Cnp1 localisation or recruitment to centromeres. Our sequence alignments show that Raf2 contains a Replication Foci Targeting Sequence (RFTS) domain homologous to the RFTS domain of the human DNA methyltransferase DNMT1. We show that the Raf2 RFTS domain is required for centromeric heterochromatin formation as its mutation disrupts H3K9 methylation but not the processing of centromeric transcripts into small interfering RNAs (siRNAs) by the RNAi pathway. Analysis of biochemical interactions demonstrates that the RFTS domain mediates an interaction between Raf2 and the CLRC component Cul4. We conclude that the RFTS domain of Raf2 is a protein interaction module that plays an important role in heterochromatin formation at centromeres.Centromeric heterochromatin assembly in fission yeast is critical for faithful chromosome segregation at mitosis. Its assembly requires a concerted pathway of events whereby the RNA interference (RNAi) pathway guides H3K9 methylation to target sequences. H3K9 methylation, a hallmark of heterochromatin structure, is mediated by the single histone methyltransferase Clr4 , a component of the CLRC complex. Loss of or defects in CLRC components disrupts heterochromatin formation due to loss of H3K9 methylation, thus an intact, fully functional CLRC complex is required for heterochromatin integrity. Despite its importance, little is known about the contribution of the CLRC component Raf2 to H3K9 methylation and heterochromatin assembly. We demonstrate that Raf2 is concentrated at centromeres and contrary to other analyses, we find that loss of Raf2 does not affect CENP-A Schizosaccharomyces pombeS. pombe due in to its genetic tractability and the fact that it encodes only single non-essential genes involved in this pathway RNAi is a widely conserved process in eukaryotes characterised by small RNAs bound by Argonaute effector proteins which act as guides to target homologous sequences for repression In fission yeast, the main domains of heterochromatin are found at centromeres, telomeres and the silent mating-type locus in vitroIn addition to Clr4, the CLRC complex consists of the cullin scaffold protein Cul4, the β-propeller protein Rik1, the RING box protein Rbx1, the WD-40 protein Raf1/Dos1 and Raf2/Dos2 Although CLRC has been the subject of extensive study, the role of the Raf2 subunit within this complex and its contribution to heterochromatin formation remains elusive.Cnp1 histone H3 variant to centromeres In addition to its association with CLRC, Raf2 has been proposed to regulate transcription within heterochromatin during S phase via its interaction with Cdc20 (DNA polymerase-ε) and the transcription factor Mms19 at replication forks DNMT1 is the major enzyme responsible for the maintenance of the typically repressive DNA modification in plant and vertebrate cytosine methylation of CpG dinucleotides We set out to characterise the RFTS domain of Raf2 and its role in centromeric heterochromatin formation. We show that the RFTS domain of Raf2 can be modelled on that of DNMT1 and that specific residues within this domain are crucial for heterochromatin integrity. We demonstrate that alteration of particular residues within the RFTS domain disrupts a direct interaction between the Raf2 and the Cul4 subunit of CLRC. Furthermore, although heterochromatin is disrupted, the generation of siRNA remains unperturbed, suggesting that Raf2 has separable roles in chromatin modification and siRNA production. Thus we have identified the RFTS domain of Raf2 as a protein interaction module crucial for heterochromatin integrity and centromere function.S. pombe strains used in this study are described in Standard procedures were used for bacterial, fission yeast and budding yeast growth and genetic manipulations Raf2-I98A and Raf2-E104A mutations were generated by mutagenizing pDONR201-Raf2 using the QuikChange XL Site-Directed Mutagenesis Kit (Stratagene). Raf2 mutant PCR products were generated using primers with 80 bp homology to the each side of the site of recombination and transformed into the FY17087 strain bearing the RFTS domain replaced with a +ura4 marker gene. Correct integrants were selected on FOA media and confirmed by PCR and sequencing of the +raf2 gene.Deletion and epitope tagging (3xFLAG) of Raf2 was achieved by homologous recombination with PCR fragments comprising resistance cassettes flanked by sequence homologous to insertion sites Raf2-S100F allele was isolated in a random UV mutagenesis genetic screen. FY 1181 cells were spread on YES plates lacking adenine, irradiated with 15000 µJ (around 50% killing) and incubated at 36°C for 5–7 days. Fast-growing colonies were picked and tested for thermosensitivity of silencing at +otr1R(SphI):ade6 and for supersensitivity to TBZ. Mutants were backcrossed at least three times. Raf2-S100F mutation was identified by complementation and sequencing of the +raf2 gene.The Sequence and secondary structure alignments were produced using Jalview version 6.1 using Muscle, a multiple protein sequence alignment method Life Technologies). Alexa Fluor 594- and 488-coupled secondary antibodies were used at 1∶1000 (Life Technologies). Microscopy was performed using a Zeiss Imaging 2 microscope using a 100× 1.3 NA Plan-Apochromat objective. Image acquisition was controlled using Metamorph software . Identical exposures were used for different strains in the same experiment. For co-staining experiments, cells were visually scored for a single interphase Cnp1 cluster at centromeres and with/without Raf2 or Swi6 co-staining were counted.Immunostaining was performed as described previously Roche) on a LightCycler 480 Instrument (Roche). qPCR analysis primers are shown in Table S2. Relative enrichments were calculated as the ratio of product of interest to control product (act1+) in IP over input. Histograms represent data from three biological replicates analyzed in parallel.Chromatin immunoprecipitation (ChIP) was performed as described with the following modifications Northern analysis of centromeric siRNAs and qRT-PCR analysis of centromeric transcripts were performed as described previously Raf2-I98A, pGAD-Raf2-E104A and pGAD-Raf2-S100F were generated with QuikChange XL Site-Directed Mutagenesis Kit (Stratagene). The yeast-2-hybrid assay was performed as described previously Yeast two-hybrid vectors were generated by PCR amplification of the Raf2 ORF with primers bearing SalI and BamHI sites. Purified Raf2 PCR products were digested and cloned into SalI/BamHI digested pGBKT7 (Clontech ‘Matchmaker’ system). Plasmids pGAD-nmt1 promoter and demonstrated that Raf2 localises to both the nucleus and cytoplasm Cnp1 at centromeres in the majority of cells examined  have been reported to mislocalise GFP-tagged CENP-ACnp1 to sites other than the centromere in approximately 20% of cells. Consequently Raf2 has been implicated in promoting CENP-ACnp1 localisation to centromeres raf2Δ cells we find that the localisation of untagged CENP-ACnp1, detected with specific anti-Cnp1 antisera, is unchanged; a single focus of CENP-ACnp1 fluorescent signal is detected in 100% of wild-type and raf2Δ cells. This suggests that CENP-ACnp1 association with centromeres is not affected by loss of Raf2  domain and an atypical C2H2 type zinc finger motif  only encode RFTS proteins devoid of a methyltransferase domain. The genomes of other fungi such as Coccoides immitis encode both RFTS only and RFTS plus methyltransferase domain proteins  from C-terminally FLAG-tagged Raf2 expressed from its own locus. However, the resulting Raf2-RFTSΔ-FLAG protein was unstable and the expected 52.6 kD band was not detectable by western . Thereforaf2-1, in a screen for mutants which disrupt heterochromatin at 36°C, but not at 25°C raf2-1 mutant expresses a protein with a missense mutation (S100F) in a conserved fungal residue within the RFTS domain, known hereafter as raf2-S100F. These three mutated residues, I98, S100 and E104, reside in a flexible looped region between the main α-helix of the RFTS domain and the β-sheet region  allele, t region .+cen1:ade6)  . Both thdoes not . Importadoes not . These acul4Δ clr4Δ, rik1Δ, raf1Δ or raf2Δ) display loss of H3K9 methylation and delocalisation of Swi6 raf2-I98A and raf2-S100F cells  assay to determine if the Raf2-I98A, Raf2-S100F and Raf2-E104 mutations affect interactions between Raf2 and other CLRC components. As previously described, we detect a direct interaction between full-length Raf2 and Cul4  for each alignment column: red (greater than 3), violet (between 3 and 1.5) and light yellow (between 1.5 and 0.5). Sequences are named according to their UniProt names. Full species names are: RAF2_SCHPO, Schizosaccharomyces pombe; S9X856_SCHCR, Schizosaccharomyces cryophilus; B6K1K8_SCHJY, Schizosaccharomyces japonicus; L8FMY4_PSED2, Pseudogymnoascus destructans; G2Q7S7_THIHA, Thielavia heterothallica; B2B7F9_PODAN, Podospora anserina; G4ML14_MAGO7, Magnaporthe oryzae; C1GEE7_PARBD, Paracoccidioides brasiliensis; J3K2M4_COCIM, Coccidioides immitis; A2Q7V8_ASPNC, Aspergillus niger; D4DHS5_TRIVH, Trichophyton verrucosum; K2RLQ8_MACPH, Macrophomina phaseolina. Residues which are subject to mutation are labeled. RFTS and C2H2 Zinc Finger domains are boxed in violet and red, respectively. B. Raf2 protein missing the entire RFTS domain does not encode a truncated protein. C. Zoom-in of RFTS structure showing the region containing the point mutations.(TIF)Click here for additional data file.Figure S2A. Western blot demonstrating that both wild type and proteins containing point mutations within the RFTS domain are produced at 36°C. B. Clr4 levels remain constant in cells containing point mutations within the RFTS domain of Raf2. TAT1 is shown as a loading control.(TIF)Click here for additional data file.Figure S3Western blots demonstrating expression of yeast-2-hybrid proteins.(TIF)Click here for additional data file.Table S1S.pombe strains used in this study.List of (DOCX)Click here for additional data file.Table S2List of primers used in this study.(DOCX)Click here for additional data file.


