Myrf is a newly discovered membrane-bound transcription factor that plays an essential role in as diverse organisms as human, worm, and slime mold. Myrf is generated as a type-II membrane protein in the endoplasmic reticulum (ER). It forms homo-oligomers to undergo auto-cleavage that releases Myrf N-terminal fragment from the ER membrane as a homo-trimer. The homo-trimer of Myrf N-terminal fragments enters the nucleus and binds the Myrf motif to activate transcription. Despite its prominent role as a transcriptional activator, little is known about the transactivation domain of Myrf. Here, we report that the N-terminal-most (NTM) domain of Myrf is required for transcriptional activity and, when fused to a Gal4 fragment, enables it to activate transcription. The transactivation function of the NTM domain did not require homo-trimerization. We also discovered that the NTM domain can be sumoylated at three lysine residues , with K276 serving as the main acceptor. K276 sumoylation repressed the transactivation function of the NTM domain without affecting the stability or nuclear localization of Myrf N-terminal fragment. In sum, this study identifies the NTM domain as the transactivation domain of Myrf and the potential regulatory impact of its K276 sumoylation. In the mammalian central nervous system (CNS), the expression of Myrf is restricted to oligodendrocytes (OLs) that make myelin sheaths1. OL-specific deletion of Myrf blocks the maturation of OLs, resulting in severe dysmyelination throughout the CNS1. Myrf is also essential for the life-long maintenance and regeneration of CNS myelin6 and its plasticity that underlies learning7. These studies left the impression that Myrf is a “myelin” transcription factor. However, Myrf orthologs are also found in organisms without myelin, where they were required for molt and synaptic plasticity (Caenorhabditis elegans)9 and prestalk cell differentiation (Dictyostelium)10. Consistent with the important roles of Myrf going beyond CNS myelination, germline deletion of Myrf in mice caused embryonic lethality1, and Myrf coding variants have been implicated in the pathogenesis of congenital heart disease and congenital diaphragmatic hernia as well as encephalopathy14.Myrf (Myelin regulatory factor) is a newly discovered pleiotropic membrane-bound transcription factor3. The Intramolecular Chaperone Auto-processing (ICA) domain of Myrf induces its homo-oligomerization in the ER membrane, and homo-oligomeric Myrf undergoes an auto-cleavage reaction to release its N-terminal fragment from the ER membrane as a homo-trimer mer Fig. 15. RemarDespite its prominent role as a transcriptional activator, little is known about the transactivation domain of Myrf. This knowledge would be essential to advance our understanding of the functional mechanisms of Myrf. This paper reports our elucidation of the transactivation domain of Myrf. We found that the N-terminal-most (NTM) domain of Myrf is required for transcriptional activity and can confer transcriptional activity to a Gal4 fragment. Further, our work shows that homo-trimerization is not critical to the transactivation function of the NTM domain. Thus, the NTM domain appears to function as an autonomous transactivator. Unexpectedly, we discovered that the NTM domain contains three consensus sumoylation motifs at K123, K208 and K276, and our biochemical analysis revealed that Myrf can be sumoylated at these lysine residues. We also found that K276 sumoylation represses the transactivation function of NTM. Overall, we elucidate the transactivation domain of Myrf and the potential regulatory impact of its K276 sumoylation.15, the NTM domain is likely to work as a transactivation domain. NTM is proline-rich (76/348 residues are prolines). Bioinformatics analysis suggests that the NTM domain has few α helices and β strands and is largely disordered (data not shown). These sequence and structural features are often found in transactivation domains18, supporting our hypothesis. To determine whether NTM is required for the transcriptional activity of Myrf N-terminal fragment, we gradually truncated its N terminus and assessed its impact on transcriptional activity. The regions of rat chr10:71034166-71034749 and chr1:23885816-23886215 (both in rn4) were identified as to be bound by Myrf in differentiating OLs2, and we cloned them into pGL3-promoter to generate the Myrf luciferase reporters Rffl and 21, respectively. The expression of Myrf effectively increased the reporter activity of Rffl and 21 in Oli-neu cells19 (a widely used mouse OL cell line)  domain, the DNA-binding domain, and the homo-trimerization motif Fig. . Given tne) Fig. . Strikinne) Fig. . Similarne) Fig. . We genene) Fig. . Westernne) Fig. . These r15. Flag-Myrf and Flag-Myrf-ΔN30 were expressed in HEK293FT cells, and cell lysates were incubated with magnetic beads coated with the Myrf motif incidence found in Rffl 3 is replaced by four alanine residues, was used as a control. We found that Myrf-ΔN30 N-terminal fragment was localized to the nucleus at a comparable rate to the wild-type one , strongly suggesting that Myrf and Sumo1 were covalently connected. To determine whether K123, K208, and K276 serve as SUMO acceptors for Myrf, we mutated them to arginine and repeated immunoprecipitation experiments. When all three lysine residues were mutated to arginine, sumoylation was completely abolished  proteins are covalently attached to the target protein276 Fig. , suggestads Fig. . When coe28 Fig. . SumoylaWestern blot analysis of primary OL and CG4 cell lysates shows that overexpressed Myrf is sumoylated at K276 by endogenous SUMOs Fig. . We wond2, and we cloned the former and a mouse genomic fragment corresponding to the latter into pGL3-promoter to generate the Myrf luciferase reporters Tpra1 and Sox10, respectively. The expression of Myrf effectively increased the reporter activity of Tpra1 and Sox10  regulatory mechanism that is hardwired into the NTM domain. Indeed, the luciferase assay results from Oli-neu cells were reproduced in HEK293FT cells  were identified as to be bound by Myrf in differentiating OLsx10 Fig. . For botx10 Fig. . Westernx10 Fig.  except fx10 Fig. . The repx10 Fig. . Westernlls Fig. . Importalls Fig. . Consistlls Fig. . Taken t10. Upon auto-cleavage in the ER membrane, its N-terminal fragment enters the nucleus as a homo-trimer and binds to the Myrf motif to activate transcription15. Despite its prominent role as a transcriptional activator15, there is scant information on the transactivation domain of Myrf, and this knowledge would be necessary to better understand the functional mechanisms of Myrf. Our thorough analysis identifies the N-terminal-most (NTM) domain of Myrf as its transactivation domain. First, we found that the NTM domain is required for the transcriptional activity of Myrf N-terminal fragment, satisfying the necessity condition. Second, when fused to a Gal4 N-terminal fragment, NTM rendered it transcriptionally active, meeting the sufficiency requirement. Residues in the N terminus of the NTM domain seem especially important for its transactivation function because their deletion almost completely abolished it. Corroborating this observation, the fusion of NTM to the Gal4 N-terminal fragment worked only when its N-terminal residues were intact. Since the deletion of the N-terminal residues did not affect the stability, DNA binding, and nuclear localization of Myrf N-terminal fragment, we think that the N-terminal residues of the NTM domain are critical to the physical interaction between Myrf N-terminal fragment and an unknown co-activator. The unknown co-activator must be a general factor that is expressed in both Oli-neu and HEK293FT cells because the deletion of the N-terminal residues had the same effect in both cell lines. Further studies will be required to identify the unknown co-activator and its role in the transcriptional activity of Myrf N-terminal fragment. Our study also shows that homo-trimerization is not essential for the transactivation function of NTM, unlike the DNA-binding and ICA domains of Myrf whose function depends on homo-trimerization30. However, homo-trimerization may enhance the transactivation function of the NTM domain either quantitatively  or qualitatively .Myrf is a pleiotropic membrane-bound transcription factor that plays an essential role in diverse organisms25. Thus, given that each SUMO molecule is about 10 kD, the polymer attached to Myrf K276 may consist of two or three internal Sumo2/3 molecules and one terminal Sumo1 molecule. This polymer may be bulky enough to disrupt the physical interaction between Myrf N-terminal fragment