We investigate the properties of Pb(In1/2Nb1/2)O3-Pb(Mg1/3Nb2/3)O3-PbTiO3 between 10 and 300 K using dielectric permittivity measurements. We compare results on single crystal plates measured in the [001] and [111] directions with a polycrystalline ceramic of the same composition. Poled crystals have very different behaviour to unpoled crystals, whereas the dielectric spectrum of the polycrystalline ceramic changes very little on poling. A large, frequency dependent dielectric relaxation is seen in the poled [001] crystal around 100 K. The relaxation is much less prominent in the [111] cut crystal, and is not present in the polycrystalline ceramic. The unique presence of the large relaxation in poled, [001] oriented crystals indicates that the phenomenon is not due their relaxor nature alone. We propose that heterophase dynamics such as the motion of phase domain boundaries are responsible for both the anomalous electromechanical and dielectric behaviour.The mechanisms underlying the anomalously large, room temperature piezoelectric activity of relaxor-PbTiO Their large piezoelectric and dielectric constants, along with low dielectric losses are desirable for a wide range of applications3. Understanding the physical mechanisms responsible for the properties of single crystal relaxor-PbTiO3 ferroelectric materials can provide useful insights for the development of new piezoelectric materials. Much of the recent effort to understand the origins of the excellent room temperature properties of relaxor-PbTiO3 materials has focused on understanding the piezo- or dielectric behaviour of the materials below room temperature. Relaxation step features in piezoelectric and dielectric properties at low temperatures have been reported in single crystal relaxor-PbTiO3 samples6, in addition to the characteristic relaxor-ferroelectric dielectric peaks above room temperature9. It has been suggested that the low temperature relaxations in relaxor-PbTiO3 crystals are related to mechanisms that produce the room temperature properties6.Single crystal relaxor-PbTiO1/3Nb2/3)O3-PbTiO3 (PMN-PT) crystals, Martin et al. and Li et al.10 have shown that at around 200 K the reduction in dielectric permittivity and piezoelectricity with temperature levels off, before dropping sharply between 100 K and 20 K. The drop in permittivity is associated with a peak in the dielectric loss, both of which show a large variation as a function of driving frequency. The step feature suggests a “freezing out” of temperature activated dynamics, which has been cited as evidence that the persistence of polar nano-regions down to lower temperatures gives relaxor-PbTiO3 materials their high room temperature properties11.For rhombohedral Pb based ceramics than relaxor-PbTiO3 single crystals18. In PZT ceramics, freezing out of the motion of domain walls is used to explain broad peaks in the dielectric loss spectra10. There are dielectric data on Fe doped PZT ceramics19 showing a step feature with frequency dispersion very similar to that seen in PMN-PT6. Arlt et al. found good agreement between this data and their domain wall dynamics model19.To explore the implications of low temperature relaxations in dielectric data we can compare data available for a range of materials. Low temperature dielectric data from different ferroelectric and relaxor-ferroelectric materials show a wide range of anomalies and features at cryogenic temperatures. The large dielectric relaxation feature highlighted by Li 3 can give dielectric data that peak to anomalously high values, then reduce close to 0 K23. The peaks are similar to those seen in PMN-PT in their shape and frequency dispersion. The presence of this low temperature relaxation behaviour in ferroelectric BaTiO3 single crystals has been shown to vary between crystals grown by different methods, and depending on the crystals’ electric field and temperature histories22. Wang et al. have shown that images of different domain states can be linked to differences in the size of the dielectric constant peak in the rhombohedral phase of BaTiO3 single crystals23.The low temperature, rhombohedral phase of BaTiO3 materials by considering polycrystalline ceramic and single crystal Pb(In1/2Nb1/2)O3-Pb(Mg1/3Nb2/3)O3-PbTiO3 (PIN-PMN-PT). We compare poled (001) and (111) cut crystals to explore the effect of an ordered domain structure with many domain walls and the effect of a state where there are nominally no domain walls. We investigate the effects of poling the material and find that the large relaxation step only becomes apparent when the single crystal is poled.In order to understand the origins of low temperature anomalies and their relationship to room temperature properties, data on materials in a range of conditions are required. Here we investigate the relaxation step in relaxor-PbTiO0.28-PMN0.40-PT0.32, was chosen to be close to the morphotropic phase boundary (MPB) and give a rhombohedral structure at room temperature. The data are from PIN0.28-PMN0.40-PT0.32 crystal plates cut with (001) and (111) faces, and from a polycrystalline ceramic pellet. Details of sample preparation and structural characterisation are available elsewhere24. The PIN-PMN-PT crystals have a rhombohedral-tetragonal transition at 420 K and depole at 440 K. The polycrystalline material has a rhombohedral-tetragonal transition at 410 K and depoles at 475 K. The (001) cut crystal has a room temperature d33 of 1150 ± 20 pC/N, the (111) cut crystal has a room temperature d33 of 75 ± 1 pC/N and the d33 of the polycrystalline material at room temperature is 163 ± 3 pC/N. The real and imaginary parts of the dielectric permittivity, 6, however, we find differences between the two crystal cuts and large differences between the depoled and poled crystals.We have studied relaxor-ferroelectric PIN-PMN-PT below room temperature. The nominal composition of the material, PINt with 00 and  crystal has the permittivity step feature seen in PMN-PT, where the rate of decrease of The imaginary part of the permittivity The behaviour with temperature of the two depoled PIN-PMN-PT crystals, (001) and (111) cut, is almost identical. There is a large variation in the relative permittivity The polycrystal behaves in a similar way to the depoled crystals. The real and imaginary parts of the dielectric permittivity, entclass1pt{minimaentclass1pt{minimaIn addition to the real and imaginary parts of the dielectric permittivity, we also show the dielectric loss tangent for all samples in Fig. For the polycrystalline ceramic the dielectric loss in Fig. 28 for the peak frequencies given byTmax, to extract a scaling frequency f0, a freezing temperature Tf and an activation energy AE. We have fitted the low temperature (001) cut crystal peak data in Fig. To investigate the low temperature relaxation mechanism we have extracted the frequencies and temperatures of the imaginary permittivity relaxation peaks for the (001) cut crystal Fig. . A commo23 and polar nano-regions11. The preparation conditions of materials, including the crystal growth method, crystalline or polycrystalline nature and the field and temperature histories are all important factors in the low temperature dielectric response.Low temperature dielectric relaxation data have been modelled and explained by freezing-out of dynamics associated with both domains11 (which has been modelled by dynamics of polar nano-regions) and in rhombohedral BaTiO323 . Comparing the dielectric permittivity versus temperature of single crystals and a polycrystal in poled and depoled states plotted in Figs The relaxation step that we measure in the PIN-PMN-PT 001) single crystal is very similar to both the step seen in PMN-PT single cIn unpoled crystals and ceramics, domains representing all crystallographically allowed polarisation directions can be present. Applying a small measurement field favours some of these domains, allowing them to expand at the expense of others. Small changes to the domain configuration give rise to an excess change in polarisation compared to the lattice response. This phenomenon, known as the domain wall contribution, appears in both the permittivity and piezoelectric charge coefficient.Poling an as-grown crystal changes the polarisation configuration so that only domains with a polarisation component parallel to the applied field remain. For a rhombohedral PIN-PMN-PT crystal poled along 111], the polarisation is aligned to [111] only, giving a nominally single domain configuration. Hence, the domain wall response is eliminated or reduced compared to the unpoled crystal, as is observed in Fig. 11, the pPoling the ceramic also results in a decrease in permittivity see Fig. , althoug1, the four domains are degenerate with respect to applied fields along [001], so there should be no permittivity contributions from domain wall motion, although contributions from static domain walls are possible30. Rayleigh studies on PIN-PMN-PT have shown that the extrinsic contributions from domain walls account for as little as 2% of the piezoelectric effect in (001) cut single crystals31. Hence, we cannot attribute the increased permittivity and its low temperature relaxation to conventional domain wall translation. According to the conventional model1, the enhanced permittivity is due to polarisation rotating towards and away from the [001] direction under applied fields. However, such a mechanism, in which the polarisation rotates within domains, is not temperature activated, so the observed low temperature relaxation is not consistent with this model.In contrast, poling an (001) cut crystal results in a large increase in the permittivity, which reduces dramatically below 100 K Fig. . For a  and hence domain wall translation contributions to permittivity would only result if poling were incomplete or “back-switching” occurred. As there is no evidence that the low temperature relaxation in [001]-poled crystals is dependent upon the magnitude of the poling field, we suggest that such a mechanism does not make a substantial contribution.Other evidence suggests that for PIN-PMN-PT36. Poling relaxor-PbTiO3 materials close to the MPB may therefore enhance nanoscale structural variations that emerge from compositional variations, giving rise to phase domains with different crystal symmetries and polarisation orientations. Theoretical work shows that rhombohedral and tetragonal regions can coexist in PMN-PT37 and there have been experimental observations of tetragonal nanodomains in PMN-PT39. In the case of both PMN-PT and PIN-PMN-PT, the monoclinic symmetry is one in which the average polarisation vector is close to [111], hence if this were a composite structure of tetragonal and rhombohedral nanodomains, the tetragonal phase would be a relatively small volume fraction.There is also evidence that the apparent monoclinic structures in materials close to the MPB may be due to the averaging of variations in local structure, such as a combination of rhombohedral and tetragonal nano-domains40 have simulated the behaviour of ferroelectric domain walls under applied fields and found that domain wall broadening contributes to the macroscale properties. In an (001) poled rhombohedral crystal, domain walls with tetragonal symmetry broaden under applied fields, whereas in a depoled crystal the effect is balanced by narrowing of domain walls with opposite polarisation at their cores. In the dynamic case, in which tetragonal fluctuations appear in the rhombohedral matrix, an [001] applied field would stabilize and increase the tetragonal volume fraction, with similar effect. For an energy landscape in which there is a barrier between the tetragonal and rhombohedral energy minima, both mechanisms would be temperature activated and consistent with the observed low temperature relaxation of the permittivity.Phase domain configurations could comprise either static or dynamic components, or some combination of both. In the case of a static tetragonal component in a rhombohedral matrix, applying a field parallel to [001] would result in growth of the tetragonal volume fraction. As the