EGFR is a key player in cell proliferation and survival signaling, and its sorting into MVBs for eventual lysosomal degradation is controlled by the coordination of multiple ESCRT complexes on the endosomal membrane. HD-PTP is a cytosolic protein tyrosine phosphatase, and is associated with EGFR trafficking by interacting with the ESCRT-0 protein STAM2 and the ESCRT-III protein CHMP4B via its N-terminal Bro1 domain. Intriguingly, the homologous domain of two other human Bro1 domain-containing proteins, Alix and Brox, binds CHMP4B but not STAM2, despite their high structural similarity. To elucidate this binding specificity, we determined the complex structure of the HD-PTP Bro1 domain bound to the STAM2 core region. STAM2 binds to the hydrophobic concave pocket of the HD-PTP Bro1 domain, as CHMP4B does to the pocket of Alix, Brox, or HD-PTP but in the opposite direction. Critically, Thr145 of HD-PTP, corresponding to Lys151 of Alix and Arg145 of Brox, is revealed to be a determinant residue enabling this protein to bind STAM2, as the Alix- or Brox-mimicking mutations of this residue blocks the intermolecular interaction. This work therefore provides the structural basis for how HD-PTP recognizes the ESCRT-0 component to control EGFR sorting. Epidermal growth factor receptor (EGFR) is a well-known cell-surface receptor tyrosine kinase, and one of the key regulators of cell survival and growth. Its aberrant expression or uncontrolled activity is directly implicated in a variety of tumors . BindingDrosophila melanogaster, are involved in the morphogenesis of MVBs and in the endosomal sorting of cargo proteins, including integrin [HD-PTP is a non-receptor type protein tyrosine phosphatase (PTP) that contains five domains . This protein has been reported to negatively regulate endothelial cell motility ,13 and tintegrin , Wntlessintegrin , and EGFintegrin ,18, and Thus far, two ESCRT components have been found to interact with the Bro1 domain of HD-PTP: the ESCRT-0 protein signal-transducing adaptor molecule 2 (STAM2)  and the KD value of 6.06 μM  contained four HD-PTP(1–361) molecules in the asymmetric unit but did not contain STAM2(350–370), which was revealed after the structure determination  in a complex with STAM2(350–370) to 2.0 Å  bound to the STAM2(350–370) peptide. Our initial trial was unsuccessful, because even though the protein and the peptide were mixed and incubated at a 1:5 molar ratio before crystallization, the resulting crystals  and STAM2(350–370) bind to the concave pocket of the Bro1 domain in a similar manner, mainly through their hydrophobic residues. We therefore superposed our HD-PTP−STAM2(350–370) complex structure onto the structure of the Bro1 domain of Alix bound to the CHMP4B peptide ). Indeed, STAM2(350–370) and CHMP4B(207–224) overlap when bound to the Bro1 domains, and the hydrophobic residues of the two proteins involved in the intermolecular interaction can be matched one by one . Thus, tet al. indicated that the Bro1 domain of Alix was not coprecipitated with STAM2 unlike that of HD-PTP [KD values of less than 8 μM; Despite the overall structural similarity among the Bro1 domains, a recent report by Ali f HD-PTP . We thusNext, the interactions in human cells between STAM2, CHMP4B, and the Bro1 domain of HD-PTP were confirmed by pull-down assays. In order to concentrate on the Bro1 domain-mediated intermolecular association and to exclude the effect of the additional binding between the SH3 domain of STAM2 and the central proline-rich domain of HD-PTP , Flag-taAs previous studies have indicated, the boomerang-shaped Bro1 domain of HD-PTP shares considerable sequence and structural similarity with that of Alix or Brox , an ESCRT-I component involved in EGFR sorting to the MVB, binds the V domain of HD-PTP but not the corresponding domain of Alix [nL motif-binding region in HD-PTP. We thus consider that, as in case of the HD-PTP Bro1 domain and the ESCRT-0 component STAM2, structural study of the V domain of HD-PTP would be necessary to elucidate the basis of its selective binding to UBAP1, which might also contribute to the understanding of the precise role and function of HD-PTP in EGFR sortingIn HD-PTP and Alix, but not in Brox, the Bro1 domain is followed by a V domain (residues 362–699 of HD-PTP). The two V domains share 17% sequence identity and 45% similarity with each other, and both were reported to interact with the Lys63-linked polyubiquitin chain –30. The ceptor 1 , the YPX budding ,33. The  budding . Interes of Alix , providiIn this work, we discovered that the residues 350–370 of STAM2 constitute the HD-PTP-binding region, and presented the crystal structure of the Bro1 domain of HD-PTP in a complex with the core fragment of STAM2. We further delineated the structural feature of the Bro1 domain of HD-PTP, discriminating it from that of Alix or Brox, as certified by structural analyses, ITC binding measurements, and coimmunoprecipitation assays. We believe that this structural information will be a rational basis for future investigations to unravel the overall working mechanism of EGFR trafficking, which has received great interest due to its direct association with cell survival and cancer signaling.E. coli BL21(DE3) RIL strain (Novagen) at 18°C and initially purified using a Ni-NTA column (QIAGEN). After the removal of the N-terminal (His)6-tag through a TEV protease treatment, the protein was further purified using a HiPrep 26/60 Sephacryl S-100 HR gel filtration column  equilibrated with a buffer solution containing 20 mM Tris-HCl (pH 7.5), 200 mM NaCl, 10% (v/v) glycerol and 10 mM dithiothreitol. The final HD-PTP protein samples were mixed at a 1:5 molar ratio with the 20-mer STAM2 peptide (residues 350–370) purchased from Peptron (Korea). HD-PTP(1–361) crystals were obtained by the sitting-drop vapor diffusion method at 18°C by mixing and equilibrating a 0.2 μL protein solution (33 mg/mL) and a 0.3 μL precipitant solution containing 0.03 M HEPES sodium (pH 7.3), 16% (w/v) polyethylene glycol 3350, 0.67% (w/v) tryptone and 0.03 M urea. Before the data collection process, HD-PTP(1–361) crystals were immersed briefly in a cryoprotectant solution, which was identical to the reservoir solution but with 42% (w/v) polyethylene glycol 3350. HD-PTP−STAM2(350–370) crystals were obtained by the sitting-drop vapor diffusion method at 18°C by mixing and equilibrating a 0.2 μL protein solution (24 mg/mL) and a 0.8 μL precipitant solution containing 0.1 M Bis-Tris (pH 7.0), 30% (w/v) polyethylene glycol 3350 and 4% (v/v) acetonitrile. Before data collection, the complex crystals were immersed briefly in a cryoprotectant solution, which was the reservoir solution plus 5% glycerol. Diffraction data were collected on the beamline 5C at the Pohang Accelerator Laboratory, Korea, and processed using the program HKL 2000 [The DNA fragment coding for the Bro1 domain of human HD-PTP (residues 1–361) was amplified by a polymerase chain reaction and cloned into the pET28a plasmid (Novagen), which was used as the template for the preparation of the mutant form of HD-PTP containing N33A and Y34A substitutions. Wild type or mutant HD-PTP protein was produced in the HKL 2000  and PHENHKL 2000  were use6-tag. Each of the DNA fragments coding for the Bro1 domain of HD-PTP containing the T145K, T145R or T145A substitutions, Alix (residues 1–359), and Brox (residues 1–374) were cloned into the pET28a plasmid. The proteins were produced in the E. coli BL21(DE3) RIL strain at 18°C and purified using a Ni-NTA column and a HiPrep 26/60 Sephacryl S-100 HR gel filtration column. Synthetic STAM2 peptides of 21-mer (residues 330-350/350-370/350-370 with Y391A substitution), 20-mer (residues 311–330), and 46-mer (residues 371–416) and a CHMP4B peptide of 18-mer (residues 207–224) were purchased from Peptron (Korea).Each of the DNA fragments coding for the core region of human STAM2 (residues 260-370/260-310/311-370) were cloned into the pET28a plasmid, designed to produce a protein N-terminally fused to maltose-binding protein together with a (His)All measurements were carried out at 2°C on an iTC200 microcalorimetry system . Protein samples were dialyzed against the solution containing 20 mM Tris-HCl (pH 7.5) and 100 mM NaCl. The samples were centrifuged to remove any residuals prior to the measurements. Dilution enthalpies were measured in separate experiments (titrant into buffer) and subtracted from the enthalpies of the binding between the protein and the titrant. Data were analyzed using the Origin software (OriginLab Corp.).Each of case of the DNA fragment coding for HA–STAM2(full), HA–STAM2(1–370), HA–CHMP4B and Flag–HD-PTP(1–712) were amplified by a polymerase chain reaction with cloning into pcDNA3.1/FRT/TO (Invitrogen). HA–STAM2, HA–STAM2 and Flag–HD-PTP were generated by site-directed mutagenesis. HEK293 cells (ATCC) transfected with these constructs were harvested after 48 hours and lysed in a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate and 1mM of EDTA supplemented with a complete proteinase inhibitor cocktail (Roche). Proteins from the cell extracts were immunoprecipitated with anti-HA agarose (Sigma Aldrich) or anti-Flag M2 agarose (Sigma Aldrich). Immunoprecipitated proteins were analyzed by western blot assay using anti-HA (1:5000) (Santa Cruz) and anti-Flag M2 (1:5000) (Sigma Aldrich). An antibody against HSP90 (1:5000) (Santa Cruz) was used to ensure equal loading. The specific protein bands were visualized by enhanced chemiluminescence detection (Amersham).The coordinates of HD-PTP(1–361) and HD-PTP in a complex with STAM2(350–370) together with the structure factors have been deposited in the Protein Data Bank with the accession codes of 5CRU and 5CRV, respectively.S1 Fighttp://bioinf.cs.ucl.ac.uk/psipred/). STAM2 residues Leu260, Val370 and Gln416 are indicated by red triangles.This prediction was obtained from PSI-PRED server ((TIF)Click here for additional data file.S2 Figα traces of four molecules of HD-PTP(1–361) in the asymmetric unit of crystals with the space group P1. (C) Crystal packing interactions. Asn33 and Tyr34 from one molecule (shown in orange) are in contact with Arg205 and four hydrophobic residues  from an adjacent molecule (shown in light blue). Dashed circle highlights intermolecular hydrophobic interactions.(A) The structure of HD-PTP(1–361) presented as a ribbon drawing. Labels of secondary structures are represented according to the order of their appearance in the primary sequence. (B) C(TIF)Click here for additional data file.S3 FigB) or mutant  HD-PTP proteins.ITC measurements were carried out by titrating the 0.5 mM indicated peptide into the 50 μM wild type ((TIF)Click here for additional data file.S4 FigThe STAM2(350–370) fragment in the (TIF)Click here for additional data file.S5 Figα atoms of Tyr34 substituted to alanine. (B) Packing of HD-PTP molecules in crystals. HD-PTP(1–361) and HD-PTP are shown in Cα traces. For clarity, STAM2(350–370) is omitted. Mol A and B of HD-PTP(1–361) are in the same asymmetric unit; Mol A, A' and A'' of HD-PTP are not, but are symmetrically related.(A) Conformational change of HD-PTP induced by the introduction of two mutations. The two residues targeted for mutation are shown in sticks and are labeled. The dashed line indicates the movements of the C(TIF)Click here for additional data file.S6 FigHD-PTP is represented in the surface model bound to the STAM2 fragment shown in green. Residues engaged in the intermolecular hydrophobic contacts Click here for additional data file.S7 FigThe sequences of the Bro1 domains of HD-PTP, Alix, and Brox are aligned based on a structural comparison. The secondary structures of HD-PTP are shown together. Conserved residues are shaded in cyan. The key residues adjusting STAM2 binding are shown in red and are highlighted by a black rectangle. Asterisks denote the HD-PTP residues involved in STAM2 binding. The sequence and structural alignment statistics are listed below.(TIF)Click here for additional data file.