and the co-activator.Unexpectedly, we found three consensus sumoylation motifs in the NTM domain , which prompted us to hypothesize that NTM may be sumoylated, and that sumoylation may modulate its transactivation function. Indeed, our biochemical assay demonstrated that Myrf N-terminal fragment can be sumoylated at the three lysine residues. Further, we found that the sumoylation of K276, the main sumoylation site, represses the transactivation function of NTM. The difference in the repressive effect among the three sumoylation sites may be due to the quantitative difference in the sumoylation level (K276 is the most sumoylated site and thus has the greatest effect) or the qualitative difference . K276 sumoylation reproducibly led to an increase of 30-40 kD in the apparent molecular weight. Sumo2/3 are known to form polymers through their internal sumoylation sites while Sumo1 works as a chain terminator due to the absence of such sites14. Heart disorder and diaphragm hernia tend to co-occur, along with other malformations such as urogenital tract defects, lung hypoplasia, and intellectual disability. So it has been suggested that Myrf-implicated congenital anomalies represent a novel type of complex birth defect14, which is consistent with the essential role of Myrf in embryonic development1. The seven missense mutations are mapped to the DNA-binding and ICA domains of Myrf. No mutation in the NTM domain has yet been reported in association with human disease. However, sequencing studies in the future are likely to uncover disease-linked missense mutations in the NTM domain. In fact, the ExAC database reveals many rare Myrf missense mutations that hit highly conserved residues in NTM31, which may significantly disrupt its transactivation function. Given the indispensable role of Myrf in diverse organisms and cellular processes, our study that identifies NTM as the transactivation domain of Myrf and the potential regulatory impact of its K276 sumoylation lays an important ground work.In line with the pleiotropic role of Myrf, ten Myrf genetic alleles  have been implicated in the genetic risk of congenital heart disease, congenital diaphragmatic hernia, and encephalopathy with reversible myelin vacuolization15. The mouse Myrf cDNA was cloned into pcDNA3. The HA-Sumo1 plasmid was a gift from Guy Salvesen (Addgene plasmid #48966)32. All mutagenesis was carried out by a PCR-based method, and sequence information was verified by Sanger sequencing.A mouse Myrf cDNA that encodes the 1139-amino-acid-long isoform was kindly provided by Dr. Ben Emery27. Primary OPCs, CG4 and Oli-neu cells were kept in a proliferative condition by supplementing the Sato media27 with PDGF (10 μg/mL), NT3 (1 μg/mL), CNTF (10 μg/mL), and NeuroCult™ SM1 Neuronal Supplement (only for Oli-neu cells). Primary OPCs, CG4 and Oli-neu cells were maintained in a humidified 8% CO2 incubator at 37 °C. HeLa and HEK293FT cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and maintained in a humidified 5% CO2 incubator at 37 °C. Transient transfection was performed by using Lipofectamine 2000 as per the manufacturer’s instructions.Animal husbandry and tissue harvest were carried out in accordance with the guidelines and protocols of the University at Buffalo  Institutional Animal Care and Use Committee . Oligodendrocyte precursor cells (OPCs) were purified from rat pups of P7~P9 by immunopanningg for 10 min at 4 °C. Cleared cell lysate was mixed with 10 ul anti-HA agarose bead slurry (Sigma #E6779) or anti-FLAG M2 affinity agarose gel (Sigma #A2220) for immunoprecipitation for 2 hr at 4 °C. The mixture was centrifuged at 7000 g for 1 min. Beads were washed with the lysis buffer 5 times for a total of 1 hour. Samples were mixed with Laemmli sample buffer and boiled at 95 °C for 5 min. Upon electrophoresis, proteins were transferred to PVDF and probed with primary and HRP-conjugated secondary antibodies. Reagents used for immunoblotting are as follows: HRP conjugated FLAG antibody , α-tubulin antibody , anti-HA tag antibody, HRP-conjugated , and goat anti-mouse antibody, HRP conjugate .Cells were lysed with a lysis buffer , and 2 mM PMSF). Cell lysate was boiled for 5 min and then sonicated with a bioruptor for 10 min. Cell lysate was centrifuged at 14,000 Cells were fixed with 4% formaldehyde and permeabilized with 0.1% Triton X-100. Upon blocking with 1% BSA, they were incubated with primary antibodies diluted in blocking buffer at 4 °C overnight, followed by incubation with fluorochrome-conjugated secondary antibodies. Fluorescence was visualized with a Leica DMi8 microscope with an ORCA-Flash4.0 sCMOS camera. Reagents used for immunofluorescence are as follows: FLAG antibody , calnexin antibody , Hoechst 33342 , donkey anti-mouse antibody, Alexa Fluor® 488 conjugate , and donkey anti-goat antibody, Alexa Fluor® 594 conjugate .2 into pGL3-promoter. The genomic coordinates of the luciferase reporters are as follows: Rffl (rn4 chr10:71034166-71034749), Tpra1 (rn4 chr4:123025896-123026295), 21 (rn4 chr1:23885816-23886215), and Sox10 (mm9 chr15:79023342-79024150).Luciferase assays were performed by using the Promega dual luciferase reporter assay kit as per the manufacturer’s instructions. Cells were transfected with Myrf-expressing plasmids, Myrf luciferase reporters, and pRL-TK . Myrf luciferase reporters were generated by cloning Myrf ChIP-seq peaksg for 20 min at 4 °C. Biotinylated duplex oligonucleotides were conjugated to Dynabeads (Invitrogen) in buffer A . Oligonucleotide-conjugated beads were washed twice with 500 µl of buffer A and three times with buffer C . 300 µg of cell lysate were incubated with oligonucleotide-conjugated beads in buffer C and sheared salmon sperm DNA  for 20 min at room temperature with rotation. The mixture was spun down to separate the bead and sup fractions. The bead fraction was washed five times with 500 µl buffer C, and both fractions were analysed by immunoblotting. The DNA sequences used are as follows. The Myrf motif15 is underlined, and the mutated portions are indicated in bold.HEK293FT cells were transfected with Flag-Myrf or its variants. Upon cell lysis, cell lysate was cleared by centrifugation at 15,000 CTGGCACTGCCTGGCGCGGCCAWT:TGACTACCCCACAAGTCAACATTGCCTGGCGCGGCCAMU:TGACTACCCCACAAGSupplementary information


Legionella that are distinct in sequence and specificity from mammalian SH2 domains. The bacterial SH2 domains are not only capable of binding proteins or peptides in a Tyr phosphorylation-dependent manner, some bind pTyr itself with micromolar affinities, a property not observed for mammalian SH2 domains. The Legionella SH2 domains feature the SH2 fold and a pTyr-binding pocket, but lack a specificity pocket found in a typical mammalian SH2 domain for recognition of sequences flanking the pTyr residue. Our work expands the boundary of phosphotyrosine signalling to prokaryotes, suggesting that some bacterial effector proteins have acquired pTyr-superbinding characteristics to facilitate bacterium-host interactions.Src homology 2 (SH2) domains play a critical role in signal transduction in mammalian cells by binding to phosphorylated Tyr (pTyr). Apart from a few isolated cases in viruses, no functional SH2 domain has been identified to date in prokaryotes. Here we identify 93 SH2 domains from  Legionella that bind to mammalian phosphorylated proteins, in some cases with very high affinity.SH2 domains bind to tyrosine-phosphorylated proteins and play crucial roles in signal transduction in mammalian cells. Here, Kaneko et al. identify a large family of SH2 domains in the bacterial pathogen  Legionella pneumophila is the causative agent for a severe form of pneumonia called the Legionnaires’ disease2. Since the discovery of L. pneumophila as a pathogenic bacterium infecting humans via the alveolar macrophage, at least 56 Legionella species have been identified, two-thirds of which are associated with human disease2. However, human-to-human transmission is exceedingly rare4. Instead, Legionella species exist as parasites of phylogenetically diverse protists in the natural environment6. To survive within the phagocytic host, the bacteria replicate inside a membrane-bound compartment called the Legionella-containing vacuole that incorporates materials hijacked from the endoplasmic reticulum and mitochondria of the host cell7. Numerous effector proteins are injected into the host cell upon Legionella infection8. For example, >330 effector proteins, accounting for ~10% of the L. pneumophila proteome, are injected into the host cell upon infection via the Icm/Dot type IV secretion system (T4SS)10. Many effector proteins contain eukaryotic motifs