identical nt with the closest V germline J-REGION, D-GENE and allele: IMGT gene and allele name of the closest germline D-REGION , D-REGION reading frame: reading frame 1, 2 or 3 , CDR_lengths: length of the 3 CDR-IMGT, AA JUNCTION: amino acid sequence of the junction, JUNCTION frame: frame of the junction (in-frame or out-of-frame), Comments: to highlight the particularities of the V-DOMAIN, if any.Each set of 19 columns for each V domain comprises the following fields:It should be noted that sequences not identified as scFv  V-DOMAIN or V-REGION is identified) are not integrated in the “12_scFv” spreadsheet or file, so this spreadsheet or file may be empty if none of the submitted sequences are identified as scFv.As the online version of IMGT/V-QUEST can analyze 50 sequences per run, the results for scFv analysis may potentially include the analysis of up to 100 V-DOMAIN. With the option “Search for insertions and deletions”, the number of submitted sequences is restricted to 10, and the results for scFv may include the analysis of up to 20 V-DOMAIN.In the IMGT/HighV-QUEST, the option “Search for insertions and deletions” is selected by default and the analysis includes all the identified V-DOMAIN. The new advanced functionality “Analysis of single chain Fragment variable (scFv)” provides the identification and characterization of, theoretically, up to one million domains for 500,000 submitted scFv sequences. This functionality has introduced, for the first time, the possibility of analysing simultaneously the two V domains of large scFv data sets from combinatorial libraries.Escherichia coli [In antibodies and T cell receptors, the antigen binding sites comprise two V-DOMAIN which are paired at the N-terminal end of the heavy and light chains for the IG and of the alpha and beta (or gamma and delta) chains for the TR –3. The phia coli , 44 and hia coli –47, by-phia coli –51.Next generation sequencing (NGS) has recently emerged as a new method for the high-throughput characterization of IG and TR immune repertoires both in vivo and in vitro. Currently available NGS platforms allow the simultaneous sequencing of millions of reads. However, two challenges remain for the NGS sequencing of scFv: first, the scFv length is > 800 bp, which is too long for most NGS platforms; and second, there is no tool for the analysis of two V domains in a single chain. Up to now, NGS methods have only provided reads encompassing one V domain (400 bp), therefore losing a critical piece of information found in scFv sequences, that of the association of two specific V domains (VH and VL for the IG) by the peptide linker. Although a few approaches have been proposed, retrieving information regarding V domain association has still not been solved –54.As reliable data depend on high-quality and long enough sequences to contain the full-length scFv, the new functionality “Analysis of single chain Fragment variable (scFv)” was implemented for providing the identification and full characterization of the two V domains in scFv sequences or NGS reads fulfilling these criteria.The functionality “Analysis of single chain Fragment variable (scFv)” provides the identification and full characterization of the two V domains of full-length scFv in IMGT/V-QUEST online or, for NGS, in IMGT/HighV-QUEST. The functionality was used to analyse >450,000 reads of about 1000 bp, obtained from a combinatorial library, generated with the Pacific Biosciences (PacBio) RS II platform using single molecule, real-time (SMRT) circular consensus sequencing (CCS). The two V domains were identified and characterized in all reads of high-quality and sufficient length. The “Analysis of single chain Fragment variable (scFv)” will facilitate and improve the description of the scFv content of combinatorial libraries, a key information in therapeutic antibody discovery, selection and development.The need for the analysis of sequences containing two V domains from expressed repertoires is also rapidly rising. NGS single-cell sequencing of paired chains have been obtained by a technology comprising flow focusing and encapsulation of single cells in emulsions containing magnetic beads for mRNA capture, reverse-transcription of mRNA transcripts, physical linkage of the partners by overlap extension PCR, and NGS sequencing . Other d