Oscillatory media can exhibit the coexistence of synchronized and desynchronized regions, so-called chimera states, for uniform parameters and symmetrical coupling. In a phase-balanced chimera state, where the totals of synchronized and desynchronized regions, respectively, are of the same size, the symmetry of the system predicts that interchanging both phases still gives a solution to the underlying equations. We observe this kind of interchange as a self-emerging phenomenon in an oscillatory medium with nonlinear global coupling. An interplay between local and global couplings renders the formation of these alternating chimeras possible. Synchronization phenomena are omnipresent in nature, and, consequently, their theoretical description has received great attention for the last two decades45678910111213141214161718The possible importance of chimera states ranges across various disciplines, pertaining to phenomena such as the unihemispheric sleep of animals21222122In contrast, in this Article we present alternating chimera states that spontaneously emerge in an isotropic oscillatory medium with nonlinear uniform global coupling, thereby tightening the connection between chimera states and unihemispheric sleep. As the chimera states found so far in this system are in phase balance, and since the parameters are uniform and the coupling is symmetric, interchanging the incoherent and coherent phases again yields a solution of the underlying equations. A combination of local and global coupling effects then triggers the alternation.W = W is a complex variable describing the dynamical state of the system at any location  at time t and η and angular frequency v. The standard CGLE is a generic model for a spatially extended system close to the onset of oscillations, and is considered one of the most important nonlinear equations in physics29143131The model we consider is a spatially two-dimensional system governed by a modified complex Ginzburg-Landau equation (MCGLE)14c1, c2, v and η (see methods section for details), we were able to reproduce different kinds of dynamics observed in the experimental silicon system, including a two-dimensional chimera statec2 we observe a transition from two-phase clusters , only two domains are left, separated by a roughly straight boundary along one of the axes of the system. Three snapshots in t = 103. We find these approximately regular alternations to persist for at least t = 3.7·107, our maximum simulation time. Moreover, the system was simulated with the same parameter values for 100 slightly differing random initial conditions, always eventually leading to an alternation, the latest occurring at t ≈ 6·104. In contrast, the system was simulated with the parameter values corresponding to the non-alternating chimera state in t = 106 without an alternation taking place.Changing The two-domain state facilitates the study of the alternation process, which always proceeds similarly: First, the turbulence in the unsynchronized domain turns into a two-phase subclustering. Then a spatial pattern emerges in the previously coherent domain, starting at the domain boundary, which acts as a nucleus for turbulence cf.  and 3b. After the turbulence has engaged the formerly synchronized domain, the now turbulent domain grows further in size. This growth process is much slower than the preceding spread of turbulence within the domain. Eventually, the turbulent domain becomes larger than the synchronized one and when a critical size is reached, another alternation takes place.In order to validate the above proposed mechanism, we used initial conditions as shown in t = 40. This rapid synchronization is followed by a spread of turbulence throughout the initially synchronized domain, and the same kind of steady domain growth as observed for the simulation where the turbulent domain is initially smaller. This corroborates that if the turbulent domain becomes too large, an alternation is triggered. Note that the growth rate of the turbulent nucleus in y-direction. This could be a manifestation of the growth being governed by two different mechanisms. The growth process in the negative y-direction is the spreading of turbulence within the initially homogeneous domain. In the positive y-direction, the domain boundary is moving, at a slower pace.When starting the simulation from a state where most of the system is covered by turbulence , an initFor parameters corresponding to a non-alternating chimera state, initializing the system out of phase balance leads to the following behavior: an initially smaller turbulent domain grows until phase balance is reached, while for an initially larger turbulent domain, an alternation takes place at first, followed by the growth of the new, smaller turbulent domain up to phase balance. Thus, the difference between alternating and non-alternating chimeras is that in the former the phase balanced state is not stable and the turbulent domain grows further.η – c2 parameter space, forming a part of the border between ordinary chimera states and two-phase cluster states.Investigating the extent of alternating chimera states in parameter space yields the phase diagram depicted in c2 = −0.66 and η = 0.66, closer to the parameter range where two-phase subclustering was detected previouslyMoreover, when carrying out simulations for In summary, our simulations of a two-dimensional oscillatory medium governed by a complex Ginzburg-Landau equation with additional nonlinear global coupling, give evidence that alternating chimera states may spontaneously occur in isotropic oscillatory media. The simulations suggest that alternations are the result of an interplay between a diffusion-driven expansion of the turbulent phase and a global restriction on its maximum size, as demonstrated in 333435363334The alternating behavior reminds of heteroclinic cycling between two attractors, similar to “slow switching” reported for two-cluster states in Refs. As unihemispheric sleep of animals is suggested to be a prominent example of chimera states emerging in biological systems2122t = 0.05. We used 256 × 256 Fourier modes, a system size of L = 400 and no-flux boundary conditions. Note that the equation is dimensionless.Simulations of Eq. (1) in the main text were carried out using a pseudospectral method, an exponential time stepping algorithmy = ±200  and choosing an appropriate section of the enlarged system.All simulations except those shown in c1 = 0.2, v = 0.1 and η = 0.66 were kept fixed for all simulations depicted in c2 was varied as described in the main text and caption of c1 = 0.2 and v = 0.1 were still left constant, while η and c2 were varied as shown in Simulation parameters t = 2·104, the dynamics were classified as an alternating chimera state. If the turbulent half remained turbulent throughout the pre-set time interval, while the synchronized half remained synchronized, or if they switched just once, the dynamics were classified as an ordinary chimera state. Two-phase cluster states were also classified correspondingly, while all other dynamics were combined into a fourth group of other dynamics (see In order to classify the dynamics for a particular set of parameter values in the phase diagram in Fig. 5, simulations were initialized from a phase balanced state consisting of a homogeneous and a spatially turbulent domain, separated by a vertical boundary. If the turbulence switched twice from one side to the other within less than


High pressure torsion is known as one of the most popular severe plastic deformation processes. However, it has certain size limitations, especially regarding the thickness of the processed samples. In this contribution incremental high pressure torsion is introduced as a novel severe plastic deformation process. This further development of conventional high pressure torsion is capable of delivering specimens having an extraordinarily high aspect-ratio of thickness to diameter. The features of this process combined with a case-study on a pure copper specimen with a deformed diameter of 50 mm and a thickness of 40 mm is presented. As an important feature it involves considerably high amounts of hydrostatic pressure which inhibits crack-formation during deformation and leads so to almost no limitation in the applicable strain. Consequently, this technique also allows SPD of intermediate and high strength starting materials, such as steels and tungsten t, to diameter, d, shortly (t/d) of the HPT disk is too large Although this technique should theoretically deliver specimens with strongly inhomogenous microstructures, homogeneity in terms of hardness can be reached along the radius. The reason is the existence of a lower limit of grain-fragmentation after applying a material and processing parameter dependent saturation strain. This leads to the minimum grain size and thereby the highest hardness and strength at low disk radii after employing an appropriate minimum number of rotations. This saturation behavior is an important requirement for achieving large scaled specimens and products with satisfyingly homogenous hardness distributions. Nevertheless, an undeniable disadvantage of HPT has always been the small specimen size. In order to overcome the status and reputation of HPT as an academic curiosity, much effort has been taken over the last years to upscale the process and to increase the sample size, see e.g. This technical extension may increase the application possibilities of materials processed by HPT such as for medical 2For the demonstration of IHPT technically pure copper (99.9%) was used. The specimens used for the deformation process had the shape of a cylinder with a total height of 70 mm and a diameter of 50 mm. Before processing the samples were annealed at 700 °C for 1 h, in order to obtain a fully recrystallized starting microstructure that can be used to distinguish between deformed and un-deformed areas. For that purpose the deformed specimens were additionally etched using a commercially available etching detergent for copper. The IHPT process that will be explained in detail in the following section, was carried out on a hydraulic machine with a load of approximately 400 t. The rotational speed was approximately 0.07 rotations per minute. A quantitative evaluation of the changes in the mechanical properties due to IHPT after deformation was performed with Vickers micro-hardness measurements using a microhardness tester from Buehler (Micromet 5104). The indents were carried out with a load of 300 gf and dwell times of 15 s. The positioning of the individual indents will be explained in conjunction with cross-sections of the deformed specimen. The microstructural investigation was performed with a light microscope from Olympus (BX51) and a scanning electron microscope (Zeiss 1525) using back scatter electron contrast.33.1The most important processing steps of IHPT are presented in 3.2In order to demonstrate the feasibility of this technique a case study on pure copper is presented. For the study technically pure copper (99.9%) was used and two different specimens were produced. The first specimen was subjected to 15 rotations to induce a high amount of shear deformation that should allow for a saturated microstructure along the radius. The second specimen was deformed using the IHPT-principle. At first it was deformed to 15 rotations again as the first specimen. Then, the deformation zone was changed in the axial direction by shifting the deformation zone in the tool and another 15 rotations were applied afterwards. In total this incremental process was repeated nine times or in other words 9 deformation steps were applied. In this way the deformed volume increased with every single step where within one step 15 rotations were applied. During one deformation step the deformation zone had a thickness of approximately 8 mm. The step size between following deformation increments was 4 mm. In this way an overlap of the deformation zones between consecutive deformation steps was guaranteed. As support cylinders disks with a thickness of 4 mm were in use and stacked together to get the appropriate heights, see also the examples in Photographs of the specimens subjected to IHPT are presented in In order to investigate the microstructure more in detail a series of micrographs using light microscopy and scanning electron microscopy is presented in A quantitative evaluation of the change in the mechanical properties due to IHPT after deformation were performed with micro-hardness measurements. The indents were carried out on the metallographic cut of the specimen deformed with 9 deformations steps, as presented in 4A reasonable explanation for the slight decrease in hardness along the axial direction may be based on a temperature rise during the deformation process. It is generally known that SPD-processed structures exhibit a rather poor thermal stability compared to coarser grained materials The maximum hardness in the singly deformed specimen is around the same level as measured on the incrementally deformed specimen regarding the area that was deformed in the last deformation step, see εvM, for saturation in the case of pure metals is around 16–32 A comprehensive inspection of the hardness distribution in εvM, of 16 and an approximate thickness, t, of the deformation zone of about 8 mm and 15 rotations as the number of rotations, n, the critical strain is reached at a radius, r, of approximately 2.5 mm. Hence, from this viewpoint saturation should be expected, which is not the case for IHPT presented here. A more likely reason for the gradient in the radial direction is connected to a strain-rate sensitivity known for UFG-materials When one assumes a minimum strain, A comparison with the maximum achievable hardness using conventional HPT with literature data is a very difficult task as it is widely known that the saturation hardness of the same metal and comparable purity can vary markedly due to the large influence of impurities on the grain boundary mobility and so on the grain size The presented deformation technique IHPT  represents a further severe plastic deformation technique with the intention to upscale the specimen size of bulk-nanostructured materials. Compared with continuous high pressure torsion (CHPT) IHPT exhibits a different deformation path To summarize, a new SPD method, called incremental high pressure torsion (IHPT) was introduced which combines the advantages of conventional HPT and overcomes the limitation of a restricted specimen thickness. As a case study, the process was performed with a pure copper specimen showing an ultrafine-grained microstructure in the entire deformed section of the specimen having a diameter of 50 mm and a thickness of 40 mm. Future work will focus on extending the process to materials having a higher saturation hardness, e.g. alloys, steels and composites. In addition a cooling system will be required preventing additional heating and so temperature rise of the specimen during deformation.