polarisation along [001] in the tetragonal phase is larger than the [001] component in the rhombohedral phase, this mechanism would significantly enhance the permittivity and piezoelectric activity, giving the enhanced properties seen in relaxor-PT crystals. Domain walls can have a different symmetry to the bulk, so may act as nucleation centres for phase domains. Rao and WangWe have presented dielectric data below room temperature for the relaxor-ferroelectric material PIN-PMN-PT. We compare PIN-PMN-PT measured in six different conditions: poled and depoled single crystal (001) cut, poled and depoled single crystal (111) cut, and poled and depoled polycrystalline ceramic.3 materials is only present in the poled single crystals, and is much more prominent in the multi-domain (001) cut than the single-domain (111). We find that in contrast to the high temperature dielectric relaxation peaks, the positions of the low temperature relaxation peaks in the (001) cut crystal fit an Arrhenius rather than a Vogel-Fulcher function, showing that the mechanism does not involve glass-like freezing dynamics.The large dielectric relaxation feature reported in relaxor-PbTiO3 materials cannot be fully explained by macroscopic polarisation rotation1, by the dynamics of polar nano-regions in a ferroelectric matrix11 or by the conventional translation of ferroelectric domain walls19. The polar nano-region model would suggest that the relaxation should be present in all of the PIN-PMN-PT samples and would lead us to expected to find a non-zero freezing temperature from the Vogel-Fulcher fit to the low temperature relaxation peak data. The domain wall motion model would suggest that the relaxation should be present in all the unpoled samples, but not in the single domain (111) crystal. Since we have shown that these mechanisms don’t account for all of our data, we suggest other temperature activated candidates: the dynamic fluctuations of nanoscale domains between rhombohedral and tetragonal phases, or the motion of phase domain walls. In the case where domain walls have a different structure to domains, the motion of phase boundaries may be linked to broadening of domain walls under applied fields.The differences between sample material under different conditions show that low temperature relaxations in relaxor-PbTiO24. Silver epoxy was painted onto the crystal and pellet main faces and cured at 770 K to form electrodes. The (001) cut crystal was 1.117 mm thick and had an electrode area of 0.2019 cm2, the (111) cut crystal was 1.200 mm thick and had an electrode area of 0.2601 cm2, and the ceramic pellet was 1.720 mm thick and had an electrode area of 0.7557 cm2. The nominal composition of all of the material is PIN0.28-PMN0.40-PT0.32, which is close to the morphotropic phase boundary (MPB) and gives a rhombohedral structure at room temperature24.We have studied crystal plates cut with (001) and (111) faces, and a polycrystalline ceramic pellet. To make the samples, powdered material was prepared by mixed oxide methods. The polycrystalline ceramic pellet was formed by sintering and the crystal was grown by Bridgman techniqueThe samples were all prepared for measurements in the poled state by first annealing to 850 K, a point well above any phase transitions or dielectric maxima, then allowing them to cool to room temperature. We poled the samples by heating them to 370 K, then applying an electric field of 1 kV/mm while the samples cooled to room temperature. The elevated temperature is high enough to lower the energy barrier for domain re-orientation, but is below any phase transitions. All samples showed piezoelectric resonance peaks at high frequency, indicating that they were properly poled. The measurements in a depoled state were taken after the samples had been annealed to a point (above 500 K) where they no longer showed a spontaneous polarisation or piezoelectric resonance peaks.The real and imaginary parts of the dielectric permittivity, 33 measurements were taken with a Berlincourt meter.The rhombohedral-tetragonal transition temperature and depoling temperature were determined by both permittivity measurements in the tube furnace and by polarisation versus temperature measurements in the cryostat. The room temperature d


Che proteins are part of the signal transduction pathway between stimulus and response, mediated by the archaellum. The structure of the chemotaxis protein CheY was determined in two different crystal forms. The novel CheF protein that connects chemotaxis signaling to the motility apparatus was expressed and crystallized, and data were acquired by X-ray diffraction. Archaea are motile by the rotation of the archaellum. The archaellum switches between clockwise and counterclockwise rotation, and movement along a chemical gradient is possible by modulation of the switching frequency. This modulation involves the response regulator CheY and the archaellum adaptor protein CheF. In this study, two new crystal forms and protein structures of CheY are reported. In both crystal forms, CheY is arranged in a domain-swapped conformation. CheF, the protein bridging the chemotaxis signal transduction system and the motility apparatus, was recombinantly expressed, purified and subjected to X-ray data collection. Our data support the conservation of the bacterial and archaeal response-regulator proteins. We observe PhCheY to have a domain-swapped, pseudo-dimeric fold, which may reflect inherent properties of the protein fold but is not likely to be of physiological relevance.In interactomic studies, we have recently identified three candidate proteins in 2.2.1.The coding sequences for PhCheF and PhCheY were provided by a synthetic plasmid carrying the ORFs PH0494 (PhCheF) and PH0482 (PhCheY) and were amplified by PCR with PH0494 specific primers  and PH0482 specific primers . In an In-Fusion cloning reaction , the gel-purified fragments were ligated with NdeI and XhoI linearized pET-22b(+) , yielding the plasmids pDW01-1 and pDW02-1 for the expression of PhCheF and PhCheY as C-terminally His-tagged proteins. For the construction of non-His-tagged variants, we removed the His tag by inserting TAG codons upstream of the XhoI restriction site via the site-directed mutagenesis method (plasmids pDW01-2 and pDW02-2).Escherichia coli, the plasmids were transformed into BL21 Gold (DE3) cells . Single colonies were used to inoculate 35 ml LB medium containing 100 µg ml−1 ampicillin and were incubated at 37°C for 16 h. This preculture was used to inoculate 2 l TB medium, which was grown at 37°C and 180 rev min−1 until the mid-to-late log phase (OD600 = 0.8–1.0) before inducing expression at 20°C with 0.5 mM IPTG. The cells were harvested after 16 h of protein expression, and the cell pellets were frozen in liquid nitrogen and stored at −80°C until further use. For purification, the cells were resuspended in appropriate buffers containing protease inhibitors  and DNaseI  and were lysed using a French press. The lysates were centrifuged for 1 h at 4°C and 47 000g. After centrifugation, the supernatants were subjected to purification protocols. The non-His-tagged proteins PhCheF and PhCheY  were purified by heating the supernatant to 80°C for 20 min followed by centrifugation of the precipitated biomolecules at 24 000g for 20 min. As analyzed by SDS–PAGE, this treatment precipitated most of the proteins of the E. coli expression host, while the thermostable proteins PhCheF and PhCheY remained soluble. Concentration of PhCheF and PhCheY and size-exclusion chromatography (SEC) using a Superdex 200 26/60 column , removing the remaining proteins and soluble biomolecules (as metabolites and nucleic acids), eventually yielded proteins that were suitable for biomolecular analysis and crystallization. His-tagged proteins were purified by nickel-chelating affinity chromatography  and SEC (buffer H) using a Superdex 200 26/60 column .For heterologous expression of proteins in in vitro pull-down assay with PhCheF and His-tagged PhCheY, 2 ml of the supernatant of each preparation (after lysis using a French press and centrifugation) were first incubated at 37°C for 15 min (in buffer W with 20 mM imidazole). The incubated protein mixture was then added to 2.5 ml equilibrated Ni–NTA beads (in 5–7.5 ml buffer W). After further incubation at 4°C for 1 h, the slurry was subjected to a gravity-flow column. The beads were washed five times with buffer W (one column volume per step) and the proteins were the eluted with buffer E . Fractions were collected and loaded onto a gel. The elution peak fractions were also subjected to SEC using a Superdex 200 10/300 column . Ni–NTA pull-downs were also performed with 5 mM BeSO4 as well as 50 mM NaF in the respective buffers to obtain the BeF3− species mimicking phosphorylated PhCheY  ethylene glycol for 1 min and was then cryocooled by plunging it into liquid nitrogen. X-ray diffraction data were recorded on a PILATUS 6M detector (Dectris) while the crystal was held in a gaseous N2 stream at 100 K.Multi-wavelength anomalous dispersion (MAD) data for PhCheF were collected on beamline PX2 at the Swiss Light Source synchrotron facility, Villigen, Switzerland. The MAD data set was collected at three wavelengths: 0.9795 Å (peak), 0.9797 Å (inflection) and 0.9718 Å (remote). For this experiment, the crystal in the droplet was transferred into a cryosolution consisting of the mother liquor supplemented with 20%. All data sets were auto-processed and merged with the 2.4.P2 and C2. The solvent-content calculations for the P2 crystal form performed with MATTHEWS_COEF  against the Protein Data Bank (PDB) with BLAST . Atomic coordinates and experimental structure factors for PhCheY in space groups P2 and C2 have been deposited in the PDB with accession codes 6er7 and 6exr, respectively. Raw X-ray diffraction data for both PhCheF and PhCheY are available from the Zenodo science data archive (https://doi.org/10.5281/zenodo.1148967).All structural figures were drawn using 3.3.1.H. salinarum have been expressed recombinantly in E. coli, but primarily as unfolded proteins that rely on the uncertainty of refolding protocols , which display sequence identities of 24 and 53% to CheF1 and CheY from H. salinarum, respectively . In addition, we expected increased thermostability of these proteins , the NMR structure of the CheY-like MTH538 from Methanobacterium thermoautotrophicum ].The structures of CheY available in the PDB typically display a compact folded structural appearance. The subunits of CheY consistently show a β1/α1/β2/α2/β3/α3/β4/α4/β5/α5 globular fold of five helices flanking a five-stranded parallel β-sheet. The subdomain in the N-terminal region  has α-helices α1–α2 packing on opposite sides of β-strands β1–β3, and the C-terminal region of the molecule  has α-helices α3–α5 packing against β-strands β3–β5. In striking contrast to all of the known CheY structures, PhCheY adopts an open conformation in both crystal forms, with the two subdomains of a given subunit directed away from one another. In both of the two PhCheY structures solved in this study, two polypeptide chains assemble by pairing the N-terminal subdomain of a chain with the C-terminal subdomain of the other chain; P2 contains three noncrystallographic symmetry (NCS) molecules in the asymmetric unit. Superposition of the three subunits shows differing orientations between the domains, leading to an inter-domain movement of up to 90° [Fig. 1b)]. The N- and C-terminal domains of PhCheY superimpose well onto each other in the crystal structure. The closest structural homolog is CheY from T. maritima  [Fig. 1c)], facilitating the inter-domain movement.The crystal form with space group C2 crystal form, there are six molecules in the asymmetric unit. Four of the subunits  interact with one another in a dimeric-type domain-swapped assembly as described above. The other two subunits, C and E, interact with their own crystallographic symmetric molecules for the dimeric assembly with swapped domains. Superposition of the N-terminal subdomain among the different subunits A, B, C, D, E and F reveals that