or domains such as the ubiquitin ligase U-box domain, the SET methyltransferase domain, and the protein kinase domain13.The bacterium 14. The tyrosine kinase signalling machinery comprises the tyrosine kinase (TK), the phosphotyrosine (pTyr) phosphatase (PTP), and the pTyr-binding module such as the Src homology 2 (SH2) or the phosphotyrosine-binding (PTB) domains. A recurring theme in pathogenic bacterium–host interactions involves host tyrosine kinases acting on bacterial effector proteins. For example, Helicobacter pylori and Escherichia coli can secrete effector proteins to function as substrates of the host tyrosine kinases. Once phosphorylated these effectors can hijack tyrosine kinase signalling in the host cell via the recruitment of host SH2 proteins16. Salmonella, Yersinia, and Pseudomonas, in contrast, secrete phosphotyrosine phosphatases to dephosphorylate host proteins19. Intriguingly, infection by L. pneumophila leads to tyrosine phosphorylation of proteins in both the host cell and the bacterium22. Regulation of tyrosine phosphorylation caused by Legionella infection was also reported for protozoan host species23. Tyrosine kinase or phosphotyrosine phosphatase inhibitors have been shown to reduce the uptake or replication of L. pneumophila in the host24, suggesting an important role for the tyrosine kinase signalling machinery in the pathogen–host interaction and Legionella lifecycle inside the host.Protein tyrosine kinases play a critical role in regulating numerous cellular functions25. The human genome encodes >120 SH2 domains dedicated to the recognition of the pTyr, thereby ensuring that the kinase signal is transduced with high fidelity and efficiency26. Intriguingly, Acanthamoeba castellanii, a natural protozoan host for Legionella, harbors 48 putative SH2 domains, suggesting that tyrosine phosphorylation may play a role in the intracellular signalling of the protozoan host27. An SH2 domain typically contains ~100 amino acid residues and shares a common fold characterized by a seven-stranded β-sheet (strands βA to βG) flanked by two α-helices (αA and αB)28. Although different SH2 domains have distinct specificity, they all contain a pTyr-binding pocket and another pocket or binding site for the recognition of flanking residues, most often a C-terminal residue to the pTyr30. The critical role of the pTyr residue to SH2-binding is underscored in the observation that it contributes ~50% of the free energy of binding for a tyrosyl phosphopeptide to an SH2 domain32. Nevertheless, the affinity of an SH2 domain for its physiological ligand peptide is generally moderate, with the corresponding equilibrium dissociation constant in the 0.1–10 μM range33.Tyrosine phosphorylation may lead to changes in activity or subcellular localization of the substrate or the creation of binding sites for proteins containing an SH2 domain34. Because the SH2 superbinder, when introduced into mammalian cells, was capable of inhibiting tyrosine kinase signaling, we wondered if pathogenic bacteria would exploit this mechanism to gain an advantage when infecting a host. To explore this notion, we conducted an exhaustive search of the bacteria genome database and identified 93 putative SH2 domains in 84 Legionella proteins. We characterized 13 Legionella SH2 domains for ability to bind pTyr-containing peptides and found 11 were capable of binding to the pTyr residue and to mammalian proteins in a Tyr phosphorylation-dependent manner. Indeed, the affinities of some Legionella SH2 domains for the pTyr or phosphopeptides derived from mammalian proteins far exceeded those of a mammalian SH2 domain, suggesting that these bacterial SH2 domains are natural pTyr superbinders. Intriguingly, unlike the mammalian counterpart, a Legionella SH2 domain displayed no apparent sequence preference beyond the pTyr residue. Structural analysis of two Legionella SH2 domains revealed the basis for this unique mode of pTyr recognition. While both Legionella SH2 domains feature a defined pTyr-binding pocket, they are devoid of a second pocket or binding site for a C-terminal residue to the pTyr that is commonly found in a mammalian SH2 domain. Furthermore, we found that the majority (8/10) of the SH2-containing proteins were capable of translocating into human macrophage cells, suggesting that they may function as effector proteins. Our findings, which expand the realm of the SH2 domain from eukaryotes to prokaryotes, imply that the tyrosine kinase–pTyr–SH2 signaling axis may play an important role in the Legionella-host interaction and pathophysiology of the Legionnaires’ disease.The moderate affinity exhibited by the mammalian SH2 domains raised the question whether the SH2 fold can support greater affinities for the pTyr residue. We addressed this question by directed in vitro evolution of the pTyr-binding pocket in the Fyn SH2 domain using phage-displayed libraries. This led to the identification of an SH2 mutant, termed the SH2 superbinder, that bound to physiological pTyr peptides with affinities two to three orders of magnitude greater than the natural domainLegionella13. Moreover, the Pfam domain database  lists eight bacterial protein sequences with a putative SH2 domain35. We analyzed these putative SH2 domains by secondary structure prediction and found that they possessed the key characteristics of a typical SH2 domain , LeSH1a, LeSH1b, LeSH2, LeSH3, LeSH4, LeSH5, LUSH (Legionella U-box and SH2), RavO , and DoSH (Double SH2), respectively. The 93 candidate SH2 domains were derived from 40 Legionella species and two Coxiellaceae species 13 that is found also in bacterial toxins38. Although the CPDs from the RavO and LeSH5 clusters showed 26–35% overall sequence identity to each other, they all contained the catalytic Cys residue  using the adenylate cyclase (Cya) reporter assay45. As expected, RavO was able to translocate to the U937 macrophages upon L. pneumophila infection in a Dot/Icm T4SS system-dependent manner  were able to translocate in the same assay. While it is possible that the L. longbeachae LeSH is not a secreted effector, it is more likely that a non-native effector may not be efficiently translocated in the L. pneumophila system employed here. Moreover, the efficient translocation of certain effectors may be facilitated by chaperones , and the L. longbeachae LeSH may require such a chaperone protein from the same species that may not be available in the L. pneumophila (strain Lp02) used in the Cya reporter assay.The SH2 domain-containing proteins are predicted effectors based on Burstein et al.ner Fig. . ImportaLegionella SH2 domains are functional pTyr binders, we determined their binding affinities for phosphotyrosine (pTyr) using the mini peptide GGpYGG that has been shown previously to bind engineered SH2 superbinders but not natural SH2 domains39. Intriguingly, the L. longbeachae LeSH, a 167-residue protein containing a single SH2 domain, bound the peptide with an equilibrium dissociation constant (Kd) of ~3.6 μM, but not to the Tyr- or pThr-containing version , 6 of the 13 Legionella SH2 domains examined exhibited <10 µM affinities for the mini pTyr peptide . The specificity of either the LeSH or RavO SH2 domain is, however, distinct from that of a mammalian SH2 domain29.To determine the specificity of the Legionella SH2 domains  for one or more pTyr peptides. The affinities for these Legionella SH2 domains were usually higher than the Src SH2 domain for the same peptides  to enrich phosphorylated proteins prior to preparing cell lysate48. Far-western blots using GST-fused proteins showed that the four Legionella SH2 domains  tested were all capable of binding the host cell proteins in a pv-dependent manner  showed a robust ability to pull down tyrosine-phosphorylated proteins  with the RavO SH2 domain crystal, we obtained the complex structure and found that the peptide was identifiable in three out of the four SH2 domain molecules in an asymmetric unit  as the top structural neighbor for LeSH, whereas the RavO SH2 domain most closely resembles the yeast Spt6 SH2 domain structurally even though the two share only 13% sequence identity  packed against a polyproline segment via favorable hydrophobic contacts Leu mutant of the Src SH2 domain even showed a marked increase in pTyr-binding affinity, due apparently to enhanced hydrophobic interaction34.Both the LeSH and RavO SH2 domains contain a defined pTyr-binding pocket enriched for positively charged residues Fig. . Of noteTo resolve these seemingly contradictory results, we analyzed the conformation of the pTyr sidechain and the pTyr-binding pocket in different SH2-peptide complexes Fig. . DependiThe