PDZ domains are protein-protein interaction modules sharing the same structural arrangement. To discern whether they display common features in their unfolding/misfolding behaviour we have analyzed in this work the unfolding thermodynamics, together with the misfolding kinetics, of the PDZ fold using three archetypical examples: the second and third PDZ domains of the PSD95 protein and the Erbin PDZ domain. Results showed that all domains passed through a common intermediate, which populated upon unfolding, and that this in turn drove the misfolding towards worm-like fibrillar structures. Thus, the unfolding/misfolding behaviour appears to be shared within these domains. We have also analyzed how this landscape can be modified upon the inclusion of extra-elements, as it is in the nNOS PDZ domain, or the organization of swapped species, as happens in the second PDZ domain of the ZO2 protein. Although the intermediates still formed upon thermal unfolding, the misfolding was prevented to varying degrees. PDZ domains are protein-protein interaction modules that are highly present in eukaryotic organisms. These domains regulate protein networks by organizing trafficking and signalling in the cell through the localization of receptors, assembly of complexes or adjustment of various sub-cellular structures. Their scaffolding role acts through the specific binding of the C-terminus of the target proteins . Members of the PDZ family share a common structure that consists of a five to six β-stranded barrel flanked by two α-helices . In spit12A universal protein folding scheme for the PDZ domains has been proposed by Gianni, Jemth and co-authors from an extensive kinetic study. Initially, they studied five different PDZ domains and detected a common on-pathway intermediate that separated two transition states, whose positions were practically identical in the reaction coordinate302–392)In the present work, we have analysed instead the unfolding equilibrium and subsequent misfolding upon thermal denaturation. Previously, we described the unfolding thermodynamics of the third PDZ domain of the PSD95 protein  traces show two calorimetric transitions The simplest thermodynamic model that may explain this unfolding behaviour (N1⇄/n I2 = 0.996 for Erbin-PDZ and R2 = 0.998 for PSD95-PDZ2) very likely indicates that both, Erbin-PDZ and PSD95-PDZ2 domains, follow the same unfolding scheme as PSD95-PDZ3302–392 and, consequently, that a relatively stable intermediate populates upon unfolding, both as a monomer (I) and also as a self-associated species (In). The stoichiometry of such an association process should be n = 3, since we performed an initial analysis that left this parameter free and floating. The best fittings corresponded to n = 2.8 and n = 3.2 for Erbin-PDZ and PSD95-PDZ2 respectively. PSD95-PDZ3302–392 showed similar results with n = 2.8 A mathematical description of the model and of the fitting procedure can be found in the To characterize the intermediate at a structural/conformational level, we used Fourier-transform infrared spectroscopy (FTIR) to detect conformational changes in the secondary structures. We recorded the amide I’ spectra of the protein solution at the highest concentration used in DSC experiments (700–800 μM), and at temperatures of either 25 °C or 60 °C wherein the most populated species are the native state and the intermediate, respectively . Our mea302–392, are very similar, the main components being those signifying the flexible and the stable native β-sheets. There is however a difference concerning the aggregation components, namely, the appearance of the amyloid component (1612 cm−1) in both the Erbin-PDZ and PSD95-PDZ2 spectra, in addition to the worm-like component (1620 cm−1). At 25 °C these bands reflect the tendency to aggregation which already has been described for PSD95-PDZ3302–392 The spectra at 25 °C, which represent the native states of Erbin-PDZ, PSD95-PDZ2 and PSD95-PDZ3−1 bands) occurs for all three domains. This is apparently generated at the cost of the rest of the bands arising from the native secondary structures, which decrease, thus indicating a structural change wherein a compact, aggregated, β-sheet predominates. These aggregates organize as worm-like fibrils in all three cases as seen by transmission electron microscopy  regular secondary structures  remain more or less unaltered upon heating; ii) the random coil component (1645 cm−1) splits from that of the flexible β-sheet (1640 cm−1); and iii) the amyloid band (1612 cm−1) disappears to the same extent in which that of the worm-like structure (1620 cm−1) increases, so that the total area for both bands reporting for aggregates remains the same. Thus, an increase occurs in the random coil component rather than in the aggregation component observed in the examples with strict PDZ fold. Therefore, amorphous aggregation is competing with fibrilation advantageously enough to achieve that the worm-like fibrils morphology is not as evident as with the previous examples  reguexamples , despiteexamples .302–402 and nNOS-PDZ, that the extra-elements do not appear to change the main unfolding characteristics, as the occurrence of a partially-folded self-associated intermediate, but are clearly modifying misfolding, thus revealing that these extra-segments not only influence binding, but also the folding landscape of the domains.In conclusion, we can affirm from both examples, PSD95-PDZ3−1) and stable β-sheets (1630 cm−1) decrease, whereas those for the irregular secondary structures (1690–1660 cm−1) increase.To complete our investigation using natural-PDZ structure variants, we chose the second PDZ domain of ZO2 (ZO2-PDZ2), as it is well known for organizing swapped structures, in which a dimer is arranged by the inter-exchange of the β2-β3 hairpin within the two molecules . Again, Overall, our results show a common thermodynamic pattern in the unfolding and misfolding routes of the PDZ fold , where mWe believe that the selected examples may be representative of the approximately 270-member collection of human PDZ domains, since this domain family has been shown to have a well-conserved structural arrangement. It might be likely that other examples would have similar behaviour, as has been suggested from the previous kinetic studies5181920302–402 , were sub-cloned into pBAT4 vector (EMBL Core Purification Facility) and expressed in Escherichia coli BL21/DE3 cells. Protein purification was carried out as previously described302–415 and purity of the protein. The extinction coefficients were 1425 cm−1·M−1 for Erbin-PDZ, 4335 cm−1·M−1 for PSD95-PDZ2, 1240 cm−1·M−1 for nNOS-PDZ, 1627 cm−1·M−1 for ZO2-PDZ2 and 2985 cm−1·M−1 for PSD95-PDZ3302–415, determined as describedThe expression-optimized DNA sequences of Erbin-PDZ , PSD95-PDZ2 (including residues 155 to 249 in PSD95 numbering), nNOS-PDZ (including residues 1 to 127 in nNOS numbering), ZO2-PDZ2 (including residues 306 to 385 in ZO2 numbering), and PSD95-PDZ3Differential scanning calorimetry (DSC) experiments were performed in a VP-DSC instrument from Microcal INC. as described previously. Fourier transform infrared spectroscopy (FTIR) and Thioflavine T (ThT) fluorescence growth curves experimental details were also carried out as it is referred in previous references8How to cite this article: Murciano-Calles, J. et al. Common features in the unfolding and misfolding of PDZ domains and beyond: the modulatory effect of domain swapping and extra-elements. Sci. Rep.6, 19242; doi: 10.1038/srep19242 (2016).


Remarkably, the Gcn- substitutions increase affinity of the HisRS domain for the C-terminal domain (CTD), previously implicated as a kinase autoinhibitory segment, in a manner dampened by HisRS domain Gcd- substitutions and by amino acid starvation in vivo. Moreover, tRNA specifically antagonizes HisRS/CTD association in vitro. These findings support a model wherein HisRS-CTD interaction facilitates the autoinhibitory function of the CTD in nonstarvation conditions, with tRNA binding eliciting kinase activation by weakening HisRS-CTD association with attendant disruption of the autoinhibitory KD-CTD interaction.The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2α. Gcn2 is activated in amino acid-deprived cells by binding of uncharged tRNA to the regulatory domain related to histidyl-tRNA synthetase, but the molecular mechanism of activation is unclear. We used a genetic approach to identify a key regulatory surface in Gcn2 that is proximal to the predicted active site of the HisRS domain and likely remodeled by tRNA binding. Mutations leading to amino acid substitutions on this surface were identified that activate Gcn2 at low levels of tRNA binding . Gcn2 also contains a C-terminal domain implicated in autoinhibition of Gcn2. Our findings identify a direct interaction between the CTD and a novel regulatory surface in the HisRS domain that is required for inhibition of Gcn2 function in non-starved cells, which is down-regulated by uncharged tRNA. The results further suggest that tRNA binding to the pseudo-active site in the HisRS domain remodels its proximal CTD-binding surface to weaken HisRS/CTD interaction and thereby release the autoinhibitory function of the CTD to activate kinase function. This study provides new molecular insights into how tRNA binding can modulate regulatory interactions among the HisRS, CTD, and kinase domains of Gcn2 to elicit kinase activation.The survival of all living organisms depends on their capacity to adapt their gene expression program to variations in the environment. When subjected to various stresses, eukaryotic cells modulate general and gene-specific protein synthesis by phosphorylating the α-subunit of eukaryotic translation initiation factor 2 (eIF2α). The yeast  The inhibition of ternary complex assembly diminishes the rate of general translation but enables translation preinitiation complexes to bypass multiple “decoy” AUG start codons in the mRNA leader of GCN4 mRNA and translate the coding sequences for Gcn4, a transcriptional activator of amino acid and vitamin biosynthetic genes in budding yeast . Phosphorylation of eIF2α reduces the function of eIF2 in recruiting methionyl initiator tRNA to the 40S ribosomal subunit by impairing the recycling of eIF2-GDP to eIF2-GTP by guanine exchange factor eIF2B and thereby reducing the cellular concentration of eIF2·GTP·Met-tRNA and HRI . PKR is  and HRI , and fun and HRI  and in l and HRI . Mammali and HRI ); and re and HRI . Hence, His for protein synthesis. Consistent with the fact that Gcn2 is activated by starvation for any amino acid . La. La11]. ino acid , the Gcnino acid . An N-teino acid , suggestin vitro , is locain vitro ,20. Our in vitro .The extreme C-terminal domain (CTD) of Gcn2 plays multiple roles in kinase regulation, both positive and negative, including dimerization, ribosome binding, and autoinhibition of the KD  1]. Act. Act1]. trans-acting factors Gcn1 and Gcn20, which form a complex that must interact with both the N-terminal “RWD” domain of Gcn2 and translating ribosomes for Gcn2 activation in starved yeast cells , wh, wh27], st cells ,31,32,33st cells , supportst cells , and it st cells .GCN2c-E803V  weakens interaction between the isolated KD and CTD and also increases tRNA binding by Gcn2 in vitro ,,18,17,18in vitro . The finin vitro .gcn2-m2 variant, which harbors substitutions in conserved residues of the predicted HisRS active site cleft that impair tRNA binding in vitro and kinase activation in vivo -methionine labeled HisRS fragment can be pulled down with immobilized GST fusions containing the Gcn2 KD, CTD or HisRS region itself, with the last interaction reflecting dimerization of the HisRS domain [Phe reduced interaction of the [35S]-HisRS fragment with all three GST fusions; however, the magnitude of the reduction was larger for GST-CTD (~5.4-fold) compared to GST-KD (~1.8-fold) or GST-HisRS (~2.1-fold). Moreover, interaction of [35S]-HisRS with GST-CTD was unaffected by an equivalent concentration of an unstructured model mRNA . This alignment produced a robust Z score of 12.9, a RMSD of 3.0 Å, and minimal clashes between tRNAAsp and HisRS. A similar alignment procedure was previously used to model HisRS interaction with tRNAHis.  