the C-terminal domain of subunits B, C, D, E and F undergoes a relative movement of up to 57° with respect to the N-terminal domain. In all cases, the rotational movement of the C-terminal domain was calculated after the initial superposition of the N-terminal domain. The connecting region between the β3–α3 loop is partially disordered for the two subunits C and E. For both crystal forms an unambiguous trace of the electron density in the β3–α3 loop was verified by a feature-enhanced map .In the case of the et al., 1999et al., 2000d)].CheY has been discussed as the evolutionary ancestor fold of periplasmatic binding proteins, such as for example the glucose/galactose-binding protein MglA. A domain swap, producing a pseudo-dimeric fold, has been suggested to be a key event in this process, and has been observed for the CheY-like protein Spo0A , and in the non-activated state  structure compares well with the structure of the non-activated CheY from M. maripaludis ]. The positions of the active Asp53, as well as Thr81 and Tyr100, involved in translating the phosphorylation signal to a physiological output ]. The accumulated negative charge at the N-terminal region of helix α4 in M. maripaludis CheY has been suggested to provide an archaea-specific interface for CheF interaction ], PhCheY may be less negatively charged than M. maripaludis CheF, since it lacks a negatively charged amino acid at the position equivalent to Asp88 (Gly84 in PhCheY), as is also the case for other archaeal CheYs [Fig. 2d)]. Glu91 is in a different rotamer position in PhCheY and may also contribute to the less developed negative surface potential [see Fig. 2c)].CheY from 3.4.M BeSO4 and 30 mM NaF to modify PhCheY with the phosphoryl analog BeF3− ]. While cross-linking with glutaraldehyde also failed in tracing interaction of the proteins under the selected conditions [Fig. 3b)], an Ni–NTA pull-down assay eventually allowed the specific interaction of the proteins to be monitored by His-tagged PhCheY retaining PhCheF [Fig. 3c)] during chromatographic elution. Both conditions, BeF3−-free and BeF3−-treated PhCheY, gave similar elution profiles. The complex observed in Ni–NTA pull-downs was not preserved during SEC. From Coomassie-stained SDS–PAGE in Ni–NTA pull-downs, PhCheY appears to be present in a molar excess, which might however result from nonsaturated PhCheY under the experimental conditions. We note that SEC showed the proteins to be monomeric after purification, with PhCheF having some tendency for the formation of a higher oligomeric species under the chosen conditions [see Fig. 3a)]. Data collected during the biochemical characterization of CheY and CheF therefore do not support a physiological relevance of dimeric CheY species, as are observed in crystal structures.A difficult aspect in interaction studies of CheF and CheY is that CheY is supposed to interact with CheF in a phosphorylated state (CheY-P), which is however not accessible from recombinant expression. As a generally accepted treatment for mimicking phosphorylated states, we therefore incubated PhCheY with 5 m4.et al., 2016Two-component systems regulate a variety of fundamental processes in metabolism and motility, as well as a set of more specialized processes such as in virulence and development ] has been suggested to have evolved from a CheY-like ancestor protein by a domain swap  is located in a positionally variable loop, it is unlikely that phosphorylation could efficiently induce the dissociation of the fold and enrich a ‘swapped’ form. Therefore, a physiological role for a domain-swapped dimeric PhCheY has to be ruled out. A physiological role of a domain-swapped dimer would also contradict the current understanding of the function of CheY ], and in SDS–PAGE of cross-linking experiments [see Fig. 3b)]. Owing to a putative cold-induced destabilization of PhCheY (P. horikoshii grows at 98°C), and putatively further induced by the crystallization conditions, the PhCheY fold disassembles into an open subdomain-dissociated conformation. At the high protein concentration in the crystallization drop, PhCheY can then reassemble from this open state with a partner polypeptide to form a domain-swapped dimeric assembly that is eventually removed from the monomer–dimer equilibrium by crystallization.Crystallization of PhCheY as a domain-swapped dimer can rather be explained as follows: PhCheY is monomeric in solution, as suggested by SEC [see Fig. 3et al., 2009et al., 2018It is surely exciting to further reveal the different structural appearances of archaeal and bacterial CheY in order to disclose the principles of diversification in bacterial and archaeal chemotaxis/motility systems, which is then further pronounced by CheY interacting with FliM in bacteria and with CheF in archaea (Szurmant & Ordal, 20045.et al. (2013et al. (2017et al. (2013et al. (2016et al. (2017et al. (1994et al. (2011et al. (2016et al. (1994et al. (2001et al. (2003et al. (2013et al. (1996et al. (2008The following references are cited in the supporting information to this article: Albers & Jarrell 2018, Banerje al. 2013, 2015 ▸,PhCheY, 6er7PDB reference: 6exrPDB reference: 10.1107/S2053230X19010896/no5164sup1.pdfSupplementary Figures. DOI: https://doi.org/10.5281/zenodo.1148967Raw diffraction data for PhCheF and PhCheY.:


Translationally controlled tumor protein (TCTP), also called histamine releasing factor, is an evolutionarily conserved multifunctional protein in eukaryotes. We previously reported that extracellular TCTP acquires its cytokine-like function following dimerization. This study aims to identify the functional domain involved in the cytokine-like function of dimerized TCTP (dTCTP). We performed X-ray crystallographic studies and a deletion mutant of dTCTP which lacks the flexible loop domain. Synthetic peptides corresponding to TCTP domains and antibodies developed against them were examined for the anti-allergic effect. In an OVA-induced airway inflammation mouse model, inhibitory effect of synthetic peptides was evaluated. dTCTP was mediated by dimers between Cys172s of TCTP monomers. Synthetic peptides corresponding to the flexible loop and helix 2 domain of TCTP, and antibodies against them inhibited dTCTP-induced IL-8 release. In particular, the TCTP mutant lacking the flexible loop domain decreased the inflammatory cytokine activity of dTCTP. We conclude that the flexible loop and helix 2 domain of TCTP are the functional domains of dTCTP. They may have the potential to be therapeutic targets in the suppression of allergic reactions induced by dTCTP. Molecular identification studies revealed that histamine-releasing factor is identical to translationally controlled tumor protein (TCTP)3, a multifunctional protein that has been highly conserved in eukaryotes. In a previous study, we reported for the first time that dimerization allows TCTP to acquire cytokine-like activity4. TCTP exhibits several intracellular functions in development6, cell cycle regulation8, cellular homeostasis9, neurotransmitter release10 and autophagy11 and also in various diseases including cancer, malaria12, and Alzheimer’s diseases13.Histamine-releasing factor (HRF) was identified in the sera of allergic patients and it showed histamine releasing activity in human basophils, which in turn provoked allergic reactions14, and a flexible loop (FL) which comprises TCTP signature 1. The evolutionarily conserved motifs of TCTP indicate the importance of their functions. The fold of the β-stranded domain classifies TCTP as a member of Mss4-like superfamily; it plays a key role in eliciting TCTP’s GDP-GTP exchange activity for Rheb15. Its helical domain was identified as tubulin16 and calcium17 binding site and is reported to be involved in the inhibition of Bax dimerization due to its similarity with H5-H6 helices of Bax18. In a study of the possible function of the FL of TCTP, Yarm8 reported that a polo-like kinase reduces microtubule stabilizing activity of TCTP by phosphorylating Ser 46 and Ser 64 residues in the loop region of TCTP. However, the overall functions of the FL domain remain unknown.Structural studies have revealed that TCTP possesses an intrinsically disordered structure consisting of three preserved motifs: a β-stranded sheet similar to that of Mss4/Dss4, a basic patch composed of H2-H3 helices172 19. Structural and functional information of the long loop is currently still very limited.Suggestions for NMR and crystal structures of TCTP have been deposited in the Protein Data Bank. These include different structures of monomeric TCTPs from several species and just one of human dimeric TCTP (dTCTP). They all share a high topological similarity. In both forms, a long middle loop located outside of the core domain of TCTP shows high flexibility. dTCTP shows a unique dimeric interface which seems to be further stabilized by the disulfide bond mediated by CysIn this study, we examined which domains of dTCTP are important for its function by X-ray crystallographic studies. We also studied the function of the FL of TCTP using a deletion mutant. Based on the structures of human dTCTP and this mutant, we predicted the mechanism of action of each functional domain.∆-TCTP , rat recombinant TCTP (RrTCTP), 11–172 residues-deleted RrTCTP (Del-N11dTCTP), and FL-deleted Del-N11dTCTP (∆-del-N11dTCTP) were amplified for cloning into bacterial expression vector. Detailed information for gene cloning is described in Supporting information  for protein expression. Overexpressed protein was purified using a HisTrap column on an ÄKTA-explorer system , followed by ion-exchange chromatography using a Hi-Trap Q column . Peptides were synthesized by Fmoc solid-phase method by AbClon or Peptron Inc. N-terminal free amine groups were acetylated, and the C-terminal free carboxyl groups were amidated to improve the stability of the peptides. Sequences of each peptide are displayed in SI  and incubated at room temperature for 30 minutes or 24 hours. For producing homogenous dimeric form, 10 μg of each protein in 10 μl was treated with 1–100 mM of tertiary-butyl hydroxide (t-BH) or H2.BEAS-2B, a human bronchial epithelial cell line, was purchased from the American Type Culture Collection . Cells were maintained in bronchial epithelial cell growth medium  at 37 °C and 5% COad libitum. 