pTyr-binding pockets in LeSH and the RavO SH2 domain are further defined by the unique structures in the region between the strand βE and helix αB, which effectively provides a roof for the pocket. Specifically, the Pro85 residue in LeSH and Thr310 in RavO sit atop of the pTyr residue, opposite to the ArgβB5 residue located at the bottom of the pTyr-binding pocket Fig. . BesidesLegionella and Src SH2 domains , is located on the opposite side of the central β-sheet to the βB5-Arg175 residue . However, the αEF helix in LeSH  and βB5-Arg46 are located on the same side  is 55% identical to the L. longbeachae LeSH, the former is characterized with a shorter (3-residue) BC loop than the latter  have never been identified in fungi or any species more distant than fungi  tyrosine kinases (which themselves prefer substrates containing specific sequence motifs). Therefore, it is possible that the Legionella SH2 domains evolved independently of tyrosine kinases and were therefore not selected for motif recognition to correlate with tyrosine kinase activation and signalling. Since the natural host species of Legionella span almost the entire eukaryotic tree of life, it is reasonable to assume that the Legionella SH2 domains have evolved to shed the specificity pocket in lieu of promiscuous binding to cope with the diverse target sequences found in distant host species.Besides the large number of 34. It is remarkable that at least two of the Legionella SH2 domains examined in this study, namely the LUSH and LeSH4 SH2 domains, bound to the pTyr (as in the pYG and GGpYGG mini-peptides) with affinities surpassing that of the Src SH2-derived superbinder. Nevertheless, unlike the Src SH2 superbinder, these bacterial SH2 superbinders did not discriminate one peptide from another based on the pTyr flanking sequence. Therefore, a Legionella SH2 domain could potentially bind any Tyr-phosphorylated proteins in the host, thereby making it a true disruptor of phosphotyrosine signaling. However, no significant defect in infection or growth efficiency was observed for L. longbeachae mutants in which either or both of the LeSH and LUSH genes were deleted. Legionella secretes hundreds of effector proteins into its host upon infection, so a large degree of redundancy in function is expected of the translocated effectors13. The remarkable sequence diversity for the different Legionella SH2 domains makes it difficult to exhaust all SH2 domains by sequence-based search; it is therefore possible that additional SH2 domain effectors may exist in a given Legionella species that have eluded our identification. Nonetheless, the preponderance of SH2 domains in Legionella and the superb pTyr-binding affinities exhibited by them suggests biological functions for the bacterial SH2-containing effector proteins.We previously created several mammalian SH2 domain mutants, called pTyr superbinders, that bind to tyrosine-phosphorylated peptides with affinities that are orders of magnitude greater than the corresponding natural domains11 reported the genome sequence of L. longbeachae and annotated the llo2327 gene as an SH2 domain-containing sequence, which we call LeSH in this paper. We first ran the PSIPRED secondary structure prediction server62 to confirm that the sequence has the signature secondary structure elements for the SH2 domain fold  and each hit sequence, and the sequence is retained if the βB5 arginine (Llo LeSH Arg46) is conserved in the hit sequence. The hit sequences were considered as SH2 domains homologous to Llo LeSH if the sequence identity is at least 40%. If the sequence identity between the query and the hit sequence is below 40%, secondary structure prediction was performed. A sequence from the BLAST search is considered a putative SH2 domain if (1) the predicted secondary structure shows the α-helix–β-sheet–α-helix SH2-fold signature and (2) the conserved arginine  is located on the predicted βB strand (the first strand after the αA helix).Cazalet et al.Legionella orthologous group) sequence clustering reported by Burstein et al.13. The repetitive BLAST searches starting from the Llo LeSH sequence identified the LOG clusters LeSH/LeSH1a/LeSH1b (LOG_02684), LeSH2 (LOG_02859), LeSH3 (LOG_07829), LeSH4 (LOG_04518), and LUSH (LOG_02977).The newly identified LeSH homologs were used as queries for a new round of BLAST searches. The same set of criteria (signature secondary structure elements and βB5-Arg conservation) was applied to identify additional bacterial SH2 domain sequences, which were in turn used as search queries in the next round of BLAST searches. The BLAST searches were repeated until no more bacterial SH2 domain homologs that satisfy the criteria were identified. The proteins were grouped according to the pairwise sequence identity as well as the LOG  was obtained from Pfam website35. The hmmsearch program in the HMMER suite63 was used to scan the bacterial protein sequences for the SH2 domain. The search identified Lpp3070 (DoSH from L. pneumophila Paris) that contains two SH2 domains that satisfy the secondary structure and arginine conservation criteria. Using the DoSH sequence as a query, repeated BLAST searches identified members of DoSH (LOG_00141), RavO (LOG_05208), and LeSH5 (LOG_04812) proteins.To identify more bacterial SH2 domains without homology to 64 was used for each homologous group to confirm that the βB5 arginine is conserved in all sequences for the group  from uncultivated species for which the species were unidentified65. We synthesized the two genes, expressed and purified proteins and conducted in-solution peptide binding assay. However, we did not detect binding to tyrosine-phosphorylated peptides, and, therefore, we did not include them in this paper.Although we did not limit the range of bacterial species in our sequence searches, we could identify SH2 domains with high confidence only from the order Dictyostelium transcription factor STAT, PDB 3GXW: yeast transcription factor Spt6, PDB 3MAZ: human BRDG1 SH2, PDB 1JYR: human Grb2 SH2, PDB 2BF9: avian pancreatic polypeptide, PDB 4WZ3: L. pneumophila effector protein LubX.The following PDB coordinate files were used for structure-guided sequence alignment and structure comparison analysis; PDB 1SPS: chicken  Src SH2, PDB 1D4W: human SAP SH2, PDB 1UUR: social amoeba L. longbeachae LeSH and LUSH SH2; L. dumoffii LeSH1a, LeSH2, and LeSH3; L. drancourtii LeSH4, and chicken Src SH2. Two of them  were associated with structure coordinates. The second alignment consists of eight sequences: L. pneumophila RavO and DoSH C-terminal SH2 domains, L. waltersii LeSH5 N- and C-terminal SH2 domains, L. brunensis Lbru_2627 N- and C-terminal SH2 domains, yeast Spt6 SH2 and chicken Src SH2. Three of them  were associated with structure coordinates. A prototype structure-guided alignment for Fig. 66 program by incorporating all the sequences . However, the program did not produce a reliable alignment for the C-terminal region, presumably due to large structural diversity and poor sequence conservation , except for the L. dumoffii LUSH, which was cloned from the genomic DNA by PCR. Full-length synthetic genes were prepared for expression of the following proteins: L. longbeachae LeSH; L. dumoffii LeSH1a, LeSH2, and LUSH; L. pneumophila LeSH2; and L. anisa LeSH1, LeSH2a, and LeSH2b. Genes corresponding to the following amino acid residue ranges were chosen for expression of the SH2 domains in E. coli. L. longbeachae LUSH SH2 domain: Ser3-Ser171, L. drancourtii LeSH4 SH2 domain: Ser3-Asp170, L. waltersii LeSH5 tandem SH2 domains: Asp3-Ala219, L. pneumophila DoSH C-terminal SH2 domain: Glu366-Pro546, and L. pneumophila RavO SH2 domain: Gly212-Asp406. The hexahistidine (His6)-tagged and His6-GST-tagged constructs were prepared by subcloning the genes into the pETM-11 and pETM-30 vectors69, respectively. The expression construct for the triple mutant Src SH2 domain (superbinder) was described previously34.The list of primer sequences used for molecular cloning and site-directed mutagenesis is provided in Supplementary Table Legionella SH2 proteins was subcloned into the pCyaA fusion vector45. L. pneumophila strains Lp02 and Lp03 expressing the Cya-fusion protein were used for the assay. L. pneumophila were grown at an initial OD600 of 0.7 with 0.1 mM of isopropyl β-d-1-thiogalactopyranoside (IPTG) until late-exponential phase with large motility. The U937 cells (ATCC CRL-1593.2) were grown in RPMI media (Sigma) supplemented with 10% fetal bovine serum, and were differentiated with 12-tetradecanoyl phorbol 13-acetate for 3 days. The differentiated cells were seeded in 12-well tissue culture plates at a