The locations of Gcn2 residues involved in this study were then projected onto the T. cruzi HisRS crystal structure based on the sequence alignment between Gcn2 and authentic HisRSs  by using primer pairs PS-3 (5’-TCTATTTGATAACTCAGTTCCAAC-3’) and PS-4 (5’- TCAGGAATATGTATAAGAAAGGTGAC-3’). The KpnI-NheI 1.8-kb GCN2 fragment encoding the HisRS-CTD was isolated from plasmid DNA prepared from a pool of E. coli transformants harboring mutagenized plasmids and subcloned into p2201. Plasmid DNA prepared from a pool of the resulting E. coli transformants was introduced into yeast strain H1149 and transformants were selected on SC-Ura medium containing 15 mM 3-AT. Resident plasmids were isolated from colony-purified transformants and subjected to DNA sequence analysis to identify the relevant mutations. As multiple mutations generally occurred, QuikChange® site-directed mutagenesis (Stratagene) was used to produce plasmids pSL501, pSL502 and pSL503, containing only single mutations in GCN2. Site-directed mutagenesis was also used to generate the novel derivatives (listed in parenthesis) of the following previously constructed plasmids: p2201 (pSL501-pSL507), p722 (pSL508-pSL525), pHQ430 (pSL535-pSL538), pHQ601 (pSL539-pSL541). Plasmids pSL526, pSL527, pSL529, pSL530 and pSL542 were generated by replacing the 3.0-kb BspEI-NheI fragment in pSL101 or pSL102 with the corresponding fragment from p722 derivatives harboring the appropriate GCN2 mutations.Plasmids employed are listed in 600 = 0.2 in SC-Ura containing 10% galactose as carbon source and grown to A600 of ∼2.5. Cells were harvested (∼25 g), washed with cold distilled water containing EDTA-free protease inhibitor cocktail (PIC) (Boehringer Mannheim) and 0.5 mM PMSF, resuspended in ice-cold binding buffer (BB)  and disrupted using SPEX freezer mill (model 6870). Lysates were clarified by centrifugation at 39,000 × g for 2 h at 4°C and mixed with 1 ml of M2-FLAG affinity resin (Sigma) overnight at 4°C. The resin was washed three times with 10 vol of BB and Gcn2 was eluted with 100 units of AcTEV protease in 500 μl of 1X TEV buffer . The eluates were concentrated with an Amicon Centricon filter  and dialyzed against 10 mM Tris-HCl [pH 7.4], 50 mM NaCl, 20% glycerol and stored at −800 C. The eIF2α−ΔC protein was purified from E. coli as previously described -HisRS domain fragments were conducted as follows. In vitro transcription/translation with [35S]-methionine was conducted using the TNT T7 Coupled Reticulocyte Lysate System (Promega) according to the vendor’s instructions. The resulting [35S]-HisRS domain fragments were partially purified by ammonium sulfate precipitation as described previously [35S]-HisRS domain fragments were added to beads (10-μL bed volume) containing 5 μg of bound GST fusion proteins along with the indicated amount of tRNAPhe , or synthetic mRNA (GGAAUCUCUCUCUCUCUCUCUGCUCUCUCUCUCUCUCUCUCUC) synthesized by T7 polymerase as described in [35S]-HisRS domain fragments, the gels were fixed with a solution of isopropanol:water:acetic acid (25:65:10), treated with Amplify , dried, and subjected to fluorography at −80°C.Pull-downs of , 50 mM NaCl, 20% glycerol for 5 min at room temperature and reactions were quenched by adding SDS sample buffer to a final concentration of 1X followed by heat inactivation at 95°C for 5 min. Digested samples were separated by SDS/PAGE and stained with Coomassie brilliant blue.S1 Figgcn2Δ strain H1149 with low-copy plasmids containing the indicated GCN2 alleles were analyzed as described in Transformants of (TIF)Click here for additional data file.S2 FigS. cerevisiae Gcn2 (residues 1030–1524). Regions of predicted motifs within the HisRSs are denoted above the S. cerevisiae sequence, based on the alignment of Gcn2 HisRS sequences with authentic histidyl tRNA-synthetases in S2A-B Fig. Substitutions conferring Gcn− phenotypes are shown in red and those conferring Gcd− phenotypes are shown in green. Residues interacting directly with histidyl adenylate in the T. cruzi structure are indicated by black letters below the sequence: H/P/S/A signify interaction with the histidyl/phosphate/sugar/adenine moieties respectively. Different portions of the HisRS domain are aligned in panels A to E, encompassing the following residues in full-length S. cerevisiae Gcn2: (A) residues 1030–1125; (B) residues 1126–1214; (C) residues 1215–1312; (D) residues 1313–1386; (E) residues 1387–1453; (F) residues 1454–1524.The multiple sequence alignment of HisRS domains from 42 fungal Gcn2 sequences, identified on the far left by abbreviations of their species of origin, was built using the MUSCLE program. Residues are colored according to evolutionary sequence variation as analyzed with the CONSURF on-line server, with magenta corresponding to the most conserved residues, and dark cyan indicating the most variable. Numbering corresponds to residue positions in full-length (PDF)Click here for additional data file.S3 FigS. cerevisiae Gcn2 (residues 1039–1502). Regions of predicted motifs within HisRSs are denoted above the alignment based on their locations in the histidyl tRNA-synthetases. Gcn2 HisRS substitutions examined in this study are shown along the top at their positions in the alignment, with those conferring Gcn− phenotypes shown in red and those conferring Gcd− phenotypes shown in green. Residues interacting directly with histidyl adenylate in the T. cruzi structure are indicated by black letters below the sequence: H/P/S/A signify interaction with the histidyl/phosphate/sugar/adenine moieties respectively. Different portions of the HisRS domain are aligned in panels A-B, encompassing the following residues in full-length S. cerevisiae Gcn2: (A) residues 1039–1304; (B) residues 1305–1502.Multiple sequence alignment of Gcn2 HisRSs from 7 fungal species with 6 different authentic histidyl tRNA-synthetases, was built using the MUSCLE program, Residues are colored according to evolutionary sequence variation as analyzed with the CONSURF on-line server, with magenta corresponding to the most conserved residues, and dark cyan indicating the most variable. Sequences are identified on the far left with abbreviations of their species of origin. Numbering corresponds to residue positions in full-length (PDF)Click here for additional data file.S4 FigThe indicated purified Gcn2 proteins were partially digested with trypsin or elastase and the reaction products were resolved by SDS-PAGE and visualized by Coomassie Blue staining. One microgram of each protein was loaded for the Undigested lanes, whereas 8μg was analyzed for each protease-digested sample. We verified that the major digestion products visible in the protease-treated samples are not visible when higher amounts of the undigested proteins are resolved.(TIF)Click here for additional data file.S5 Fig(A & B) Two views of the region surrounding the conserved HisA motif (cyan) in the model of the Gcn2 HisRS domain/tRNA complex, colored as in (TIF)Click here for additional data file.


Major histocompatibility complex class I (MHCI) proteins present antigenic peptides for immune surveillance and play critical roles in nervous system development and plasticity. Most MHCI are transmembrane proteins. The extracellular domain of MHCI interacts with immunoreceptors, peptides, and co-receptors to mediate immune signaling. While the cytoplasmic domain also plays important roles in endocytic trafficking, cross-presentation of extracellularly derived antigens, and CTL priming, the molecular mediators of cytoplasmic signaling by MHCI remain largely unknown.PSD95/disc large/zonula occludens-1) ligands. PDZ ligands are motifs that bind to PDZ domains to organize and mediate signaling at cell-cell contacts. PDZ ligands are short, degenerate motifs, and are therefore difficult to identify via sequence homology alone, but several lines of evidence suggest that putative PDZ ligand motifs in MHCI are under positive selective pressure. Putative PDZ ligands are found in all of the 99 MHCI proteins examined from diverse species, and are enriched in the cytoplasmic domain, where PDZ interactions occur. Both the position of the PDZ ligand and the class of ligand motif are conserved across species, as well as among genes within a species. Non-synonymous substitutions, when they occur, frequently preserve the motif. Of the many specific possible PDZ ligand motifs, a handful are strikingly and selectively overrepresented in MHCI’s cytoplasmic domain, but not elsewhere in the same proteins. Putative PDZ ligands in MHCI encompass conserved serine and tyrosine residues that are targets of phosphorylation, a post-translational modification that can regulate PDZ interactions. Finally, proof-of-principle in vitro interaction assays demonstrate that the cytoplasmic domains of particular MHCI proteins can bind directly and specifically to PDZ1 and PDZ4&5 of MAGI-1, and identify a conserved PDZ ligand motif in the classical MHCI H2-K that is required for this interaction.Here we show that the cytoplasmic domain of MHCI contains putative protein-protein interaction domains known as PDZ  contains supplementary material, which is available to authorized users. The major histocompatibility complex class I (MHCI) is a multi-gene family known for its roles in the immune response. Classical MHCI proteins . MHCIeceptors ), and iseceptors . DespiteMHCI proteins are generally single pass transmembrane proteins comprised of an extracellular domain, a transmembrane domain, and a short (~20–40 amino acid) cytoplasmic domain. The extracellular domain of MHCI binds to peptides, immunoreceptors and coreceptors to participate in immune surveillance. Although the cytoplasmic domain is dispensable for MHCI delivery to the cell surface and for effective presentation of endogenous antigens in vitro , 13, subPSD95, disc large, zonula occludens-1) ligands. Multiple bioinformatic analyses suggest that PDZ ligand motifs in the cytoplasmic domain of MHCI are under positive selective pressure, and biochemical assays demonstrate that the cytoplasmic domain of MHCI can bind directly and specifically to PDZ domains in vitro. These results reveal that MHCI proteins may participate in PDZ interactions with transmembrane or cytoplasmic proteins, and suggest a possible mechanism by which MHCI could mediate known and novel functions at both immunological and neuronal synapses.Here we identify potential mediators of cytoplasmic signaling by MHCI: cryptic putative protein-protein interaction motifs known as PDZ  motifs that are recognized by larger (~80–90 amino acid) binding pockets known as PDZ domains. PDZ domains are found in more than 200 proteins in humans, and have been identified in viruses, bacteria, yeast, plants, and animals. PDZ interactions regulate protein localization, binding specificity, and modification (reviewed in ), and foMost PDZ ligands characterized to date lie at the extreme carboxy terminus (C-terminus) of transmembrane or cytosolic proteins. These C-terminal PDZ ligands have been divided into classes, based on the properties of the residues in specific positions in the ligand motif, although the exact composition and number of consensus motifs is a topic of ongoing research . Despite variation in the length of the cytoplasmic domain, significant conservation was seen throughout the aligned sequences. All of the sequences have several basic residues immediately adjacent to the hydrophobic transmembrane region, a common feature of transmembrane proteins. These basic residues may interact with the negatively charged phospholipid head groups at the inner face of the membrane, and help prevent movement of the protein perpendicular to the plane of the membrane  –51). In membrane . In addiHLA-A is one of three classical MHCI genes in the human genome, which also encodes several non-classical MHCIs and MHCI-like proteins. Similarly, in mice, there are three classical MHCI genes and several non-classical MHCI