5 weeks old BALB/c female mice (Orientbio) were sensitized with OVA by i.p. injection of 1.3 mg of aluminum hydroxide (Sigma) and 100 µg of ovalbumin (Sigma) twice a week, followed by challenge with intranasal instillation of OVA on day 14, 16, and 18. Ten minutes before challenge, PBS, dexamethasone, and synthetic peptides were intraperitoneally injected. On days 15, 17, and 19, i.p administration was performed without antigen administration. On day 19, the animals were sacrificed 2 hours after the i.p injection, followed by BALF and lung tissue preparation.All animal studies were approved by Ewha Womans University’s Institutional Animal Care and Use Committee . All methods and experimental procedures were conducted according to the guidelines of the Ewha Womans University’s IACUC. The animals were housed under pathogen-free conditions with a 12-h light/12-h dark cycle, and were fed with standard diet and water Details for structure determination are described in SI Table .Data were analyzed using GraphPad Prisms 5 software and expressed as mean ± standard error of mean (SEM). Statistical significance was determined using Student’s two-tailed unpaired t-test for comparison between two groups. For three or more groups, one-way ANOVA analysis was performed.23. These structurally diverse proteins can produce biological responses by binding to or providing structured hub proteins with potential partner proteins24. Considering the intrinsically disordered structure of TCTP15, this structural characteristic may be important to elicit the biological function of TCTP. In order to understand the role of FL domain in dimeric TCTP, we constructed full length human TCTP (f-TCTP) and the FL domain deleted mutant TCTP (∆-TCTP) expression vectors and produced recombinant f-TCTP and Δ-TCTP proteins. Purified proteins were analyzed by SDS-polyacrylamide gels both in reducing and non-reducing conditions. We found that the molecular weights of most of the expressed proteins corresponded to the monomers (25 kDa for f-TCTP and 17 kDa for Δ-TCTP), but two weak bands were also observed  and DTT. When both f-TCTP and ∆-TCTP were treated with 1 mM DTT for 24 hours, homodimers were reduced to monomers, but monomers with intramolecular disulfide bonds remained. However, at 10 mM, DTT completely reduced both proteins regardless of incubation time.Several studies have investigated the role of the intrinsically disordered proteins (IDPs) in cellular interactions under the physiological conditionsved Fig. . These b2O2  secreted the inflammatory cytokines, IL-8 and GM-CSFCTP Fig. . To undeCTP Fig. , suggestCTP Fig. . We furtCTP Fig. . These f25. Therefore, we deduced that the FL and H2 domains could be important for the function of dimerized TCTP. To verify this hypothesis, peptides corresponding to the FL, H2, and helix 3 (H3) domains and polyclonal antibodies against them were tested for their inhibitory effect on del-N11dTCTP-induced cytokine release. IL-8 release by del-N11dTCTP was significantly reduced in BEAS-2B cells pretreated with 0.07, 0.7, and 7 nM FL peptides and 0.07 nM H2 peptides  determined by X-ray crystallogrphy or NMR have been reported. Interestingly, three of these suggested possible dimeric forms have been based on the packing in the different unit cellslts Fig. . Our cry2 per each subunit without a hydrophobic core or a salt- bridge network. Therefore, the dimeric forms of dTCTP may be more stable when the the disulfide bond is formed. Second, dTCTP takes an extended conformation and shows a butterfly like shape. As a result, two coiled-coil like helices (H2 and H3) are located far apart from each other, and two Fab binding sites 33 become surface exposed in each molecule. Third, the flexible loops are positioned in the opposite sides of their N- and C-terminals. Consequently, if a dimer formed, the movement of the FL is restricted at one side. In contrast, the movement of the FL of the monomeric form is expected to reach the region forming the dimeric interface and thus obstructs dimer formation. The NMR structures explicitly support this possibility34. Therefore, to sustain the dimeric state, the disulfide bond may be critical to overcome the pressure from the movement of the FL hampering dimer formation. Conversely, the limited flexibility of the FL at the confined space in the dimeric form may be necessary for its molecular function.Several structural properties of the dimeric forms are of particular interest Fig. . First, 172 residues and show the same dimeric archtecture found in the f-dTCTP. Therefore, Δ-dTCTP is also superimposed on f-dTCTP with RMSD values for 286 Cα atoms of ~0.8 Å. The uneven distribution of electrostatic potential caused by ten lysine and two arginine residues concentrated on H2 and H3 is evident as shown in Fig. In order to investigate the influence of the FL over the overall structure of f-dTCTP, we determined the X-ray crystal structrue of Δ-dTCTP at 1.90 Å resolution Fig. . The oveThis study found that the mutant TCTP lacking the FL domain (∆-del-N11dTCTP) exhibits impaired cytokine-like activity of del-N11dTCTP, even if it becomes a dimer. In addition, the synthetic peptide that competes with the FL domain of dTCTP for its putative receptor, and anti-FL antibody which can mask the loop domain of dTCTP, could effectively inhibit the dTCTP-induced cytokine release. The reported structure of dimeric fTCTP indicates that the shapes of the dimeric forms are like a butterfly in which the interface is formed with a weak interaction between edge of β-sheet. The crystal structure of the dimeric Δ-TCTP determined in this study shows no overall structural difference except for the presence of loop domain. Therefore, the function of the dTCTP appears to be largely due to the FL domain of the dimeric TCTP.Schizosaccharomyces pombe, p23fyp 35, suggested that molecular interactions of this loop would play a crucial role in the function and regulation of this protein family. It is now well accepted that IDPs perform important biological functions without stable tertiary structures36. One of the important functions of IDPs is molecular recognition, and there is growing evidence that IDPs mediate ligand-receptor binding37. Examples of receptors involved in ligand-receptor interactions include glucocorticoid receptor38, growth hormone receptor39, and retinoid X receptor40. Multiple phosphorylations of IDP have been reported to cause protein-protein interactions by modulating folding of IDP42. These reports suggest that the phosphorylation of Ser46, Ser64 and Thr65 by plk-1 not only affects the microtubule-stabilizing activity of TCTP, but also induces conformational change in the loop region, thus providing the possibility of interacting with other targets.The FL domain of TCTP contains one of the conserved sequences of the TCTP protein family and has been reported to be highly mobile and disordered. The first identified backbone structure of the TCTP of 35. But when it forms a dimer, the free movement of the FL is restricted in a certain confined space. This structural difference provides an explanation for the previously reported higher affinity of dTBP2 for dimers than for monomers25. The FL of mTCTP can move freely and, to some extent, prevent dTBP2 from binding to the H2 region. However, for dTCTP, the loop region is directional and does not affect the binding of dTBP2.Monomeric TCTP (mTCTP) also contains the FL domain, but it does not exhibit cytokine-like function probably due to the differences in the orientations of the loop domains in mTCTP and dTCTP. In mTCTP, the FL domain is thought to be disordered and free to move43, the H2 domain was assumed to be another functional domain. We examined the effect of a synthetic peptide corresponding the H2 domain and antibody against it on dTCTP-induced cytokine secretion. As expected, IL-8 secretion decreased when the H2 region of dTCTP was interrupted, suggesting that the H2 region plays an important role in the cytokine-like function of dTCTP. To more clear illustrate the function of the H2 region, construction of a deletion mutant lacking the H2 domain was considered. However, the H2 domain was found to be an important axis for the overall structure of dTCTP, forming a coiled-coil structure with the H3 region. Therefore, the mutant would not reflect H2 domain-specific functions, but rather overall mutation in TCTP structure.Based on the fact that the dTBP2 peptide, interacting with the H2 site, showed anti-allergic effects in mouse modelsThis research, for the first time, demonstrated the important role of the FL and H2 domains of TCTP in dTCTP-induced inflammation. In conclusion, we hereby propose that the FL and H2 domains are the functional domains of dTCTP and promising therapeutic targets for allergic diseases.supplementary information.


This recognition mode resembles the molecular mechanism evolved in the coupling of ubiquitin conjugation to endoplasmic-reticulum (ER) degradation (CUE) domain family, where an invariant proline, usually following a phenylalanine, is required for ubiquitin binding. Interestingly, this novel UBD, which is not evolutionary related to CUE domains, shares a 40% identity and 47% similarity with cullin binding domain associating with NEDD8 (CUBAN), a protein module that also recognizes the ubiquitin-like NEDD8. Based on these features, we dubbed the region spanning the C-terminal 50 residues of N4BP1 the CoCUN domain, for Cousin of CUBAN. By performing circular dichroism and 15N NMR chemical shift perturbation of N4BP1 in complex with ubiquitin, we demonstrate that the CoCUN domain lacks the NEDD8 binding properties observed in CUBAN. We also show that, in addition to mediating the interaction with ubiquitin and ubiquitinated substrates, both CUBAN and CoCUN are poly-ubiquitinated in cells. The structural and the functional characterization of this novel UBD can contribute to a deeper understanding of the molecular mechanisms governing N4BP1 function, providing at the same time a valuable tool for clarifying how the discrimination between ubiquitin and the highly related NEDD8 is achieved.Ubiquitin binding domains (UBDs) are modular elements that bind non-covalently to ubiquitin and act as downstream effectors and amplifiers of the ubiquitination signal. With few exceptions, UBDs recognize the hydrophobic path centered on Ile44, including residues Leu8, Ile44, His68, and Val70. A variety of different orientations, which can be attributed to specific contacts between each UBD and surface residues surrounding the hydrophobic patch, specify how each class of UBD specifically contacts ubiquitin. Here, we describe the structural model of a novel ubiquitin-binding domain that we identified in NEDD4 binding protein 1 (N4BP1). By performing protein sequence analysis, mutagenesis, and nuclear magnetic resonance (NMR) spectroscopy of the  Ubiquitin (Ub) and ubiquitin-like (Ubl) proteins are small polypeptides that are covalently conjugated to various target proteins, leading to proteasome-dependent or independent degradation and changes in their localization and/or in their activity . In ordeNeural precursor cell expressed developmentally downregulated protein 8 (NEDD8) is the closest relative to ubiquitin (58% identity and 80% similarity). Both molecules present a similar asymmetric surface distribution of charges with a predominantly acidic face opposed to a hydrophobic one. Notably, the Ile44-centered patch is identical in ubiquitin and NEDD8 molecules [The NEDD4 binding protein 1 (N4BP1) was originally identified as a target for the proteasomal degradation by the homologous to the E6AP carboxyl terminus (HECT) E3-ligase Nedd4 . Indeed,By screening protein arrays with the human ubiquitin precursor UbC, Fenner and collaborators identified novel polyubiquitin binding proteins potentially regulating the NF-κB activity. Among them, the region of N4BP1, including amino acids (aa) 209–896, was identified in the screening, and the interaction was experimentally confirmed ,11, in aCousin of CUBAN. The observed similarity, both at the primary sequence and the structural level, supports the identification of a novel domain family having the CUBAN and the CoCUN domains as unique members. Finally, we show that both domains are poly-ubiquitinated when expressed in cells and that a functional ubiquitin/NEDD8 binding property is required for the domain in order to be post-translationally modified.We present here the structural model of the novel ubiquitin-binding domain identified in N4BP1, both alone and in complex with ubiquitin. Structural analysis performed by nuclear magnetic resonance (NMR) spectroscopy revealed a three alpha-helix bundle domain. Using biochemical assays, we demonstrate that the interacting surface involves the hydrophobic patch in ubiquitin and a key motif in N4BP1 resembling the Phe-Pro (FP) motif typified in the coupling of ubiquitin conjugation to ER degradation (CUE) domain family. Nevertheless, the structural analysis shows that key differences in the ubiquitin-binding domain of N4BP1 do not support the inclusion of this novel domain either in the CUE or in other three-helix bundle domain families, such as ubiquitin-associated domains (UBA). Conversely, the UBD of N4BP1 shares common features with the cullin binding domain associating with NEDD8 (CUBAN), recently identified in the evolutionary related KHNYN protein ,11. BaseFenner and collaborators recently identified N4BP1 as a potential polyubiquitin chains binding protein. A previous analysis performed with the phage display approach enabled us to identify the region spanning residues 813–896 in N4BP1 as the one showing ubiquitin binding features . The bioCousin of CUBAN.Interestingly, the minimal ubiquitin-binding region in N4BP1, including residues 847–896, shares 40% identity with the CUBAN domain, recently identified in the evolutionary related KHNYN protein . The