density of 1.0 × 106 cells/well and challenged with bacteria strains at a multiplicity of infection of 5. After incubation at 37 °C for 1 h, the U937 cells were washed three times with phosphate-buffered saline (PBS), lysed by adding 200 µl lysis solution , and incubated on ice for 10 min. Lysed samples were boiled for 5 min, and then neutralized with 10 µl of 1 M NaOH. After addition of 400 µl of cold 95% ethanol and incubation on ice for 5 min, insoluble material in the extracts was removed by centrifuging at 4 °C at 16,000 × g for 5 min. Supernatants were vaporized under vacuum and pellets were resuspended in EIA buffer . The levels of cyclic AMP were measured using Cyclic AMP EIA Kit . The CyaA fusion proteins in bacterial lysate were detected by western blot, using mouse anti-CyaA antibody  diluted 1:5000 and goat anti-mouse IgG-HRP conjugate  diluted 1:3000.The full-length gene encoding 6- or His6-GST-fusion proteins were induced with 0.3 mM IPTG in Escherichia coli BL21(DE3) at 18 °C, overnight, except for the Src SH2 domains expressed at 30 °C for 6 h. Bacterial cells were lysed in 20 mM sodium phosphate, 20 mM imidazole, 100 mM NaCl, 1 mg/ml lysozyme, 0.5% Triton X-100, pH 8.0, by incubating the cells on ice for 30 min. The Ni-NTA (Qiagen) nickel affinity chromatography was conducted by following the manufacturer’s instruction. Samples eluted from the column were dialyzed against 20 mM Tris-HCl, 150 mM NaCl, at 4 °C for 48 h (supplemented with 0.5 mM Tris(2-carboxyethyl)phosphine for the wild-type Src SH2 domain as the reducing agent).Expression of the HisL. longbeachae LUSH protein fused with the His6-GST tag was used for the assay. The L. pneumophila ubiquitin ligase LubX with C-terminal truncation (residue 1–186) was used as the positive control for the in vitro ubiquitination assay. The human E2D2 was used as the ubiquitin-conjugating enzyme E2. The experiment was conducted in 50 mM Tris-HCl, 100 mM NaCl, 10 mM ATP, 10 mM MgCl2, 0.5 mM dithiothreitol, overnight at 25 °C. See Quaile et al.41 for detailed experimental procedure.The full-length 47. In addition to the first 20 peptides, four more peptides were identical between Array-1 and Array-2. These 24 identical peptides were used for normalizing signal intensities between the two membranes for each probe . Either a Gly residue  using the standard Fmoc protocol. The peptides were N-terminally labeled with fluorescein, with either two glycines (Gly–Gly) or 6-aminohexanoic acid (ahx) as a spacer between the peptide and fluorescein. The final peptide concentration was adjusted between 1 and 5 nM. Fluorescein polarization was measured on an EnVision multilabel reader 2103 (PerkinElmer) using the wavelength 480 nm for excitation and 535 nm for emission. Average readings from two replicate experiments were used for binding curve fitting assuming one-site peptide binding , equilibrated in the lysis buffer, were added for incubation for 20 min at room temperature, followed by GST pulldown and western blot analysis. The following antibodies were used: 4G10 Platinum mouse anti-pTyr antibody , mouse anti-SHC1 antibody , rabbit anti-VCP antibody , goat anti-mouse IgG-HRP conjugate , and goat anti-rabbit IgG-HRP conjugate . Uncropped scans of the western blots that correspond to Fig. 6-tagged LeSH eluted from the nickel affinity column was dialyzed against 20 mM Tris-HCl, pH 7.0, 50 mM NaCl, 1 mM DTT, 0.5 mM EDTA, at 4 °C. The affinity tag was cleaved by TEV protease at room temperature for 2 days. The cleavage reaction product was further purified by Superdex 75 size exclusion chromatography  in 20 mM Tris-HCl, 150 mM NaCl, pH 7.0. Crystallization was conducted by either hanging or sitting drop vapor diffusion method by mixing an equal volume of the LeSH sample (with or without a ligand) and the mother liquid. The apo crystal was grown in the following condition: 43 mg/ml LeSH, 0.1 M Tris-HCl, pH 9.0, 20% PEG3000, 0.2 M sodium acetate, at 5 °C. The phosphotyrosine complex crystal was grown in the following condition: 25 mg/ml LeSH, 10 mM pTyr, 20% PEG3000, 0.2 M sodium acetate, 0.1 M Tris-HCl, pH 8.9, at 5 °C. The DnaJ-A1 pTyr381 peptide complex crystal was grown in the following condition: 1.3 mM LeSH, 2.8 mM peptide, 16% PEG3000, 150 mM malic acid, pH 7.0, 0.1 M Tris-HCl, pH 7.8, at room temperature. The IL2Rβ pTyr387 peptide complex crystal was grown in the following condition: 1.5 mM LeSH, 3.1 mM peptide, 12% PEG3000, 0.2 M sodium malonate, at room temperature. Parrafin oil was used as the cryoprotectant for all crystals. Data collection was conducted at 114 K using the RUH3R X-ray generator (Rigaku) and the mar345 detector (MarResearch) with CuKα radiation. Diffraction datasets were processed with iMosflm71. A dataset from 741 images with the oscilation angle of 2°  was used for phasing of apo LeSH with the sulfur single-wavelength anomalous diffraction (sulfur SAD) method. The Auto-Rickshaw webserver was used for phasing72. The ligand complex structures were solved by molecular replacement. The Coot73 and the Phenix74 program suites were used for model building and refinement.For LeSH crystallization, the Hislpg1129 coding for this region of the protein was cloned into the p15Tv-LIC vector. For purification of native RavO SH2 domain, full-length lpg1129 was cloned into the p15Tv-LIC vector, coding for an N-terminal His6 tag and TEV protease cleavage site. After structure determination of the apoprotein RavO SH2 domain, the termini of the expression construct were modified to include the region of lpg1129 coding for RavO residues 225–344 into the pMCSG53 vector, also coding for an N-terminal His6 tag and TEV protease cleavage site; this construct was utilized for expression of native RavO SH2 domain for crystallization with the Shc1 pTyr317 peptide (acetyl-DPSpYVNVQNL-amide). For native protein purification, E. coli BL21(DE3)-RIL or BL21(DE3)-Gold cells were transformed with the appropriate expression plasmid, grown to an OD600 of 0.6 at 37 °C, chilled to 16 °C, and induced overnight with 500 mM isopropyl β-d-thiogalactopyranoside. Selenomethionine-substituted RavO[209-406] was expressed in BL21(DE3)-RIL cells according to the manufacturer’s instructions (Shanghai Medicilion). All cells were harvested via centrifugation at 5000 g and pellets resuspended in binding buffer (50 mM Hepes (pH 7.5), 100–300 mM NaCl, 10 mM imidazole and 2% glycerol (v/v)), lysed by sonication, and cell debris removed via centrifugation at 30,000 g. Cleared lysates were loaded onto a 5 ml Ni-NTA column (QIAGEN) pre-equilibrated with binding buffer, extensively washed with binding buffer containing 30 mM imidazole, and protein was eluted using the above buffer with 250 mM imidazole. The His6-tags were removed by cleavage with TEV protease overnight at 4 °C in dialysis buffer , 5% glycerol, and 0.5 mM tris[2-carboxyethyl]phosphine), followed by binding to Ni-NTA resin and capture of flow through. All RavO crystals were grown with the sitting drop vapor diffusion method. SeMet-substituted RavO[209-406] was crystallized by mixing an equal volume (1 μL) of the protein solution at 55 mg/mL and reservoir solution  and the crystal was cryoprotected using paratone oil before flash freezing under liquid nitrogen. Apo RavO was crystallized by mixing an equal volume (1 μL) of the protein solution at 90 mg/mL and reservoir solution  PEG 5K MME), and the crystal was flash frozen under liquid nitrogen. The RavO SH2 domain + Shc1 phosphopeptide complex was determined by co-crystallization using 0.2 mM pTyr317 peptide, and mixing an equal volume (1 μL) of the protein:peptide solution (protein at 90 mg/mL) and reservoir solution  PEG 5K MME), and the crystal was cryoprotected using paratone oil. X-ray diffraction data were collected at the Structural Biology Center, Advanced Photon Source, beamline 19-ID at selenium absorption peak. Data were processed using HKL3000 (ref. 75). For SAD phasing, Phenix.autosol detected four of the six selenomethionine sites in the asymmetric unit of the RavO[209-406] crystal. Phenix.autobuild and Coot73 were used for all model building. All B-factors were refined and TLS parameterization was included in final rounds of refinement. All geometry was verified using Phenix validation tools and the wwPDB server.The RavO SH2 domain apoprotein form was determined from selenomethionine-derivatized RavO[209-406] solved by SAD methods to a resolution of 3.30 Å, and subsequently used to phase crystals of native, full-length RavO that spontaneously degraded into the SH2 domain, using molecular replacement, to a resolution of 1.95 Å. For purification of selenomethione-derivatized RavO[209-409], the region of 50. PyMOL (Schrödinger) was used for structure drawing. The APBS program76 were used for molecular surface potential calculation  was used for Supplementary Fig. 80 was used for the normal mode analysis (Supplementary Fig. The structure-based sequence identity Fig.  was deriion Fig. . The Mulres Fig. . The bur 78 Fig. . MODELLESupplementary InformationPeer Review FileReporting Summary