genes Fig. . To deteSeveral notable patterns emerged. First, the short cytoplasmic domains of nearly all of the MHCI proteins examined contain one or more putative PDZ ligands. Of the seven human sequences examined, all but one  contain at least one potential PDZ ligand motif Fig. . SimilarOne challenge of identifying short, degenerate motifs based on sequence alone is that they are likely to occur at a high frequency by chance. Thus the motifs we identified in the cytoplasmic domain of MHCI may or may not be true PDZ ligands. The fact that they occur in identical positions in multiple genes Fig. , supportWe first compared the frequency of PDZ ligand motifs in the cytoplasmic versus extracellular domains, since PDZ interactions are only known to occur in the cytoplasm. In the extracellular domain, the frequency with which class 1 PDZ ligand motifs occurred was comparable to what was seen in scrambled amino acid sequences (~1 per 100 amino acids), consistent with the idea that PDZ ligands are not selected for in the extracellular space. In contrast, the number of PDZ ligands observed in the cytoplasmic domain was roughly three times this baseline value Fig. . The sigOne unusual set of MHCI-like proteins which may be freed from this selective pressure is viral MHCI mimics. These mimics have been adopted from host genomes, and can allow infected cells to evade NK cell-mediated lysis after viral down regulation of endogenous host MHCI, as part of the viral immuno-evasion strategy –67. CytoHowever, it is possible that the enrichment of these short motifs in the cytoplasmic domain simply reflects a cytoplasmic enrichment of the specific amino acids that make up the consensus motifs, which would increase the probability that they would occur there by chance. To examine this possibility, we searched the same mouse MHCI sequences for the same class 1 PDZ ligand consensus motif [X (S/T) X (V/L)] , but invUnlike true PDZ ligand motif sequences, inverted PDZ ligand motifs were not enriched in the cytoplasmic domain Fig. . Indeed,http://string-db.org/)  X (V/L)] . To obtarg/) see . A custoAll four class 1 PDZ ligand motifs were detected in both domains of the MHCI proteins examined. However, their relative frequencies differed widely in the two compartments. In the extracellular domain, serine-containing motifs represented 33 % of the total, and each threonine-containing motif occurred at a nearly identical frequency . In the cytoplasmic domain, however, there was a striking skewing of the distribution Fig. . Serine-A similar analysis was performed for class 2 PDZ ligand motifs, using the consensus definition XΦXΦ, where Φ = primarily hydrophobic residues  . Likely b; Fig. b are encoded in single exons. Exon 6 encodes the overlapping ligand motifs GDYA, DYAL, and YALA, exon 7 encodes the overlapping ligand motifs TSDL and DLSL, and exon 8 encodes the C-terminal motif HSLA. Only the overlapping motifs DCKV and KVMV span an exon/intron boundary. Notably, KVMV is also among the least conserved putative PDZ ligand motifs in H2-Kb, and is not detected in this position in any other mouse MHCI examined  differed from intact H2-Kb peptide only at the mutated residue. Diagnostic spectra are shown in Additional file b. A band of the appropriate size is detected in Western blots .b bound directly and specifically to PDZ1 of MAGI-1, and less intensely to PDZ4+5 , a multi-PDZ domain synaptic scaffolding protein that interacts with NMDA receptors and is required for specific aspects of associative learning and memory –75, and 4+5 Fig. , but not4+5 Fig. . The speb, which we find can bind directly to PDZ1 of MAGI-1, contains a single sequence that conforms to this class 1 PDZ ligand motif: TSDL. Several other MHCI proteins contain sequences that conform to this motif in an identical or similar position in amino acid sequence alignments . Strikingly, this single point mutation is sufficient to disrupt the direct interaction between the cytoplasmic domain of H2-Kb and PDZ1 and PDZ4&5 of MAGI-1 in vitro  together encode a polypeptide segment of only ~40 amino acids , class 2 ), or class 3 [X (D/E) X Φ] PDZ ligand motifs . The totThe Logos Look Amazing (LOLA) program was used to visualize amino acid sequence conservation.http://baderlab.org/Software/LOLA) [LOLA was created by the Bader lab and is freely available on their lab website (re/LOLA) .b were amplified using PCR with the primer sequences shown in Additional file All procedures were performed in accordance with protocols approved by the Princeton University Institutional Animal Care and Use Committee (IACUC). Mice were maintained on a 12 h light-dark cycle with food and water access ad libitum. C57BL/6 mice were obtained from The Jackson Laboratory . Postnatal day 14 C57BL/6 mice were deeply anesthetized to a surgical plane via inhalation anesthesia, decapitated, their brains rapidly removed, and hippocampi microdissected. Tissue was homogenized via mortar and pestle and the RNA was extracted and purified using a Qiagen RNeasy Mini Kit . RT-PCR of total RNA was performed using the Quanta Biosciences qScript cDNA Synthesis Kit . C-terminal sequences of the classical MHCI H2-KE. coli  using 1.2 L (MHCI peptides) or 10 ml (PDZ peptides) of Overnight Express Instant TB Media . Cell lysates were applied to columns (equilibrated glutathione columns (Pierce GST Spin Purification Kit) for GST-tagged MHCI cytoplasmic domains, or Pierce HisPur Cobalt Spin Purification columns  for 6xHis-tagged PDZ domains) to bind the expressed peptides, and washed according to the manufacturer’s protocols. Recombinant proteins were desalted using Thermo Fisher desalting columns, following the manufacturer’s protocol, and quantified using a Pierce BCA Protein Assay kit.GST-tagged C-terminal MHCI peptides and 6xHis-tagged PDZ domain constructs for MAGI-1 and SAP97  were expressed in BL21(DE3) competent Binding assays were performed as previously described . BrieflyE. coli  using 1.2 L of Overnight Express Instant TB Media . Cell lysates were applied to equilibrated glutathione columns (Pierce GST Spin Purification Kit) for GST-tagged MHCI cytoplasmic domains. Recombinant GST proteins were desalted using Thermo Fisher desalting columns, following the manufacturer’s protocol, and quantified using a Pierce BCA Protein Assay kit. His-tagged MAGI-1 were expressed in BL21(DE3) competent E. coli  using 5 mL of Overnight Express Instant TB Media . Cell lysates were bound to HisPur Cobalt Spin Purification columns  and incubated overnight at 4 °C. Columns were washed 3 times according to manufacturer’s protocols and preincubated with respective competitor GST MHCI peptide (GST-H2-T22 or GST-H2-D) at a concentration of 200 nM in manufacturer’s wash buffer for 2 h at 4 °C. Following preincubation, 200 nM of GST-H2-K was added to the column for 2 h at 4 °C. Columns were washed 5 times in manufacturer’s wash buffer at 700×g for 2 min, and bound peptides were eluted according to manufacturer’s protocols . Samples were prepared as follows: 30 μL of eluate + 10 μL of 4× sample buffer were boiled for 10 min and run on a 4–20 % gradient gel (BioRad), followed by western blotting (below) to visualize proteins.GST-tagged C-terminal MHCI peptides were expressed in BL21(DE3) competent b was subjected to SDS-PAGE on a 4–20 % gradient gel (Bio-Rad) and transferred to an Immobilon-P PVDF transfer membrane (Millipore) as previously described [To verify the identity of the recombinant GST-tagged MHCI cytoplasmic domain peptide, 1 μg of purified GST-H2-Kescribed . PVDF meFor competition experiments, samples were prepared and run on a SDS-PAGE 4–20 % gradient gel (Bio-Rad) and transferred to a PVDF membrane (Millipore). Membranes were incubated in an antibody raised against exon 8, in the cytoplasmic domain of H2-K (P8 (GeneScript), 1:5,000 dilution in 5 % organic dry milk), followed by 12×5 min washes in TBST. Membranes were then incubated in HRP-conjugated goat anti-rabbit secondary  for 1 h at RT, and washed 7×5 min in TBST.Membranes were incubated in stripping buffer  for 15 min and washed 3×5mins in TBST. The membranes were blocked overnight at 4 ° C in 3 % BSA in TBST and re-probed with anti-HIS antibody  overnight at 4 °C. Membranes were washed 12×5min in TBST and incubated in HRP-conjugated goat anti-rabbit secondary antibody  for 1 h at RT and washed 7×5min in TBST. Blots were treated with ECL as above and used to expose film. The density of the bands on Western blots (below) was quantified using ImageJ software and calculated as the ratio of the density of H2-K over the density of HIS-PDZ.b fusion peptide or the GST-H2-Kb T329A point mutant peptide. Protein solutions were subjected to buffer exchange into 8 M urea, followed by thiol reduction and alkylation, and tryptic digestion using the FASP procedure [m/z 445), which maintained absolute mass accuracy to within 2–3 ppm.We performed mass spectrometry on samples that contained high concentrations of the GST-H2-Krocedure . Peptiderocedure  and subjN-terminal protein acetylation as variable modifications and carbamidomethylation of cysteines as a fixed modification. Aggregate search results for each sample were imported into Scaffold software , for consolidation and visualization.Resultant LC-MS/MS data were searched against custom databases consisting of MHCI fusion protein construct sequences concatenated to either the UniProt Mouse proteome or a PDZ-domain-containing protein subset of the UniProt Mouse proteome, using ProteomeDiscoverer  with the Mascot search engine node . Search parameters allowed for a mass error of 6 ppm for precursor species and 1.2 Da for fragments, ≤ 3 missed trypsin cleavages, methionine oxidation and N-methyl D-aspartate; PDZ, PSD-95/discs large/zonula occludens-1; PVDF, polyvinylidene fluoride; RT, room temperature; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TBS, tris-buffered saline; TBST, tris-buffered saline + Tween 20C-terminus, carboxy terminus; CTL, cytotoxic T lymphocyte; GST, glutathione S-transferase; HLA, human leukocyte antigen; HRP, horseradish peroxidase; MHCI, major histocompatibility complex class I; NCBI, National Center for Biotechnology Information; NMDA,


SWI/SNF complexes use the energy of ATP hydrolysis to remodel chromatin. In mammals they play a central role in regulating gene expression during differentiation and proliferation. Mutations in SWI/SNF subunits are among the most frequent gene alterations in cancer. The INI1/hSNF5/SMARCB1 subunit is mutated in both malignant rhabdoid tumor, a highly aggressive childhood cancer, and schwannomatosis, a tumor-predisposing syndrome characterized by mostly benign tumors of the CNS. Here, we show that mutations in INI1 that cause schwannomatosis target a hitherto unidentified N-terminal winged helix DNA binding domain that is also present in the BAF45a/PHF10 subunit of the SWI/SNF complex. The domain is structurally related to the SKI/SNO/DAC domain, which is found in a number of metazoan chromatin-associated proteins. •INI1 and its metazoan homologs contain a variant winged helix DNA binding domain•A homologous domain is present in the BAF45a/PHF10 subunit of the SWI/SNF complex•Structurally related domains are found in other metazoan chromatin-associated proteins•INI1 mutations that cause schwannomatosis map to the winged helix domain Allen et al. show that the INI1/hSNF5/SMARCB1 subunit of the human SWI/SNF chromatin-remodeling complex contains a variant winged helix DNA binding domain. Mutations in INI1 that cause the tumor-predisposing syndrome schwannomatosis perturb the fold or disrupt the function of this domain. INI1 gene also cause the tumor suppressor syndrome schwannomatosis, which is characterized by mostly benign tumors of the CNS that typically develop in the second or third decade of life  spectra characteristic of a globular protein, confirming that these residues compose an autonomously folded domain. To determine whether this domain is present in other proteins, we undertook a detailed sequence profile search using the PSI-BLAST program . A reciprocal search with this portion of