CUB15N labeled protein. The 15N heteronuclear single quantum coherence (HSQC) spectrum is reported in 15N-HSQC-TOCSY  and 3D 15N-HSQC-NOESY (nuclear Overhauser spectroscopy) spectra. The cross-peaks were labeled, and the assignments were further confirmed by 1H TOCSY and 1H NOESY spectra obtained with different mixing times as reported in Materials and Methods  using the two-dimensional (2D) and the three-dimensional (3D) NMR methodology was performed, leading to the assignment of 47 out of 50 amino acids in the  Methods B. As shohis size C, indicand Sykes  led to tBased on the NOEs observed, the structural model of the CoCUN domain was calculated using the conventional software package XPLOR . Simulathttp://agadir.crg.es) [To gain more information on changes in the secondary structure of the N4BP1 UBD upon binding, the CD spectra were obtained in the region from 195 to 260nm, as reported in .crg.es)  which giCD spectroscopy is widely used for the extraction of elements of secondary structure of polypeptides. This practice uses a fitting that often does not give very good results depending on the database applied. On the contrary, CD remains an optical physical chemistry measure, which is a sensitive monitor of secondary structure changes of proteins and of their interactions ,24,25. W15N labeled CoCUN domain upon addition of unlabeled ubiquitin at different molar ratios. The typical chemical shift perturbations (CSPs) plot is reported in 15N labeled CoCUN and ubiquitin were A14, L15, I18, S22, I28, Q30, and D40 at both molar ratios. Residues N9, K27, and H35 were only perturbed at the 1:1 molar ratio. The complementary experiment performed using 15N labeled ubiquitin and the unlabeled CoCUN domain showed that a large number of residues were perturbed following the addition of the unlabeled protein at both 1:0.5 (15N-labeled ubiquitin/unlabeled CoCUN. Fixing a threshold of 1σ (indicated by a line), a view of the residues involved in the interaction could be highlighted. As shown, the hydrophobic residues mapping in the β-sheet region of ubiquitin were the main determinants of the interaction, followed by a large number of polar and neutral residues that were also involved, thus indicating a complex recognition mechanism between the two proteins upon the early hydrophobic recognition step. In order to determine the regions involved in the interaction, CSPs detected by both experiments were analyzed by the HADDOCK procedure as reported in Materials and Methods [We then investigated the CoCUN/ubiquitin complex by monitoring the chemical shift changes(s) of th 1:0.5 C and 1:1th 1:0.5 D of 15N- Methods .The model reported in 15N backbone chain caused by conformational changes of either the side chain or the secondary structure tract itself, the CSP observed for these residues could be considered as an indicator of magnetic environment perturbations of the 15N of the amide bond due to a secondary structure perturbation in that tract. Based on this premise, a possible explanation for the relevant perturbations of K27, I28, and Q30 upon binding with ubiquitin could be that the tract of helix-2 containing these residues was subjected to helical geometry distorting forces generated by the pulling down effects of the two flanking loops 1 and 2 toward the ubiquitin-interacting surface.D = 49.7 ± 3.6 µM and ∆H = −12.6 ± 0.3 kJ/mol, respectively  with the purified N4BP1 polypeptide (aa 847–896) A. The diectively A. We alssurement B. ComparThe CUE domains are closely related to UBA ,12. IndeDifferences and similarities were clearly visible; while the FP motif was positioned at the C-terminal end of helix-1 in the CUE domain of AMFR, both the FP and the FW pairs in CoCUN and CUBAN occupied a central position inside the extended loop1. Nevertheless, the Trp647 pointed towards the core of the domain and, together with flanking residues, constituted a sort of pivot that reduced the flexibility of loop1 in CUBAN . On the Ct-W647A and N4BP1Ct-P866A fused to the GFP, and we performed the immunoprecipitation with anti-GFP antibody. The immunoprecipitated samples were analyzed by immunoblotting with the anti-ubiquitin antibody  protein. This domain, called CUBAN for cullin binding domain associating with NEDD8, shows a clear preference for monomeric NEDD8 and mediates the interaction of KHNYN with neddylated cullins ,11. Althto N4BP1 .Cousin of CUBAN) domain, is more similar to CUBAN with respect to CUE and UBA domains. Intriguingly, despite the high level of similarity, these domains show significant differences in their binding preferences. The circular dichroism spectroscopy confirmed that the interaction of CoCUN with ubiquitin occurs with slight mutual changes in the dichroic profiles of both molecules in line with the structural stability of the ubiquitin molecule, characterized by β-strands flanked by one another with a network of hydrogen bonds. The same experiment performed with the close relative NEDD8 did not report any variation in the dichroic profiles, thus confirming that N4BP1 does not bind to NEDD8. Although tiny these differences indicate that small changes occurred in both CoCUN and ubiquitin, thus suggesting a mutual conformational change [15N labeled ubiquitin and in the CoCUN domain are located in regions corresponding to the loops connecting secondary structure elements, indicating that changes in the conformational freedom could be the basis of the CD effects.The sequence analysis of this novel domain revealed a 40% identity and 47% similarity with CUBAN ,11. Morel change ,24,25. O15N of the backbone for this residue in our NMR binding experiments. Nevertheless, as expected, the mutational analysis confirmed a clear involvement of this residue in the interaction with N4BP1 . Tak. Tak15N h N4BP1  and hKHNYN (598–678) were previously described. The minimal ubiquitin binding region of N4BP1, including amino acids 847–896, was cloned in pGex2TK vector using primers R2326 (forward) CAAGGATCCGCTCAGAGATCTTCTGCA and R2473 (reverse) CTCTCGAGTCAATCCAACACCATGGCAG. The T-REx flag-NEDD8 cell line was generated as previously described [® Site-Directed Mutagenesis Kit  according to the manufacturer’s instructions.escribed . Site-diProtein expression and purification. The C-terminal fragment of hN4BP1 (aa 847–896) was cloned in pGex2T vector, and the recombinant protein was prepared as previously described [escribed . The bacescribed .Cell extracts, immunoprecipitation, and in vivo ubiquitination assay. Whole-cell extracts were prepared from mammalian cells by lysis in 25 mM Tris pH 7.5, 125 mM NaCl, 1% glycerol, 1 mM MgCl2, 1 mM orthovanadate, 5 mM NaF, 0.5% NP-40, 0.5% Triton X- 100, complete proteases inhibitor cocktail , and complete phosphatase inhibitor (Sigma) for 30 min on ice before clarification by centrifugation. To detect protein in cell lysates, protein samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membrane. Proteins were detected by immunoblotting and visualized by treating the blots with enhanced chemiluminescence (ECL) . For the pull-down experiments, equimolar amounts of GST-fusion proteins corresponding to the selected clones and GST alone linked to glutathione-Sepharose 4B beads were incubated 1.5 h with 1 mg of cell extract. Beads were washed three times in washing buffer , and bound proteins were electrophoresed on polyacrylamide gel, transferred to polyvinylidene difluoride (PVDF) or nitrocellulose membranes, and detected with specific antibodies. For the coimmunoprecipitation assay cells were plated in 100 mm dishes and grown to 60–70% confluence in culture medium supplemented with 10% fetal bovine serum (FBS). Twenty hours post-transfection, cells were solubilized in lysis buffer. An equal amount of each protein lysate was incubated with anti-Flag M2 affinity gel beads for 2 h at 4 °C and then washed three times with washing buffer supplemented with fresh inhibitors. The protein lysates and the immune complexes were analyzed by Western blot analyses with rabbit anti-GFP and anti-ubiquitin antibodies.Antibodies and reagents. Polyclonal anti-flag was from Sigma, anti-tubulin and anti-ubiquitin P4D1 from Santa Cruz Biotechnology , anti-NEDD8 was from Abcam , and antirabbit and anti-mouse peroxidase-conjugated were from Jackson Immunoresearch .Cell culture. HEK293 and Flp-In T-REx-293-Flag-NEDD8 were grown on DMEM  supplemented with 10% FBS (GIBCO) and 100 units/mL penicillin and 100 μg/mL streptomycin . Stably transfected Flp-In T-REx-293-Flag-NEDD8 cells were generated as in [Nuclear magnetic resonance spectroscopy. The experimental conditions were similar to those previously reported [1H) and 15N NMR spectra were obtained at 298 K on a Bruker Advance instrument operating at 700.132 MHz. Nuclear magnetic resonance studies were carried out in 95% H2O–5% D2O. Two-dimensional and 3D experiments (15N-HSQC-TOCSY and 3D 15N-HSQC-NOESY spectra with mixing times TOCSY from 0.040 to 0.080 s and for NOESY from 0.080 to 0.3 s) [15N-HSQC-NMR. Spectra of the 15N labeled N4BP1 domain alone and in presence of NEDD8 or ubiquitin at different molar ratios were run [15N labeled amide groups in the different conditions, TOPSPIN 3.1  and NMRView software packages [reported . Proton o 0.3 s) ,35,36,37were run . This puwere run . To monipackages ,42.Interaction of N4BP1 in complex with ubiquitin and NEDD8 performed by circular dichroism CD. The circular dichroism spectroscopy (CD) measurements were performed on a Jasco 810 spectropolarimeter  using a 0.1 cm path length cell cuvette. The concentration of N4BP1 was 50 μM in H2O. The CD spectra were obtained in the far UV spectral region from 195 to 260 nm using a step resolution of 0.2 nm and a speed scan for spectra collection of 20 nm/min. Spectra obtained were the average of four scans at room temperature. The estimate of secondary structure elements content was obtained using the web server for CD data prediction as described in the text. Circular dichroism studies were performed at a protein concentration of 50 µM and by adopting the scheme described below. In the first step, the CD spectra of N4BP1 and ubiquitin alone were obtained and expressed as rotatory power. Then, the CD spectrum of N4BP1 in the presence of an equimolar amount of ubiquitin (or NEDD8 for comparison as reported below) was performed. In order to identify the possible conformational changes due to protein–protein interactions, the CD spectra, expressed as rotator power and obtained with the components alone, were subtracted point-by-point from the CD spectrum of the protein complex. Specifically, from the mixture N4BP1+ubiquitin, the first subtraction (mixture-ubiquitin) allowed the evaluation of the conformational changes induced on N4BP1 in complex with ubiquitin with respect to N4BP1 alone. Subsequently, by subtracting the CD spectrum of N4BP1 alone from the one of the mixture N4BP1-ubiquitin (mixture-N4BP1), we obtained the conformational changes induced on ubiquitin in complex with N4BP1. Finally, in order to better detect the tiny changes in the CD profiles, the traces of the obtained differences transformed in mean residue ellipticity were vertically amplified. An identical procedure was applied to the study of the interaction of N4BP1 upon binding with NEDD8. In all these experiments, great attention was devoted to maintaining the same protein concentration, buffering system, and ionic strength.HADDOCK procedure. The HADDOCK protocol of ambiguous interaction restraints (AIR) [ts (AIR)  was usedIsothermal titration calorimetry (ITC). The ITC measurements were performed on a MicroCal PEAQ-ITC  instrument in ITC buffer . First, ubiquitin was titrated from a stock of 3.3 mM to 246 µM of N4BP1 provided in the reaction cell. The reverse experiment was performed by titrating from a stock of 2.78 mM N4BP1 to 200 µM ubiquitin in the cell. For each experiment, we used 38 injections of 1 µL with 150 s spacing at room temperature with 750 rpm stirring speed. Raw data were analyzed with the integrated analysis tool, and heat production was fitted to a one-site binding model. Plots are displayed with the GraphPad Prism 8.1.2 for macOS .