AbstractTermitinae, soldiers have long and slender mandibles that cannot bite but, instead, snap and deliver powerful strikes to their opponents. Here, we use morphological and molecular evidence to describe Roisinitermesebogoensis Scheffrahn, gen. & sp. n. from near Mbalmayo, Cameroon. Soldiers of R.ebogoensis are unique among all other kalotermitid soldiers in that they possess snapping mandibles. The imago of R.ebogoensis is also easily distinguished from all other Kalotermitidae by the lack of ocelli. Our study reveals a new case of parallel evolution of snapping mandibles in termites, a complex apparatus responsible of one of the fastest biological acceleration rates measured to date.Termites have developed a wide array of defensive mechanisms. One of them is the mandibulate soldier caste that crushes or pierces their enemies. However, in several lineages of  Termites are extremely abundant  and coloTermitinae . A few species of Cryptotermes . Measurements Tables –2 were oR.ebogoensis, after removal of the digestive tract. The full mitochondrial genome was amplified with TaKaRa LA Taq in two long PCR reactions using primers specifically designed for termites . The reconstruction of Maximum Likelihood phylogenies was carried out with RAxML .The lack of visible ocelli is unique among all other PageBreakMonomorphic Figs , 5. Eye Roisinitermes leads to couplet 17 (Allotermes). In dorsal view, the mandibular blades of Allotermes, especially A.denticulatusRoisinitermes as those of the former are long, rather narrow and with rudimentary dentition. In lateral view, however, the Roisinititermes mandibles differ from all other kalotermitids with projecting mandibles in that the Roisinitermes mandibles arch downward. Although the Roisinitermes imago venation and dentition is very similar to those of Epicalotermes, the soldier of Roisinitermes shares no major characters with the Epicalotermes soldier.Stick-like mandibles unique among all other kalotermitid soldiers. In The genus is named in honor of Dr. Yves Roisin for his many contributions to the study of termites.PageBreakTaxon classificationAnimaliaIsopteraKalotermitidaeScheffrahnsp. n.http://zoobank.org/129573FB-E5DE-4673-9E1B-EF062D413FEBHolotype. Soldier from colony UF no. AFR3327. CAMEROON: Ebogo II, , 647 m elev., 18FEB18, col. Raphael Onana, AFR3327 ca. 500 alates, 50 soldiers, and many pseudergates, nymphs, larvae, and eggs. Paratypes. CAMEROON, Ebogo II , 664 m elev., 10DEC2016, col. Jan Šobotník and collaborators, AFR2982 4 soldiers (1 damaged), one female dealate, and 46 brachypterous nymphs.See generic diagnosis above.Winged Imago  and long (over 3 m) broad-leaf tree branch suspended from the canopy approximately 2 m above the ground. The colony contained roughly 2,000 members. A second colony of R.ebogoensis was collected in a nearly pristine rain forest near the village of Ebogo II. The colony was taken from a dead liana branch (ca. 15 mm diam.) hanging from the canopy at a height of approx. 1 m above the ground. Liana stems have been generally overlooked as a colonization site for Kalotermitidae . In C.cryptognathus from Jamaica, the mandibles are reduced to small stubs that do not project beyond the frontogenal boundaries of the head capsule, and therefore cannot be used to bite opponents  possess long mandibles with robust dentition (crushing mandibles sensuEpicalotermespakistanicus has particularly long and serrated mandibles (Roisinitermes soldiers does not match any of these; instead, Roisinitermes employs a unique strategy of snapping, achieved by long and slender mandibles pressed against each other in a defensive encounter. When this potential energy is released, the left mandible springs over the right and the resultant snap is forced onto the opponent if it is in the path of the strike. This singular mandibular modification was previously known in several lineages of Termitinae (Roisinitermes represents the first undisputable evidence of parallel evolution of snapping soldiers.Kalotermitids inhabit a single woody item and are largely unable to move to a new food source once the original is exhausted. The lone exception is d pieces . The abid pieces  and soldpponents . Some otandibles . The defrmitinae , and wasRoisinitermes on a long branch, next to N.insularis. Neotermesinsularis is a large termite species from Northern Australia with soldiers endowed with biting mandibles of crushing type. The smaller Roisinitermes shares no obvious similarity with N.insularis, supporting its generic status. Currently, the number of mitochondrial genomes available for Kalotermitidae is limited to a handful of genera, and there is a possibility that future phylogenetic analyses will support affinities between Roisinitermes and yet-to-be sampled taxa. In any case, the highly unusual morphology of Roisinitermes suggests that it shares no close relatives among modern Kalotermitidae. Future studies should focus on whether the mechanisms used by soldiers of Roisinitermes to snap are like those of the distantly related Termitinae.Our phylogenetic analyses consistently placed


N), and a binding domain (HC). The HC is responsible for targeting the BoNT to the neuronal cell membrane, and each serotype has evolved to bind via different mechanisms to different target receptors. Most structural characterisations to date have focussed on the first identified subtype within each serotype . Subtype differences within BoNT serotypes can affect intoxication, displaying different botulism symptoms in vivo, and less emphasis has been placed on investigating these variants. This review outlines the receptors for each BoNT serotype and describes the basis for the highly specific targeting of neuronal cell membranes. Understanding receptor binding is of vital importance, not only for the generation of novel therapeutics but also for understanding how best to protect from intoxication.Botulinum neurotoxins (BoNTs) are categorised into immunologically distinct serotypes BoNT/A to /G). Each serotype can also be further divided into subtypes based on differences in amino acid sequence. BoNTs are ~150 kDa proteins comprised of three major functional domains: an N-terminal zinc metalloprotease light chain (LC), a translocation domain (H Clostridium botulinum, under anaerobic conditions . E. EC. botN and LC c 11,12],12C. bot complex ,14.Polysialogangliosides consist of a hydrophilic complex polysaccharide with many sialic acid residues, bound to a hydrophobic ceramide tail. Different forms of these gangliosides can be found embedded in the cell membrane with the various sugar moieties displayed on the cell surface. The most common examples found on neuronal membranes include GT1b, GD1a, GD1b, and GM1. Two types of BoNT protein receptors have been identified to date: three isoforms of synaptic vesicle glycoprotein 2 (SV2A-C) and two isoforms of synaptotagmin (SytI-II). Both types are involved with the regulated secretion of neurotransmitter from synaptic vesicles ,16. SV2AN forms a pore through which a partially unfolded LC passes into the neuronal cytosol . Al. Al54]. C .C domain, BoNT/C1 is still able to bind gangliosides [C domain in complex with sialic acid revealed two potential binding sites that are independent of the GBS identified in other BoNTs [BoNT/C  is predominantly associated with botulism in animals rather than humans ,73. Therliosides . Indeed,liosides . Crystaler BoNTs ,83.Like BoNT/C, there are no subtypes of BoNT/D, of which there are multiple sequences that share a primary sequence identity of >96%. BoNT/D appears to recognise all three isoforms of SV2 ,82. CellThere are currently twelve known BoNT/E subtypes (BoNT/E1 to /E12) whose amino acid identities vary by up to 12% . The proC/A was introduced as a competitor molecule [C domain from BoNT/F1 in complex with GD1a confirmed the existence of a GBS [In addition to BoNT/F1, there are eight other BoNT/F subtypes (BoNT/F2 to /F9) which differ by up to 30% sequence identity . The examolecule , whereasmolecule . For ganor GD1a) ,100. Theof a GBS .C domain was successfully engineered to improve human SytII binding, and a similar approach would be worth investigating here [Only two protein sequences of BoNT/G are currently known to exist, and they share 99.9% amino acid