BAF45a retrieved the N-terminal domain of INI1 and metazoan INI1-like proteins. BAF45a is a member of a family of mutually exclusive SWI/SNF subunits , unique to metazoans, all of which contain tandem PHD fingers at their C termini. Complexes contain BAF45a appear to be associated with dividing cells, during neural development; for example, BAF45a is switched for BAF45b or BAF45c as proliferating neural stem and progenitor cells transition into post-mitotic neurons  measurements show is highly mobile (Z score of 6.2). The SnoN domain is a member of a family of structurally related domains found exclusively in metazoan chromatin-associated proteins that includes the DHD domains of DACH1 and the SKI proto-oncogene DNA sequence by recording 2D BEST-TROSY spectra of residues A. AnalysGermline mutations in the gene coding for INI1 are found in approximately 50% of inherited cases of schwannomatosis . The mosMissense mutations have also been identified in sporadic schwannomatosis; it is not clear whether a particular substitution is pathogenic . One of The appearance of a variant winged helix domain in both INI1 and BAF45a is presumably the result of the need for more intricate regulation of the SWI/SNF complex as it began to direct complex gene expression programs during the development of multi-cellular animals. The finding that structurally related domains are also present in a number of other metazoan chromatin-associated proteins suggests that domains with this fold may have a conserved role related to some aspect of chromatin biology. The data presented here argue strongly that the INI1 domain has a function involving binding to DNA. A sequence-specific DNA binding role appears to be unlikely, as the SWI/SNF complex is thought to be directed to specific sites within the genome by interacting with transcription factors. It is more probable that, as has been proposed for the homologous domain from DNTTIP1 , it acts6-tagged lipoyl domain of Bacillus stearothermophilus dihydrolipoamide acetyltransferase. The resulting plasmids were transformed into E. coli C41 (DE3) cells. Cells were grown in 2XTY media at 37°C to mid-log phase and induced with 1 mM isopropyl β-D-1-thiogalactopyranoside. The temperature was reduced to 22°C and the cells were grown for a further 16 hr. Isotopically labeled domains were prepared by growing cells in K-MOPS minimal media containing 15NH4Cl and/or [13C]glucose. Cells where lysed by sonication, and the fusion protein was purified by Ni2+-NTA affinity chromatography and then dialyzed overnight in the presence of TEV protease, which cleaves the fusion protein after the lipoyl domain. A second Ni2+-NTA affinity chromatography step was carried out to remove the lipoyl domain, and the IN11 domain was further purified by gel filtration using a HiLoad 26/60 Superdex 75 column . The elution volume of the domain was consistent with it being monomeric.The DNA encoding the first 115 residues of human INI1 was amplified from human cDNA by PCR and cloned into a modified pRSETA (Invitrogen) expression vector that produces proteins fused to the N-terminally His2O, 10% D2O, containing 20 mM potassium phosphate (pH 6.5), 100 mM NaCl, and 5 mM β-mercaptoethanol. Assignments were obtained using standard NMR methods. Heteronuclear NOE spectra were obtained using the standard Bruker pulse program.NMR measurements were made using either a Bruker DRX800 or DRX600 spectrometer equipped with triple-resonance cryoprobes at 25°C. NMR samples were typically 1.5 mM in 90% H5aj1 and r5aj1mr, respectively.Distance restraints were obtained from the analysis of 2D and 3D NOE spectra. Backbone phi/psi dihedral angle restraints were obtained from chemical shifts using TALOS+ . The thr15N-labeled INI1 winged helix domain sample was titrated with unlabeled 24-bp dsDNA. A palindromic oligonucleotide 5′-GGAATTGTGAGCGCTCACAATTCC-3′ was chemically synthesized (Integrated DNA Technologies), heated to 98°C for 10 min, and annealed at room temperature to form dsDNA. 1H,15N-BEST-TROSY spectra of the INI1 domain were collected at each point of the titration to monitor changes in 1H and 15N resonance frequencies of the domain induced by dsDNA binding.Chemical shift perturbation analysis was used to characterize the interaction of the INI1 winged helix domain and dsDNA. A


The generic architecture of a semaphorin–plexin complex is characterized by the dimeric semaphorin cross-linking two copies of the plexin receptor. For specific family members, the co-receptor neuropilin serves to bolster this architecture, but in all cases, the dimeric interaction lies at the core of the ligand receptor complex, providing the essential trigger for signalling.The cell-to-cell signalling mechanisms of multi-cellular organisms orchestrate human development during embryogenesis and control homeostasis in adult tissues. These are mechanisms vital to human health and perturbation of cell-to-cell signalling is a contributing factor in many pathologies including cancer. The semaphorin cell guidance cues and their cognate plexin receptors exemplify a cell-to-cell signalling system for which insights into mechanistic principles are emerging. X-ray crystallographic data from Diamond beam lines have enabled us to probe the inner workings of semaphorin–plexin signalling to atomic-level resolutions. Importantly, we can complement protein crystallographic results with biophysical and cellular studies to dovetail structural information with functional impact. The signature seven-bladed  In these drawings, Cajal records observations he made using the cutting-edge microscopy techniques of his day. The drawings show the intricate networks formed by the dendrites and axons extending from cells of the nervous system in, for example, a pigeon cerebellum . The com2.The semaphorins were first identified in the grasshopper , and, wi3.ecto) [a). The sema domain β-propeller topology is distinguished by an elaborate insertion (of some 70 residues between β-strands C and D of blade 5) that we termed the extrusion [a). In total, there are now crystal structures for class 3, 4, 6 and 7 semaphorins [In 2003, my laboratory (in Oxford) and that of Prof. Dimitar Nikolov  contemporaneously determined crystal structures of the semaphorin sema domain ,11, the ecto)  revealedxtrusion . The firxtrusion ,11. The aphorins –15. All 4.Semaphorins typically trigger signalling by interaction with members of the plexin family of cell surface receptors . The ple5.Escherichia coli. In 2003, to produce the protein sample for our first crystal structure of a semaphorin ectodomain, hSema4Decto, we engineered stable high level expression in mammalian (CHO) cells [ecto construct took several months. Over the past decade we have developed, and refined, a transient expression system which uses mammalian cells  to produce, within weeks rather than months, secreted glycoproteins for structural and biophysical studies [− [ecto, human plexin B1, for structural and functional studies [The complex, cysteine-rich architectures of glycoprotein ligands such as the semaphorins, or the ectodomains of receptors such as the plexins, are not conducive to high-level expression in O) cells . The gen studies . This syudies [− . We haveudies [− . Equippe studies .6.The capabilities of third-generation synchrotron beam lines have played a critical role in structure determinations of semaphorins, of the semaphorin-binding region of plexin ectodomains and of semaphorin–plexin complexes. For work in my laboratory, the macromolecular crystallography beamlines at the Diamond Light Source have allowed a succession of structure determinations to be brought to successful conclusions despite the challenges presented by the nature of our glycoprotein targets.ecto) crystallized and diffracted to 2.3 Åresolution at Diamond beamline I03. The structure, one semaphorin dimer of 145 kDa per asymmetric unit, showed the classical sema domain topology and dimeric structure [a). Sequence analysis of plexin ectodomains indicates a highly conserved architecture comprised of an N-terminal sema domain, followed by a combination of three cysteine knot and six β-sandwich type folds, termed PSI and IPT domains, respectively. A construct comprising the sema domain, PSI, IPT and second PSI domain of murine plexin A2 (mPlxnA21−4) crystallized and we collected a 2.3 Åresolution X-ray diffraction dataset at the European Synchrotron Radiation Facility beam ID23-1. In parallel, we grew crystals of the mPlxnA21−4–mSema6Aecto complex. These crystals required extensive optimization, and it was necessary to deglycosylate both mPlxnA21−4 and mSema6Aecto. Ultimately, the complex crystals diffracted to 2.2 Å resolution at Diamond I03. These data were phased by molecular replacement using as search models the newly determined mSema6Aecto structure and the distantly related Met-receptor structure (PDB code 2UZX). After several cycles of refinement and manual rebuilding, the electron density for mPlxnA21−4 in the complex structure was used successfully to phase the uncomplexed structure revealing one plexin monomer of 85 kDa per asymmetric unit. In order to provide a representative structure of a semaphorin–plexin interaction involving a class B plexin, we then crystallized and collected data (Diamond I03) for hSema4Decto in complex with the two N-terminal domains of human plexin B1 (hPlxnB11−2).A construct comprising essentially the entire ectodomain of the mouse Sema6A  reveals that, as expected from sequence analysis, the N-terminal domain of the plexin ectodomain also conforms to the seven-blade β-propeller topology of the sema domain and again includes the distinctive elaboration that we have termed the extrusion [1−4 is monomeric. It appears that unlike its counterpart in semaphorins the plexin sema domain shows little or no propensity to homodimerize. We and others have been unable to detect any measureable interactions  in biophysical analyses of secreted plexin constructs comprising sema–PSI or sema–PSI1–IPT1–PSI2 [The crystal structure of mPlxnA21−4 b revealsxtrusion . Sequencxtrusion ). Detailxtrusion . Crystalxtrusion ,24. LikePT1–PSI2 ,13.1−4 crystal structure provides information on four domains from what is in total a 10 domain ectodomain. It reveals a relatively elongated structure comprising the N-terminal sema domain followed by the three domains of the PSI1–IPT1–PSI2 region. These three domains are arranged sequentially, pointing away from the sema domain, and exhibit the predicted cysteine knot and β-sandwich type folds (b).Overall, our mPlxnA2pe folds b.8.ecto dimer appears stable in analytical ultracentrifugation and multi-angle light scattering measurements, consistent with the extensive dimer interface (2600 Å2 buried surface area) present in the crystal structure [1−4–mSema6Aecto and hPlxnB11−2–hSema4Decto we determined using X-ray diffraction data collected on Diamond beamlines allowed us to address this question. These structures revealed semaphorin–plexin complexes for both classes of plexin comprise the dimeric semaphorin interacting with two essentially separate plexin molecules through side-to-face binding of the sema domains (c). Each side-to-face interaction between semaphorin and plexin sema domains buries a surface area of 2000–2500 Å2 and requires no major conformational change in either the semaphorin or the plexin. The extensive interface is mediated by residues which show high levels of sequence conservation within the vertebrate classes of semaphorins and plexins, thus it appears that our complex structures reveal an architecture that is generic for this signalling system. However, there are significant variations in the detailed structures of the mPlxnA21−4–mSema6Aecto and hPlxnB11−2–hSema4Decto interfaces consistent with the specificity of these particular ligand–receptor pairings.To recapitulate, the semaphorin ectodomain dimerizes through a sema-to-sema domain interaction in all crystal structures of the isolated ligands determined to date –15. Thistructure . What th domains c. Each s9.Kd of 2.5 μM for the monomeric mPlxnA21−4 binding to a