Vigna subterranea (L.) Verdc.) (BG) flour from red and black-eye varieties were studied. Functional properties were water and oil absorption, gelation, pasting, emulsification and foaming capacity. Pre-treatment of seeds  improved dehulling efficiency of BG varieties. Protein content of flour ranged from 15.6–19.6%, starch from 47.8–52.0% and sucrose from 1.9–5%. An improvement was observed for protein and ash content of pre-treated flour compared to raw flour. Heat treatments increased onset gelatinization temperature of flour. Black-eye BG flours that had higher starch content, also had better gelation capacity than red BG flours. All pre-treatment methods decreased flour emulsification capacity and stability. Dry-roasting caused a greater decline than other methods, whereas soaking had little effect on emulsion stability. Further, soaking increased foaming capacity, whilst a decline was observed in roasted flour. All pre-treatment methods increased oil absorption capacity of both BG flour varieties. Overall, soaked and combined soaked and roasted flour is recommended for BG flour to be incorporated in food products.Variability in dehulling efficiency, colour, chemical composition and selected functional properties of raw and pre-treated bambara groundnut ( Vigna subterranea (L.) Verdc.), which features in subsistence farming systems throughout semi-arid regions of SSA x m X-ray microfocus and nanofocus CT scanner.Flour was dispersed in ethanol and sonicated according to Kaptso, Njintang  before mColour was measured using Hunterlab colourflex EZ. Differences were determined by calculating chroma, hue and ΔE*.Dry matter, fat and ash content of BG flour were determined in duplicate according to methods described by Association of Official Analytical Chemists . AnalysiFlour (2.5 g) was dispersed in 25 ml Milli-Q/ethanol mixture (50–50% (v/v)) and centrifuged . The supernatant was filtered  and centrifuged . A calibration curve was made for D-fructose (Merck) and D-sucrose (Sigma Aldrich). Quantification of carbohydrates (sucrose and fructose) was by HPLC using an Evaporative Light Scattering Detector (ELSD) [Thermal properties of flour were analysed using a Differential Scanning Calorimeter (DSC) equipped with a thermal analysis data station  according to Evageliou, Richardson . Flour (Least gelation concentration (LGC) was determined in triplicate according to .Pasting properties were determined according to Afolabi  using a Foaming capacity (FC) and stability (FS) were determined in triplicate as whip-ability of flour dispersed in water according to Coffmann and Garciaj . Flour (Emulsification capacity (EC) and emulsion stability (ES) were determined according to Chaparro Acuña, Gil González  with somWater and oil holding capacity (WAC and OAC) were determined using the method of Diedericks and Jideani .Results were analysed and expressed as mean values with standard deviations. Two-way ANOVA statistical analysis was performed to determine significant differences amongst means. When significant differences (p < 0.05) were found, results were compared using a post-hoc test (Tukey). All statistical analyses were run using SPSS v 23.0 software.According to Malik and Saini , a decreRaw seeds are very difficult to mill (HTM properties) as shown by a DE (%) of less than 15%, which increased to 83–88% when a dehulling pre-treatment was adopted. 10, d50 and d90, which represent the diameter of 10, 50 and 90% of the population of the particles, are represented in Milling efficiency of raw coarse milled seeds increased after pre-treatment of seeds from 65% to 70–75% showing little distinction between varieties and methods, implying the applicability of all pre-treatments in circumventing HTM properties. ab, chroma and hue values of flour made from red and black-eye varieties using different pre-treatment methods. Colour changes during roasting are ascribed to Maillard reactions occurring during roasting [ab, chroma and hue values of flour. The ΔE*ab specifies differences between colour of control sample (raw) and pre-treated flour [roasting , influened flour . The ΔE*Chroma indicates the degree in which a colour differs from the neutral colour of the same value whilst hue is defined as perception of colour of an object  . Just liab, chroma and hue values of flour made from red and black-eye varieties using different pre-treatment methods. Colour changes during roasting are ascribed to Maillard reactions occurring during roasting [ab, chroma and hue values of flour. The ΔE*ab specifies differences between colour of control sample (raw) and pre-treated flour [roasting , influened flour . The ΔE*ed flour . Just liProtein content of flour from both varieties ranged from 15.6–19.6 g/100 g DW using a factor of 5.7 to convert nitrogen to protein. In previous studies 6.5 was used , 38. TheHowever, as expected, no significant differences between pre-treatments were observed (p = 0.98) as thermal treatments do not change the amount of proteins.The fat content of flour, which ranged from 7.0 to 8.6 g/100 g DW, agrees with the 6.0–10.4 g/100 g reported by Yusuf, Ayedun . Fat conThe starch content in flour from raw red and black-eye seeds, i.e. 50.4 and 49.8 g/100 g DW, respectively, is comparable to the 39–50% reported by Poulter , but lowThe starch content of the red variety decreased significantly (p = 0.000) due to pre-treatment contrary to trends in pre-treated black-eye flour. The exhibited opposite behaviour in varieties is attributed to differences in the rate of starch hydrolysis due to presence of fibre, possible presence of natural enzyme inhibitors during analysis and inherent differences in starch structure and composition , 48. InsFructose and sucrose contents ranged from 0.18 to 0.33 g/100 g DW and 1.9 to 5.0 g/100 g DW, respectively. Soaking decreased sucrose in both varieties, but the effect was more pronounced in the black-eye variety, which initially had a higher sucrose content. The current findings agree with Adebowale, Awolala , who alsThe ash content, which ranged from 2.5 to 3.2 g/100 g DW, agrees with the 2.8 g/100 g and 3.6–3.8 g/100 g DW reported by Abiodun and Adepeju  and Kapt0) of raw flour, i.e. 77.1 and 76.9°C, was comparable to 76.8°C reported by Sirivongpaisal [Gelatinization temperatures and enthalpies of flours are presented in ngpaisal , but higngpaisal .0) and peak (Tp) temperatures were obtained in the study as compared to cowpea (Tp = 86°C) [The substantial differences observed indicate differences in the starch granule, such as a more rigid granular structure in the red variety . Lower o = 86°C) . Enthalp = 86°C)  and Kapt = 86°C) .p) temperatures for both varieties, indicating that heat treatment hardly affected (Tp). Pre-treated flour increased enthalpy of gelatinization (ΔH) and the same statistical trend as for gelatinisation temperatures was observed.Pre-treatments increased gelatinization temperatures, indicating alterations in starch granule characteristics. In all cases, a significant effect of variety on gelatinization temperatures (p > 0.05) was observed. Basically, flour from the red variety had higher gelatinization temperatures than flour from the black-eye variety. Furthermore, a significant effect of pre-treatments on gelatinization temperatures (p > 0.05) was revealed. Pre-treatments resulted in similar values peak (Te-T0) were small, reducing the importance of this flour characteristic for food applications.Gelatinization improves stiffening abilities of flour. In food applications, good gelatinization properties are important for thickening of soups and porridge . During Generally, black-eye flours had the least LGC values as compared to the red variety flours. This is credited to thee higher starch content of flours from the black-eye variety. Moreover, differences are also ascribed to variations in the relative ratio of protein and lipids and the interaction between these components . In bothRelative to simple sugars, Evageliou, Richardson  showed tPasting properties of raw and pre-treated bambara groundnut varieties are presented in Peak viscosity indicates the water-binding capacity of the starch whereby the gelatinized starch reaches its maximum viscosity during heating in water . CurrentThe final viscosity indicates the ability of the starch to form a viscous paste or gel after cooling. Final and setback viscosity decreased significantly in both varieties due to roasting, while an insignificant decrease was recorded in soaked flour of both varieties. Low final viscosity implies that flour will form a low viscous paste rather than a thick gel on cooking and cooling. Nutritionally, this means a high calorific density for a low volume . The setPasting temperature provides an indication of the minimum temperature required to cook a flour . High paEmulsification capacity (EC) of raw and pre-treated flour ranged from 29 to 50% as shown in ES of flours from raw seeds, i.e. 47.9 and 45.2%, were lower than the 70% reported for soaked BG flour by Adebowale and Lawal . All preGood emulsification properties are important in many fat-containing food products, such as sausages, vegetable milk and milk-based products, as well as cake batters . The negFoams can be defined as a colloidal dispersions in which gas is the dispersed phase and liquid is the continuous phase . FoamingFC of the black-eye variety was significantly higher than of the red variety. FC was increased by soaking, whilst a decline was observed for roasting, agreeing with Obatolu, Fasoyiro  for roasFC has been related to the amount of protein, with native protein having better foaming abilities than denatured protein . This isWater absorption capacity (WAC) of raw and pre-treated flours varied from 0.51 g/g to 1.12 g/g DW , which iOil absorption capacity (OAC) of flours ranged from 0.56–0.68 g/g , which iAll pre-treatment methods improved dehulling and milling efficiency of BG varieties cementing applicability in flour production. The decisive factor in selecting the preferred method for a specific food application has to focus on the sensorial, functional and nutritional properties of the flour. Colour was affected by roasting, such that roasted flour had a darker colour. Nutritionally, bambara groundnut flour compares with other legumes such as cowpea and chickpea. Varietal differences in chemical properties were minor, except for protein content and ash content, which were much higher in the red variety, making it considerably more nutritious. Consequently, pre-treatment is expected to have more effect on the functional properties of the red variety.Differences in thermal properties due to varietal differences and pre-treatment methods were observed. The flours from the red variety had higher gelatinization temperatures as compared to the black-eye variety, whilst roasting caused a higher onset gelatinization temperature. Starch granule alterations are credited in the increase in gelatinization temperatures. Black-eye flours had lower LGC values than the red variety flours, which can be attributed to the higher starch content in the former.Concerning food applications, the low emulsification and low foaming properties reported, suggests that BG flour is not suitable for food products that require a high percentage of porosity. Therefore, the solution to this will be blending with wheat to improve the properties. The high WAC of dry-roasted flour and combined soaked and roasted flour indicates usefulness of flour especially in food formulations involving dough due to its ability to absorb water and swell for improved consistency in food.In conclusion, based on functional properties, both varieties are recommended for flour processing. No single processing method is optimal, but is determined by the intended application. Based on functional properties, the soaked and combined soaked and roasted flours are recommended for further research in product development and consumer acceptance of locally consumed food products such as porridge, soups, bread, cakes and fritters as illustrated in