identity. The protein receptor for BoNT/G is either SytI or SytII, although interestingly the interface diverges from BoNT/B and it has a lower binding affinity ,106,107.ing here . BoNT/G ing here . In addiing here ,106, anding here .BoNTs composed of domains from different serotypes also exist in nature. The most common of these chimeric toxins are discussed below.N domain that is most similar to BoNT/C and a HC domain that is most similar to BoNT/D. Interestingly the binding domain of BoNT/CD binds synaptosomes more tightly than BoNT/D [BoNT/CD is a mosaic toxin composed of a LC domain and a Hn BoNT/D . This man BoNT/D . Proteinn BoNT/D .N with 96% sequence identity to BoNT/D and a HC domain similar to that of BoNT/C (74% sequence identity) [The BoNT/DC chimera possesses an LC domain and a Hdentity) ,112. Botdentity) ,112. Desdentity) . The BoNdentity) ,115. Howdentity) ,114.bont sequences placed the gene in a lineage distinct from other serotypes [N domain similar to that of BoNT/F1, and an HC domain similar to that of BoNT/A1 [BoNT/FA was recently identified in 2014 from a case of infant botulism ,117. At erotypes ,119,120. BoNT/A1 . Direct  BoNT/A1 , and cry BoNT/A1 ,123. Alt BoNT/A1 . The BoN BoNT/A1 . The eff BoNT/A1 . UnderstC. botulinum that was already known to express BoNT/B was recently found to contain the gene for another BoNT molecule that shared low primary sequence identity to other serotypes (<30%)—this was named BoNT/X [N and HC domains, and considering the novel characteristics of LC, attempts are underway to determine the specific receptor(s) that it targets and how it functions in vivo. The BoNT/X HC does contain an SxWY sequence motif, indicating that it potentially shares similar ganglioside binding characteristics with other BoNTs. Due to its divergence and low sequence similarity to existing BoNTs, structural and functional characterisation could lead to new insights into receptor binding that could be exploited for future therapeutics.A strain of d BoNT/X . It is ud BoNT/X . This sud BoNT/X . Little Clostridium species. The first was found in 2015 and is referred to as “BoNT/Wo”, named after the bacterium that produced it, Weissella oryzae SG25 [Enterococcus faecium, which is a ubiquitous commensal microorganism commonly found in the gut of mammals. The BoNT-like protein, referred to as BoNT/En or eBoNT/J, possesses many traditional BoNT motifs, including a HExxH zinc-binding motif in the LC and a ganglioside-binding SxWY motif in the HC domain [Considering that BoNTs are the deadliest biological agents that exist, it was surprising to find BoNT-like proteins produced by non-zae SG25 ,128. BoNzae SG25 , but it C domain ,131. EarC domain .bont gene cluster, their ability to be transferred between species, the potential implications for biosafety, and the need for an agreed-upon consistent naming convention to avoid confusion and ambiguity [BoNTs are highly specific and potent exotoxins that are being exploited for therapeutic gain. Our knowledge of the molecular aspects of botulinum neurotoxin, such as mechanism of cell targeting and internalisation, is incomplete and mostly limited to only one or two serotypes . We have yet to fully understand the binding mechanism of others and also how subtle amino acid differences may result in differences of intoxication . From what we know so far, X-ray crystallography has suggested that the mechanism of binding is more complex than was initially thought. It is possible that BoNTs may accommodate heterogeneous glycosylation of their protein receptors and target a variety of gangliosides to ensure successful binding to their target cell type. It is not a trivial task to determine how BoNTs bind to their receptors on neuronal cell membranes, especially when trying to replicate the conditions in vivo. With the recent discovery of new BoNTs and BoNT-like molecules in other bacterial species, this raises questions regarding the evolution of the mbiguity ,133. Fasmbiguity . In summ


In vitro lipid-binding experiments showed that the FARP1 FERM domain binds specifically to several types of phospholipid, which is dependent on the positively charged surface patch. We further determined through cell-based analyses that this surface patch on the FERM domain underlies the localization of FARP1 to the plasma membrane, and that FERM domain interactions recruit it to postsynaptic sites in neurons.FARP1 is a multi-domain protein that is involved in regulating neuronal development through interacting with cell surface proteins such as class A Plexins and SynCAM 1. The N-terminal FERM domain in FARP1 is known to both promote membrane localization and mediate these protein interactions, for which the underlying molecular mechanisms remain unclear. Here we determined the crystal structures of the FERM domain of FARP1 from zebrafish, and those of FARP2 (a close homolog of FARP1) from mouse and zebrafish. These FERM domains adopt the three-leaved clover fold that is typical of all FERM domains. Our structures reveal a positively charged surface patch that is highly conserved in the FERM domain of FARP1 and FARP2.  FARP1 has been shown to interact directly with the neuronal guidance receptors PlexinA1 and PlexinA4 and regulate signaling6. Activation of the Plexin receptor by its semaphorin ligand leads to signaling that drives morphological changes of neuronal axons and dendrites7. FARP1 is enriched in dendrites of lateral motor column neurons, where it serves as an effector of PlexinA4 to promote dendritic growth5. More recently, FARP1 has been shown to regulate synapse number and dendritic spine morphology8. This function of FARP1 is mediated, at least in part, by a direct interaction with the synaptogenic adhesion molecule SynCAM 18. FARP2 also interacts with PlexinA family members, contributing to both Plexin-mediated repulsive axon guidance and dendritic development10. Recently, FARP2 has been found to be involved in Plexin-mediated regulation of bone homeostasis12.FARP1  and its close homolog FARP2 were identified as guanine nucleotide exchange factors (GEFs) for RhoGTPases that play regulatory roles in neuronal development13. Crystal structures of the DH-PH-PH domains of both FARP1 and FARP2 show an autoinhibited conformation where the RhoGTPase-binding site in the DH and the first PH (PH1) domain is blocked by the second PH domain (PH2) as well as several other structural elements in the protein14. Therefore, FARP1 and FARP2 cannot act as GEFs for RhoGTPases unless the autoinhibition is released through a conformational change. Alternatively, it has been suggested that FARP1 and FARP2 may be “pseudo-GEFs”, which have lost their GEF activity but regulate RhoGTPases through an indirect mechanism14.FARP1 and FARP2 share a conserved domain architecture, containing a N-terminal 4.1, ezrin, radixin and moesin (FERM) domain, which is followed by a long linker (~200 residues) that connects to a Dbl-homology (DH) domain and two pleckstrin homology (PH) domains Fig. . The two8. The molecular details of these interactions are unknown due to lack of structural analyses. In addition to membrane proteins, many FERM domains can interact with phospholipids, which targets them to lipid membrane and facilitates their binding to cell surface proteins15. It is unclear whether the FERM domains in FARP1 and FARP2 have a similar ability to bind lipid membranes. To address these questions, we determined the crystal structures of the FERM domain of FARP1 from zebrafish as well as those of FARP2 from mouse and zebrafish. These structures and the associated biochemical and cell-biological analyses provide mechanistic insights into the interactions of these FERM domains with the plasma membrane and cell surface receptors, supporting FERM domain roles in the synaptic recruitment of FARP1 in neurons.The FERM domain in FARP1 mediates interactions with the intracellular sequences of the transmembrane proteins PlexinA4 and SynCAM 115. The F1 subdomain exhibits a ubiquitin-like fold and consists of a five-strand β sheet capped by an α-helix. The structure of the F2 subdomain is constituted entirely with α-helices and structurally similar to acyl-CoA-binding proteins. The F3 subdomain is a β sandwich capped by a C-terminal α-helix, similar to PH and PTB (phospho-tyrosine binding) domains.We determined crystal structures of the FERM domains of zebrafish FARP1 (zfFARP1), mouse FARP2 (mFARP2) and zebrafish FARP2 (zfFARP2) Fig. , charact15. The cytoplasmic tail of cell surface proteins adopts an extended β-strand conformation to pack against β5 in F3 of the FERM domain. The FERM domains in FARP1 and FARP2 have been proposed to use this