To enable high-speed underwater wireless optical communication (UWOC) in tap-water and seawater environments over long distances, a 450-nm blue GaN laser diode (LD) directly modulated by pre-leveled 16-quadrature amplitude modulation (QAM) orthogonal frequency division multiplexing (OFDM) data was employed to implement its maximal transmission capacity of up to 10 Gbps. The proposed UWOC in tap water provided a maximal allowable communication bit rate increase from 5.2 to 12.4 Gbps with the corresponding underwater transmission distance significantly reduced from 10.2 to 1.7 m, exhibiting a bit rate/distance decaying slope of −0.847 Gbps/m. When conducting the same type of UWOC in seawater, light scattering induced by impurities attenuated the blue laser power, thereby degrading the transmission with a slightly higher decay ratio of 0.941 Gbps/m. The blue LD based UWOC enables a 16-QAM OFDM bit rate of up to 7.2 Gbps for transmission in seawater more than 6.8 m. Transmission in pure seawater revealed a higher performance level than the other available carriers didet al. employed a double freeform reflector to collimate the LED light for improving the throughput power and SNR of the UWOC systemet al. demonstrated an LED-based single-input-multi-output UWOC system that enabled an effective transmission distance of over 60 mTo satisfy the requirement of high-capacity underwater machine-to-machine data transmission, a high-speed underwater optical communication (UWOC) system is highly effective compared with currently available carriers such as microwaves and acoustic waves. In contrast to acoustic wave and microwave carriers, light emitting diodes (LEDs) with lower attenuation were proposed for implementation in the UWOC system to provide a transmission data rate of up to several tens of Mbps1314151618et al. employed a frequency-doubled green laser at 532 nm to implement a UWOC link at 1 Gbps over a 2-m long water pipeet al. applied a directly OFDM-modulated blue LD at 405 nm to enact a 4.8-m underwater transmission at 1.45 Gbpset al. demonstrated a UWOC system with a nonreturn-to-zero on-off-keying (NRZ-OOK) data format at 2.3 Gbps over 7 m by utilizing a TO-can packaged LD at 520 nmet al.et al. demonstrated a 450-nm LD-based UWOC system for carrying 4.8-Gbps 16-QAM OFDM dataet al. proposed a 4.883-Gbps UWOC system for transmitting 32-QAM OFDM data over 6 mAs usual, the proposed LED-based UWOC system inevitably suffered from an insufficient modulation bandwidth, thereby decreasing the allowable data rate and the reachable transmission distance. Therefore, a laser diode (LD) with a high coherence for providing large modulation bandwidths and small beam divergence was proposed to construct high-speed UWOC networks24252627In this study, the OFDM subcarrier amplitude pre-emphasized data format was employed to directly modulate the blue LD at 450 nm and optimize its transmission performance to more than 10.2 m in both tap-water and seawater environments. The blue LD was observed to be overly biased for carrying 16-QAM OFDM data. After over-biasing and pre-emphasizing, the transmitted error vector magnitudes (EVMs), SNRs and bit-error-rates (BERs) of the received data format at different biased currents and pre-leveling slopes were compared and discussed. With the reflector-folded underwater path scheme, three underwater transmitting distances of 3.4, 6.8, and 10.2 m were selected to characterize the maximal allowable transmission data rates before and after OFDM subcarrier amplitude pre-leveling.P-I) response illustrated in dP/dI slope and external quantum efficiency were calculated as 0.7 W/A and 0.25, respectively. From the frequency response of the 450-nm blue LD shown in th). To carry 16-QAM OFDM data at 8 Gbps covering a bandwidth of 2 GHz for transmission over the 1.7-m long UWOC system, the bias current was increased from 70 to 95 mA. The received constellation plot revealed an EVM reduction from 13.3% to 12.6% resulting from significant RIN suppression and appropriate data-clipping, as shown in The temperature-controlled package of a 450-nm blue LD in TO-can is illustrated in –3, respectively, were obtained with corresponding subcarrier SNR spectra and constellation plots, as shown in 35−3 to 2.9 × 10−3, verified the blue LD-based UWOC system for 12.4-Gbps QAM-OFDM transmission over 1.7 m.To enlarge the allowable transmission bit rate of the 450-nm blue LD-based 1.7-m UWOC system, the 16-QAM OFDM data bandwidth was gradually increased to evaluate the trend of EVM degradation, as shown in −3. The penalty for expanding the underwater distance to 6.8 m is a