Klebsiella pneumoniae carries a thick polysaccharide capsule. This highly variable chemical structure plays an important role in its virulence. Many Klebsiella bacteriophages recognize this capsule with a receptor binding protein (RBP) that contains a depolymerase domain. This domain degrades the capsule to initiate phage infection. RBPs are highly specific and thus largely determine the host spectrum of the phage. A majority of known Klebsiella phages have only one or two RBPs, but phages with up to 11 RBPs with depolymerase activity and a broad host spectrum have been identified. A detailed bioinformatic analysis shows that similar RBP domains repeatedly occur in K. pneumoniae phages with structural RBP domains for attachment of an RBP to the phage tail (anchor domain) or for branching of RBPs (T4gp10-like domain). Structural domains determining the RBP architecture are located at the N-terminus, while the depolymerase is located in the center of protein. Occasionally, the RBP is complemented with an autocleavable chaperone domain at the distal end serving for folding and multimerization. The enzymatic domain is subjected to an intense horizontal transfer to rapidly shift the phage host spectrum without affecting the RBP architecture. These analyses allowed to model a set of conserved RBP architectures, indicating evolutionary linkages. Klebsiella pneumoniae is a Gram-negative bacillus. In spite of being part of the natural human and animal flora, K. pneumoniae is also the widespread cause of both nosocomial and community acquired infections. Since 2013 K. pneumoniae has been marked as a prominent member of the carbapenem-resistant Enterobacteriaceae (CRE), featured by a multidrug-resistant phenotype and labeled as a class of antibiotic-resistant bacteria for which novel ways of therapy are most urgent  to a specific site in the middle of the K1-5 adapter protein, whereas the second tailspike  binds to a different specific site in its C-terminal part through an undecapeptide (MIQRLGSSLVK) , demonstrating exchange of the domain across members of the Podoviridae and Myoviridae families with highly different tail structures  . The hepLGSSLVK) . Notablyructures , 2015.E. coli N4-like podovirus G7C . In a large proportion of these phages  we could predict an RBP with depolymerase activity. The observed large diversity of depolymerase domains accommodates the high diversity of capsular serotypes among Klebsiella strains. Based on an integrated analysis, we propose diverse RBP architectures in Klebsiella phages.In this study we present an extensive bioinformatic analysis of the structural and genetic organization of depolymerase-containing RBPs in Klebsiella phages were collected from the GenBank database (retrieved at 15.08.2018). A number of 59 phages were finally analyzed  theKlebsiella phages were then grouped based on gene homology and a conserved gene synteny into KP32viruses, KP34viruses, and KP36viruses and into groups containing only Klebsiella-specific phages similar to phage JD001 (belonging to Jedunavirus), similar to phage Menlow (belonging to Ackermannviridae), similar to phage ΦK64-1 (belonging to Alcyoneusvirus). Within each group, further subdivisions were proposed for the purpose of this study, based on the organization of the RBP gene cluster .All selected 5  over at least 166 residues starting from the N-terminus of the corresponding protein among phages belonging to the same group. These parameters were set empirically based on the shortest identity region found among all RBPs, specifically in the first RBP of phage IL33, belonging to KP32viruses group B (166 amino acids) and the identity% of the first RBP of phage Kp1. When more than one RBP was present, the anchor domain was annotated in the RBP in which also a T4gp10-like domain was detected. In the other RBP(s) the N-terminal conserved sequence was called ‘conserved peptide,’ which was also generally shorter than the anchor domains. To define consensus sequences of the anchor domains and conserved peptides, multiple sequence or pairwise alignment were used, since these structures are highly conserved among phages from the same group. To identify domains involved in the branching of RBPs, the sequences were analyzed by HHPred performing protein structure prediction5  in searc5 . WebLogo5  that may explain the RBP architecture of the particular phage. To further refine this architecture, we analyzed the sequence for the presence of conserved peptides that may mediate attachment to putative T4gp10-like domains. We integrated all these data to model the RBP apparatus of an extensive and diverse set of Klebsiella phages with (predicted) depolymerase activity.Taxonomically closely related phages are characterized by a synteny of conserved structural genes interrupted by divergent RBP genes, which are subject to intensive horizontal transfer. We therefore inspected the region of structural genes across different Podoviridae and have tail fibers attached to a short, non-contractile tail. A similar synteny of highly conserved structural genes is observed across twenty-one KP32viruses  is as an opposite example of truncation. The first RBP lacks an enzymatic or receptor binding domain but contains an N-terminal anchor including a fragment of a T4gp10-like domain, while the second tail fiber is a full-featured RBP that contains a conserved N-terminal peptide and a depolymerase domain .E. coli phages K1E and K1-5 and Salmonella phage SP6  contains no predicted enzymatic domain. Group B KP34viruses also encode a second RBP with a predicted enzymatic activity and the same conserved heptapeptide motif as in the second RBP of group A KP34viruses (MALxxLV). The organization of the RBPs in group B KP34viruses is thus similar to that of group A KP32viruses. We found two incongruences in this genus, specifically viruses KP-Rio/2015 and myPSH1235. They both share the gene synteny of KP34viruses but no RBPs were annotated in their genomes. Further genome analysis revealed two open reading frames that presumably fulfill the role of RBPs. We found that phage myPSH1235 follows the RBP organization of group A KP34viruses, while phage KP-Rio/2015 encodes a large first RBP with a predicted enzymatic activity (and a fragment of a T4gp10-like domain) and a second protein that is only 61 aa long, which likely represents a truncated, non-functional RBP. Therefore, phage KP-Rio/2015 forms a different group C with an RBP organization analogous to KP32viruses group C.Klebsiella phages analyzed in this study belong to three different myovirus groups  with the latter two groups having a potentially broad host spectrum since they encode between five and nine   but with a different distal end of the tail, which ends with “stars” or “prongs,” being identified as tailspikes  protruding from the baseplate. Nine ge RaK2)  or even ge RaK2)  (Supplemge RaK2) , necessinviridae . Ackermailspikes .The putative RBP genes of the viruses of the JD001 group  were ideThe viruses of the Menlow group encode, amid a conserved synteny of structural genes, four non-conserved putative RBPs and one conserved RBP, all with putative depolymerase activity . Phages via a conserved N-terminal anchor and that its T4gp10-like domain probably provides an attachment site for at least two RBPs . Together they constitute a unit of branched tail fibers. The highly conserved fifth RBP (protein 8) may be the central tail fiber that protrudes from below the plane of the baseplate  and RBP 3, whereas RBPs 3, 5, 7  contain aseplate . More stKlebsiella phages belonging to ΦK64-1 group  and phage 1513 (group C) represent exceptions from the general structure of group A . Instead, a small domain (residues 4–63) homologous to domain B (92–155 aa) of the distal tail protein (Dit or T5pb9) of siphovirus T5 has been detected. Dit is located in the T5 tail tip at the junction between the tail tube and the ultimate conical structure and is composed of two domains. Domain A forms a hexameric structure and connects to the end of the tail tube, whereas domain B constitutes the attachment site for three L-shaped tail fibers . These LPhage PKP126 and 1513  have an additional RBP with putative depolymerase activity. Its exact role is difficult to predict and typical elements hinting at a specific structural organization such as a conserved peptide or anchor domain are missing. We hypothesize that those enzymes are not incorporated in the phage particle, but rather are produced as soluble proteins. Upon cell lysis the neighboring cells are sensitized for infection through enzymatic removal of the capsule by the soluble, diffusible depolymerase. This mechanism would be especially beneficial for phages lacking depolymerase activity in the their first RBP .An additional preceding RBP . An altein silico analysis of the RBPs of Klebsiella phages genomes with a focus on RBPs with depolymerase activity. The tripartite relationship between depolymerase specificity, capsular serotype and phage host spectrum has now extensively been demonstrated for Klebsiella phages  that produce two RBPs encode the structural elements for an anchor-branched mechanism as reported for phage G7C. Here, the first RBP contains a conserved N-terminal anchor serving for attachment to the virion, followed by a specific fragment of a T4gp10-like domain providing the docking site for a second RBP. Notably, the fragment encoding the T4gp10-like docking site in those Klebsiella phages is shorter compared to the corresponding domain in T4 and may therefore correspond to a single attachment site. The second RBP is presumably attached via a conserved peptide . This conserved peptide is different for each group of phages, varies in length and can be as short as seven amino acids. Such attachment via a short peptide is in line with the RBP complex of K1E/K1-5/SP6-like phages where both RBPs carry either a 7- or 11-residue conserved peptide at their respective N-terminus. In the case of E. coli phage G7C the shorter G7Cgp63.1 RBP carries a positively charged surface that binds to the T4gp10-like domain of G7Cgp66, yet, the conserved peptides in Klebsiella phages lack this positive charge, inferring that different interacting forces take place between the first and second RBP. Similar to the RBPs of phage K1-5, the two experimentally verified depolymerases of phage KP32 target two different capsular serotypes. In both cases the double RBP system thus expands the host spectrum.In this study, we propose that RBPs of  systems . The simKlebsiella phages belonging to Podoviridae show similarity with the well-characterized N-terminal domain of T7 tail fiber and their first 300 amino acids are conserved across different podoviruses analyzed in this study. The T7 tail fiber attaches with its N-terminal anchor domain to the region where the adaptor (gp11) interacts with nozzle (gp12) of the short tail complex , a remarkable horizontal transfer event has taken place between the distal tail protein and the tail fiber of KP36viruses when comparing to the siphovirus T5 model. Domain B of the