mSema6Aecto-coated surface, independent of the coating density; Kd of 0.048 μM for the dimeric mSema6Aecto binding to a surface densely coated with mPlxnA21−4 versus a Kd of 0.28 μM for a more sparsely coated surface). Structure-guided design allowed us to generate mutant forms of the semaphorin ectodomains which were monomeric. These mutant molecules were stable and were still capable of binding plexin, but showed no avidity effect in SPR measurements. Significantly monomeric Sema4Decto could no longer elicit a response in the standard cellular assay for plexin B1 signalling, the cell collapse assay [We used surface plasmon resonance (SPR) equilibrium binding studies to demonstrate that the bivalent nature of the semaphorin interaction can result in a substantial (up to 50-fold), avidity-based, increase in the binding affinity . The12.1−4) and our previously characterized mPlxnA21−4. We also detected binding between mSema3AS−P (a form of mouse Sema3A comprising the sema and PSI domain) and mNrp11−4 but, in agreement with all the previously reported cell-based studies, found no measurable binding between mSema3AS−P and mNrp11−4. On combining mSema3AS−P and mNrp11−4, we could show an additive effect for interaction with mPlxnA21−4.The neuropilins, neuropilin 1 and 2 (Nrp1 and Nrp2), are type 1 cell surface receptors with ectodomains comprising five distinct domains, a single transmembrane span, and a short, unstructured, cytoplasmic region. Class 3 semaphorin binding has been reported to require the N-terminal four domains of the neuropilins ,30–33; t1−4  [1−4 at 2.3 Åfrom our earlier studies [1−4 and one copy each of the Sema3A sema domain and Nrp11−4 . Given the low resolution of the diffraction data, the model was only subjected to rigid-body refinement, with each domain treated as a rigid group  and a single B factor per domain. Twofold crystallographic symmetry generates the biologically relevant 330 kDa complex which comprises the semaphorin dimer, two neuropilin a1 domains and two plexins  , and so  studies , and a 2 studies ). These  studies . After e plexins .Figure K108N mutant mice show severe defects in the development of their peripheral nervous system [Our semaphorin–neuropilin–plexin crystal structure reveals that the arrangement of dimeric semaphorin separately interfacing two plexin receptors is identical to the architecture we, and others, have found in all other semaphorin–plexin complexes –14. So hs system  is now e13.In this review, I have focused on the results which now form the basis of our understanding of the structure and function of the extracellular components of the semaphorin–plexin system. These results highlight the key role played by synchrotron radiation in enabling structural studies of challenging glycoprotein complexes. In summary, our structural and functional data suggest a model in which the bivalent interaction of the semaphorin ligand with two plexin receptors provides an essential trigger for cell guidance signalling. For the secreted class 3 semaphorins, the co-receptor neuropilin is necessary to cement together this core complex. Further work can now build on these insights to probe the architecture and role of the entire plexin ectodomain, the requirements for signal transduction, and whether the formation of the initial semaphorin–plexin interface generates a standalone signalling complex or provides the first step in a larger-scale clustering of receptors.We and others have also made progress in the analysis of the molecular mechanisms controlling plexin signal transduction within the cell (reviewed in ). Again,14.Semaphorin–plexin-mediated cell guidance was first characterized in the nervous system, but is now known to function in the development and homeostasis of myriad tissues and organs. This broad swath of biology includes numerous areas in which semaphorin–plexin signalling has emerged as a potential target for therapeutic intervention. For example, the recent finding that Sema3A acts as a biological regulator of bone homeostasis, able both to reduce bone resorption and increase bone synthesis, is of potential importance for the development of therapeutics to combat osteoporosis . As a re


DSG) to yam flour might increase its nutritional value, with the aim of reducing nutritional diseases in communities consuming yam as a staple. Hence, yam flour was fortified with DSG at 5–35%. The effects of this fortification on the nutritional, chemical, and functional properties of yam flour were investigated. The result showed a significant increase (P ≤ 0.001) in fat, ash, protein, total amino acids, total dietary fiber, and insoluble dietary fiber contents of the blends as DSG increased except for starch and soluble dietary fiber contents, which decreased. The functional properties showed a significant (P ≤ 0.001) reduction with DSG inclusion. The inclusion of DSG increased both the tryptophan and methionine contents of the blends. Therefore, the DSG fortified yam flour could contribute to quality protein intake in populations consuming yam as a staple, due to its indispensible amino acid content.It was envisaged that the inclusion of treated distiller's spent grain ( However, because of the low protein content of Dioscorea spp. .Therefore, this study was aimed at evaluating the effect of DSG fortification on the nutritional composition and functional properties of yam flour produced from water yam , Ibadan, Nigeria and its tubers were used for the investigation. The DSG was obtained from United State Department of Agriculture‐Agricultural Research Service (USDA‐ARS), North Dakota, USA.A suspension was made from 100 g of DSG containing alcohol and residual sugar in 400 mL of water and fermented with yeast (0.8 g) for 1 h to convert residual sugar to alcohol, which was then removed by distillation. The pH of the suspension was then adjusted to about 6.0 to 7.0 by adding sodium hydroxide (7 mL). The resulting suspension was dried to about 5–10% moisture content as reported by Awoyale et al. .The yam tubers were peeled, washed, sliced into cubes, and dried in a hot air oven at 65°C for 48 h. The dried yam chips were milled into flour using an attrition mill, packaged in polyethylene bags, and stored until needed  and insoluble dietary fiber (IDF) fractions using the enzymatic‐gravimetric procedure  with UV detection at 254 nm as reported by Cohen et al.  at the SBulk density was determined using a standard laboratory method AOAC, . Flour sWater absorption capacity (WAC) and oil absorption capacity (OAC) were determined using the method as described by Beuchat . Flour sThe method described by Ukpabi and Ndimele  was usedThe least gelation concentration was determined by the method described by Coffman and Garcia . Ten sus°C) (the temperature at which there is a sharp increase in the viscosity of the flour suspension after the commencement of heating), and peak time (min) (time taken for the paste to reach the peak viscosity) were taken.Pasting characteristics was determined using a Rapid Visco Analyzer  interfaced with a personal computer equipped with the Thermocline software supplied by the same manufacturer  using Statistical Analysis System (SAS) package  SAS, . Means wP ≤ 0.001) in the ash content of the fortified yam flour. The fortified yam flour moisture content was also significantly (P ≤ 0.001) higher in 65% yam flour: 35% DSG blend (4.6%) and lower in 100% yam flour (3.6%) (Table P ≤ 0.001) decrease in the starch content of the blends from 62% for 100% yam flour to 48% for 65% yam flour: 35% DSG blend (Table P ≤ 0.001) increase in the fat content from 0.43% (100% yam flour) to 4.30% (65% yam flour: 35% DSG blend) (Table P ≤ 0.001) from 7.2% for 100% yam flour to 15.10% for 65% yam flour: 35% DSG blend  content of the fortified yam flour increased from 6.02% for 100% yam flour to 11.7% for 65% yam flour: 35% DSG blend, out of which the insoluble dietary fiber (IDF) had the highest value (23.50%) and the soluble dietary fiber (SDF) the least (0.90%) (Table P ≤ 0.001) decreased oil absorption capacity (OAC) of 100% yam flour from 213 to 183% for 90% yam flour: 10% DSG blend (Table P ≤ 0.001) were observed for bulk density of the fortified yam flour; which decreased as the amount of added DSG increased. It ranged from 57% for 80% yam flour: 20% DSG blend to 70% for 100% yam flour  or water yam . However, because of its low protein content (Onayemi and Potter Yam flour is a fermented or unfermented flour produced from either white yam (The result of this investigation showed that the protein content of the flour blends increased as the quantity of DSG increased. This could be attributed to the high protein content of DSG (29.80%) compared to that of the 100% yam flour (7.15%). Similar results for the protein content of the 100% yam flour had been reported for the flour produced from different yam varieties Bokanga . FurtherThe increase in fat content of the flour blends with DSG inclusion could be attributed to the high fat content of DSG. Comparable value (0.42%) with the fat content of the 100% yam flour was reported by Bradbury and Holloway  and SoucDioscorea alata flour reported by Lebot et al. , as starch is a very important factor for gel formation. However, similar observations with the starch content of the 100% water yam flour were made by other researchers  for storage of flour FAO, . This imn Pierre , if propamala) produced from all the yam blends with total indispensable amino acid content (g/100 g protein) range of 28.09–34.34 (with histidine) and 25.89–31.72 (without histidine) by the target group (children and adults) might increase their total indispensible amino acids level required for growth and repair of worn‐out tissue.Amino acid assay of foods is an important quality index, from which useful information on the nutritional quality and authenticity of food products and sources of raw materials used in food manufacture could be revealed , water absorption capacity (WAC), swelling capacity (SWC), bulk density (BD), amylose content, and pasting properties would be of importance to the end users.The OAC is important as oil acts as a flavor retainer and improves the mouth feel of foods Kinsella . The corFurthermore, the decrease in the SWC of the flour blends with increase in DSG may be due to the low starch content of DSG  before consumption, the pasting properties become important in predicting its behavior during and after cooking. Final viscosity is the most commonly used parameter to determine a particular starch‐based samples quality as it indicates the ability of the material to form gel after cooking  compared to the others due to its high final viscosity. Additionally, the higher the setback viscosity, the higher the rate of syneresis or weeping, and the easier it is for the food to be digested (Shittu et al. mala produced from 65% yam flour: 35% DSG might keep longer before “weeping” and as well digest fast when consumed due to its low setback value compared with that of 100% yam flour (Shittu et al. amala at a temperature of <63°C and time of <8 min., this implied low energy cost (Fasasi et al. As the flour blends would be reconstituted to a thick paste (amala and consumed with preferred soup due to its high indispensible amino acid content.The fat, ash, protein, sugar, total amino acids composition, total dietary fiber, and insoluble dietary fiber contents of the fortified yam flour increased while the starch and the soluble dietary fiber contents decreased as the DSG increased. In addition, aspertic and glutamic acids contributed the highest percentage of amino acids while tryptophan and methionine contributed the least in the flour blends. However, the inclusion of DSG to the yam flour increased both the tryptophan and methionine contents. There was also a decrease in the peak, final, and setback viscosities of the fortified yam flour. The WAC, OAC, swelling capacity, bulk density, and amylose content of the flour blends decreased with DSG inclusion. However, the DSG fortified yam flour could serve as a quality protein source when prepared to None declared.