decrease in the data rate to 8.8 Gbps, with corresponding SNR of 15.2 dB and BER of 3.8 × 10−3. Under the FEC criterion, 5.2-Gbps data with an average SNR of 15.2 dB and a BER of 3.6 × 10−3 was obtained after propagation over 10.2 m. With the OFDM subcarrier intensity pre-leveling slopes of 0.51, 0.51, and 1 dB/GHz, the 3.4-, 6.8-, and 10.2-m UWOC links were able to further increase their data rates to 12, 9.2, and 5.6 Gbps, respectively, thereby providing improved EVMs, SNRs, and BERs, as shown in To enhance the applicability of the 450-nm blue LD-based UWOC system, the underwater distance was increased in turn from 1.7 to 3.4, 6.8, and 10.2 m to analyze the maximum allowable transmission capacities at different distances. Images of the blue LD-based UWOC system with the longest distance of 10.2 m is presented in To perform undersea transmission, the tap water was mixed with commercially available sea salt purchased from an aquarium store to replicate seawater, as shown in −3. The added sea salt polluted the tap water to induce the scattering of impurities and attenuate the transmitted power of the blue LD carrier. Under an identical distance of 6.8 m, the maximal transmittable data rate of 6.8 Gbps in seawater was less than that of the 8.8 Gbps recorded in tap water. The encodable data bandwidth was suppressed from 1.7 to 0.9 GHz as the underwater distance increased from 6.8 to 10.2 m, thereby achieving a raw data rate of 3.6 Gbps with an average EVM of 15.7%, an average SNR of 16.1 dB, and a BER of 1.6 × 10−3. Similarly, the OFDM subcarrier intensity pre-leveling technique was adopted to optimize the transmission capacity of the seawater-based UWOC system at different transmission distances. Pre-leveling the OFDM subcarriers somewhat improved the 6.8- and 10.2-m transmissions from 6.8 to 7.2 Gbps and from 3.6 to 4 Gbps, respectively, as shown in −3, as shown in For 6.8-m transmission in seawater, the affordable OFDM bandwidth was degraded to 1.7 GHz to allow a raw data rate of only 6.8 Gbps with an average EVM of 16.8%, as shown in –3, respectively. For the proposed UWOC system in the tap-water environment, under the limits of the FEC criterion, a further increase of the underwater distance from 3.4 to 10.2 m revealed that the maximal allowable transmission capacity was slightly reduced from 12 to 5.6 Gbps. Moreover, the bit rate-to-distance decay ratio on the transmission capacity was 0.847 Gbps/m for the UWOC data transmission in tap water. If reflector-induced loss can be released, the transmitted carrier can retain higher power for a longer propagation distance and a higher transmission capacity. By replacing tap water with seawater in the water tank, the proposed UWOC system achieved data rates of 7.2 Gbps over 6.8 m and 4 Gbps over 10.2 m after introducing the OFDM subcarrier pre-leveling technique, which indicates a bit rate-to-distance decay ratio of 0.941 Gbps/m. Note that the seawater-based UWOC system exhibited a higher bit rate-to-distance decay ratio on the transmission capacity than the tap water-based system did. This is because the impurities in the seawater induced light scattering, thereby attenuating the transmitted blue LD power. The UWOC transmission performance exhibited a strong correlation with the blue LD carrier power and the underwater propagation loss. A transmission capacity above 10 Gbps was expected for the blue LD-based UWOC link after excluding reflector loss. This is a breakthrough for exploiting high-speed underwater communication.By directly pre-leveling the 16-QAM OFDM encoded data, the blue LD-based high-speed UWOC system was successfully demonstrated in tap-water and seawater environments at 5.6–12.4 Gbps over 10.2–1.7 meters, and 4–7.2 Gbps over 10.2–6.8 meters, respectively. The pre-leveled OFDM subcarrier power spectrum facilitated the compensation of the significant SNR decline of the received QAM data at high OFDM subcarrier frequencies. After pre-leveling, the tap water-based UWOC system after 1.7-m underwater transmission enabled 12.4-Gbps data delivery with an average EVM, SNR, and BER of 16.8%, 15.5 dB, and 2.9 × 10The experimental setup of the proposed UWOC system based on a 450-nm blue LD  for pre-leveling 16-QAM OFDM data transmission over 10.2 m is illustrated in How to cite this article: Wu, T.-C. et al. Blue Laser Diode Enables Underwater Communication at 12.4 Gbps. Sci. Rep.7, 40480; doi: 10.1038/srep40480 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.