distal tail protein has been transferred to the N-terminus of the tail fiber protein in KP36viruses. Whereas in phage T5 protein–protein interaction occurs between the N-terminus of the RBP and domain B of the distal tail protein, novel interactions between domain A of the minor tail protein and domain B embedded in the tail fiber must have been evolved to compensate for the loss of interaction by a direct covalent bond as in phage T5.The presence of either an anchor- or an anchor-branched system is not directly linked to the taxonomic organization. In addition, there is no sequence homology between functionally similar, structural building blocks across those phage groups. E.g., the first 140 amino acids of the N-terminal anchor domains of RBPs encoded by  complex . The corE. coli phage G7C has acquired a full T4gp10-like domain (similarity to both subdomain D2 and D3 of T4gp10), offering a potential second attachment site for a different RBP. This second site is not occupied in phage G7C, whereas the E. coli model phages K1-5, SP6, and K1E have effectively two RBPs attached to the same intermediate protein that also comprises both subdomain D2 and D3. In K1-5, SP6 and K1E, this intermediate protein with the full T4gp10-like domain has lost its C-terminal receptor-binding domain, resulting in an ‘adapter’ system – a short protein with two sites for binding two different RBPs and no domain beyond these two domains  cannot be excluded as well. The success of the modular build-up of the RBP apparatus and the extensive number of horizontal transfer events have obscured possible insight in the direction of this evolution. The assumption that evolution generally takes place from simple to more complex systems, hints at the first direction (from anchor to adapter). KP32viruses group C and D, and KP34viruses groups A and C may have lost a second intact RBP by retrograde evolution when thriving in a new environment that is dominated by a single serotype Klebsiella strain. Having a truncated second RBP may provide a fitness advantage in such a situation. The truncated RBP may remain as a temporal docking site to acquire a new RBP for host range expansion by horizontal transfer when moving to a niche with different Klebsiella serotypes. Phages belonging to the Menlow group and ΦK64-1 group carry multiple RBPs that obviously recycle established structural elements such as a conserved N-terminal domain, short conserved peptides and a T4gp10-like domain (or fragments thereof). Yet, more experimental  studies are required to make a plausible prediction on the structural organization of these elaborated RBP systems.Interestingly, several phages with a single enzymatic RBP do not follow the anchor system as described for phage T7, but use the anchor-branched system of G7C with either the first  or second RBP  being truncated. In the case of KP34viruses it is even the predominant RBP system. The occurrence of these intermediate RBP systems suggests evolutionary linkages between the different RBP architectures. Starting from the simplest organization with a single RBP , the acquisition of a fragment of a T4gp10-like domain allowed for the attachment of a second RBP (KP32viruses group A and KP34viruses group B). The first RBP from  domains . It shouKlebsiella phages. The modular composition and re-use of established structural domains for anchoring and branching provide the phages the full potential to rapidly shift capsular serotype specificity or to expand the spectrum. We expect that the increasing amount of (meta)genome sequencing data will reveal further evolutionary relationships between some of the groups we describe in this analysis, but the main groups will remain in place. The data available to us today clearly show that the architecture of RBP systems is dominated by horizontal transfer events of modules that can be as small as short peptides to as large as multiple domains. Although our analysis was based on experimentally confirmed interactions of E. coli phage RBPs (In summary, we have modeled the organization of diverse RBP systems in age RBPs , furtherThe datasets analyzed for this study can be found in the GenBank. All genome and protein accession numbers are listed in the tables and in silico search approach of RBPs domains and modeled the RBP assembly systems. PL introduced the concept of branching RBPs. AL performed in silico analysis and collected the data. AL, PL, ZD-K, and YB analyzed the data and worked on the manuscript. All authors read and accepted the final version of the manuscript.AL, ZD-K, and YB designed the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Intracellular transport is one of the most confusing issues in the field of cell biology. Many different models and their combinations have been proposed to explain the experimental data on intracellular transport. Here, we analyse the data related to the mechanisms of endoplasmic reticulum-to-Golgi and intra-Golgi transport from the point of view of the main models of intracellular transport; namely: the vesicular model, the diffusion model, the compartment maturation–progression model, and the kiss-and-run model. This review initially describes our current understanding of Golgi function, while highlighting the recent progress that has been made. It then continues to discuss the outstanding questions and potential avenues for future research with regard to the models of these transport steps. To compare the power of these models, we have applied the method proposed by K. Popper; namely, the formulation of prohibitive observations according to, and the consecutive evaluation of, previous data, on the basis on the new models. The levels to which the different models can explain the experimental observations are different, and to date, the most powerful has been the kiss-and-run model, whereas the least powerful has been the diffusion model. The structure of the ER–Golgi interface and the Golgi complex (GC) is well-known and has been described many times  observation is IGT of megacargoes. For example, the VM poses that IGT is carried out by COPI vesicles. However, COPI vesicles have a diameter of 52 nm, and thus megacargoes cannot be transported by COPI vesicles. However, they are transported  of any cargo, and especially of megacargoes, are the prohibitive observation for the CMPM. Also, the speed of the cargo delivery from The main principle of the KARM is fusion before fission, and even that fusion results in fission. Fusion/ fission might occur at the same site  or at different sites . Within the framework of the asymmetrical KARM, fusion would be between the edges of the proximal and distal compartments whereas fission would be somewhere within the proximal compartment where rows of pores or thin tubules should be localized and SNAREs should be concentrated over the cargo domains , which connects a cargo domain with the compartment domains Pores should be consumed during transport.per se under all conditions.There should be a negatively exponential regression line for the process of emptying of the GC For EGT and post-Golgi transport, the bolus-like mechanism should be optimal.All of the compartments should always be connected. However, the existence of many SNAREs already denies this falsification observation.S. cerevisiae, where all of the Golgi compartments are localized far away from each other. However, we then described connections between different Golgi compartments 1, BST2  or BST3 did not cause cell death, although the removal of just Sec13 was lethal for the cells. In the absence of Sec23 and also of p24, which regulates the behavior of COPI, transport occurs in yeast. Also, in the absence of cargo transport, the GC in yeast disappears  after a 15°C temperature block. However, the existence of ER–Golgi connections was not confirmed by Koga and Ushiki  that are contradictory to their logic, whereas the KARM should explain corner-stone observations that support the VM, DM, and CMPM.The main problem for the VM of IGT Palade, , 1984 isIt is established now that the vast majority of cargoes are absent from COPI vesicles. We previously presented a list of cargo proteins that are excluded from COPI vesicles , and 0.9 μm3 of Golgi volume . These considerations suggest that they observed the movement of carriers, that were much larger than COPI vesicles. Also in Figure 2f by Dunlop et al. .” To demonstrate that round profiles represented separated vesicles, they showed very small sized serial electron microscopy tomography images of only one round profile. However, this round profile showed a visible neck that connected it with a Golgi cisterna. This neck is visible on frames 45–55 of Rizzo et al. . If we tDuring synchronous IGT, the number of COPI vesicles should be sufficient for the recycling of all of the resident proteins. However, when a large amount of cargo moves across the GC, even if we take into consideration the maximal possible speed of COPI vesicle formation . However, there are no buds coated with COPI on the TMC  if pores inside cisternae are consumed, there should be the need for resting of the Golgi stack; (2) recycling of Ykt6 is improbable. As such, there should be one use of this SNARE, and after consumption of the cytosolic pool of Ykt6, there should be the need for the resting of the whole Golgi complex in the entire cell. Thus, there could be several waves of cargo, and only then would the pores be consumed. Fission and then fusion of COPI vesicles might induce the formation of tubules and restoration of the number of pores along cisternal rims ; chylomicron-containing distensions formed during transcytosis of lipids through enterocytes; and cisternal distensions filled with VLDL particles in hepatocytes. The presence of a row of pores behind the cargo domain during IGT and the concentrating of SNAREs over the cargo domain favor the KARM. Our observation that there is no cargo diffusion between VSVG-GFP domains formed during IGT   of COPI-coated buds on the trans-most cisterna  elimination of excessive membrane curvature , and this should induce a bolus-like delivery of ER-to-Golgi carriers. At the post-Golgi stage, the tubule or endosome cis-compartment displaces Sec31 from the cargo domain. (4) After arrival of the cis-compartment at ERES, the size of the cargo domain should increase (C). (5) The transient phase of maturation when domains of two different Golgi compartments are situated within the same Golgi cisterna should be shorter than other  phases when only one Golgi compartments is present within the same Golgi cisternae . (6) The cargo should be concentrated into two cargo domains. However, if other cargoes (which are invisible to the observer) are transported simultaneously, this multiplication should be slightly lower than 2-fold. (7) During the transitional phase, the initial co-localization would be between cargo and trans and not between cis and trans markers. (8) The cargo-domain and the Golgi domain should be in the same membrane disk with pores, but within their own domains. The cargo domains should be separated from the rest of the Golgi cisterna by pores.Thus, the KARM give the following predictions. (1) The cargo should be organized in the domain more or less clearly separated from the domains, which are formed by Golgi-resident proteins. (2) Initially, there should be lateral contact between the cargo domain and the Golgi domain. Then the cargo has to go together with the Golgi compartment. (3) After its arrival at ERES, the All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.