binding mode to interact with SynCAM 1 and class A Plexins, respectively10. A crystal structure of the C-terminal tail of SynCAM 1 bound to the FERM domain of DAL-1 (PDB ID: 3BIN) shows the detailed interactions in this mode  motif found in cytoplasmic tails of cell surface proteins that bind FERM domains. While the FERM domains in FARP1 and DAL-1 only share ~40% sequence identity, the residues in the binding groove of these two FERM domains are nearly identical as shown by the structural superimposition -trisphosphate (IP3) (PDB ID: 1GC6) shows that the phosphatidylinositol binding site is located at the junction between subdomains F1 and F3, where several positively charged residues interact with the phosphate groups on IP3 , suggesting that it cannot serve as a phosphatidylinositol binding site in these FERM domains  (See methods). These observations suggest that these FERM domains in solution may oligomerize through electrostatic interactions. Interestingly, both the FERM domains of mFARP2 and zfFARP2 form similar head-to-tail packing patterns in the crystal lattices, as a result of interactions between the positively charged and the negatively charged faces of the proteins Fig. . These p2, PIP2, PIP2, PIP3, and Phosphatidylserine (PS), but not Lysophosphatidic Acid (LPA), Lysophosphocholine (LPC), PI, Phosphatidylethanolamine (PE), Phosphatidylcholine (PC), Sphingosine-1-phosphate (S1P), or Phosphatidic Acid (PA) P2, PIP3, and PS. In contrast, the binding to PI3P, PI4P, PI5P and PIP2 was not substantially affected by the mutations. These results together suggest that the FARP1 FERM interacts specifically with certain types of phospholipid, and the KRKR motif is important for some of these interactions. Of particular note, the KRKR motif is essential for binding to PIP2, the most abundant type of phosphatidylinositol on the plasma membrane22.The results showed that the FERM domain of hFARP1 bound robustly to a variety of phospholipids, including phosphatidylinositol (PI)-3-phospate (PI3P), PI4P, PI5P, PIP8. Another study showed that FARP1 was constitutively localized to the plasma membrane23. To further analyze the membrane localization of FARP1 and the role of the FERM domain in this process, we used immunofluorescence to determine the subcellular localization of FLAG-tagged full-length human FARP1 expressed in HeLa cells with an anti-FLAG antibody. Confocal microscopy images showed that full-length FARP1 is localized predominantly on the plasma membrane. In contrast, FARP1 with the FERM domain truncated distributed evenly in the cytosol, suggesting that the FERM domain is essential for the membrane targeting of FARP1. The double mutation (R273E/K274E) of the KRKR motif in the FERM domain also abolished the membrane localization of FARP1  to disrupt FARP1-SynCAM 1 interactions. According to the FERM/SynCAM 1 binding mode discussed above, Trp306 (corresponding to Trp308 in zebrafish FARP1) is important for interacting with the “GXY” motif in SynCAM 1  a pronounced enrichment in dendritic protrusions8. We compared the spine enrichment of wild-type FARP1 and its FERM domain variants by measuring the ratio of GFP signal in spines versus the adjacent dendritic shaft, normalized by the mCherry signal. The W306E mutant showed a 50 ± 5.9% reduction in spine enrichment compared to wild-type FARP1  for protein expression, induced by 0.2 mM IPTG at 16 °C overnight. Bacterial lysates were cleared by centrifugation and the supernatants were passed through a 1 mL HisTrap column  and washed with Buffer A containing 10 mM Tris (pH 8.0), 1 M NaCl, 5% glycerol (v/v), 20 mM Imidazole, and 3 mM β-mercaptoethanol. On-column digestion of the His6-SUMO-tag was carried out by injecting recombinant SUMO protease in Buffer A to the HisTrap column and incubating at 4 °C overnight. The FERM domain proteins were eluted with Buffer A and further purified by a Superdex 200 HiLoad 16/60 gel filtration column  equilibrated with Buffer B containing 20 mM Tris (pH 8.0), 500 mM NaCl, 10% glycerol (v/v), and 2 mM DTT. Purified proteins were concentrated and stored at −80 °C. These FERM domains needed NaCl at 500 mM or higher concentrations to remain soluble. For the hFARP1 FERM domain, the N-terminal His6-SUMO tag was uncleaved and used for Western blotting detection in the protein-lipid overlay assay.The coding regions of the FERM domain of hFARP1 (residues 36–329), zfFARP1 (residues 38–322), mFARP2 (residues 38–324), and zfFARP2 (residues 49–378), were cloned into a modified pET-28(a) vector that encodes an N-terminal His24. The structure of the mFARP2 FERM domain was solved by molecular replacement using the Phaser program in the Phenix package with the structure of the DAL-1 FERM domain (PDB ID: 2HE7) as the search model26. The structure of the mFARP2 FERM was then used as the search model to solve the structures of the FERM domains of zfFARP1 and zfFARP2. Iterative model building and structure refinement were performed by using the programs Coot and Phenix, respectively27. Statistics of data collection and refinement are listed in Table 28 and rendered with ESPript29.Initial crystallization trials were set up in sitting-drop 96-well plates at the 1:1 volume ratio between the protein and crystallization solutions, at 4 °C for the zfFARP1 FERM domain and at 20 °C for the FERM domains of mFARP2 and zfFARP2. High quality crystals of the mFARP2 FERM for X-ray data collection were obtained in 0.1 M sodium acetate trihydrate pH 7.0 and 12% w/v Polyethylene glycol (PEG) 3350 (w/v). Crystals of the zfFARP2 FERM domain were grown in 0.2 M potassium sodium tartrate tetrahydrate and 20% PEG3350 (w/v). The zfFARP1 FERM domain was crystallized in 0.1 M Bicine pH 9.0 and 16% (+/−)-2-Methyl-2,4-pentanediol (MPD) (v/v). Crystals were cryo-protected using the crystallization solution supplemented with 20–30% glycerol and flash cooled in liquid nitrogen. Diffraction data were collected at −173 °C on beamline 19ID at the Advanced Photon Source . Data were indexed, integrated and scaled by using HKL200030.HeLa cells purchased from American Type Culture Collection were cultured in DMEM supplemented with 10% fetal bovine serum (HyClone) and 1x penicillin/streptomycin solution. Cells were plated on 8-well Lab-Tek chambered cover glass (Nunc) at a low density the day before transfection. 100 ng of C-terminal Flag-tagged hFARP1 WT, FERM deleted, and R273E/K274E in the pCDNA3.1(+) vector (Invitrogen) were transfected to HeLa cells using Fugene HD (Promega). 12–14 hrs after transfection, cells were fixed using 4% formaldehyde (Thermo scientific), immunostained with an anti-FLAG primary antibody  and an Alexa-488 conjugated secondary antibody . Cells were washed with PBS and imaged in PBS at room temperature with a 60x objective on a spinning-disc confocal system built around a Ti-E Perfect Focus microscope (Nikon) with an EM camera  controlled by Micro-Manager software21 was used to examine the interaction between the hFARP1 FERM (D212N and D212N/R273E/K274E) and phospholipids. The N-terminal His6-SUMO tag in the proteins were used for detection by an anti-His6 tag antibody  and an HRP-conjugated secondary antibody . PIP strips (P-6001) were purchased from Echelon.The protein-lipid overlay assay as described previouslyet al., 2007) and scanned with a FluorChem M Imaging System . Primary antibodies used for immunoblotting were an anti-FARP1 antibody raised in guinea pig against the C-terminal peptide8 and a monoclonal antibody against GAPDH .Proteins from cell lysates were subjected to immunoblotting using standard procedures . In brief, dissected hippocampi were incubated in 0.05% trypsin at 37 °C for 20 minutes and plated at a density of 60,000 cells per 12 mm coverslip coated with poly-l-lysine (Sigma P1274). Cells were incubated in a cell culture incubator maintained at 37 °C with 5.0% CO2. Cytosine arabinoside  was added at a final concentration of 2 μM at 2 days in vitro (DIV) to prevent glia cell overgrowth before the medium was replaced with Neurobasal without Ara-c at 4 days in vitro (div). Neurons were transfected at 7 div using Lipofectamine LTX and Plus Reagent (ThermoFisher). For neuronal transfections, the FARP1 cDNA was subcloned into a pCAG-BGH-pA and tagged with N-terminal GFP tag. The W306E mutation was introduced by PCR mutagenesis and the FARP1 ΔFERM mutant generated by PCR amplification of an open reading frame beginning at amino acid 441 in human FARP1. The mCherry vector has been previously described . At 21 div coverslips with neurons were quickly washed two times with PBS, followed by fixation for 15 min with 4% PFA/4% sucrose in PBS at RT, washed 3 times with PBS, and mounted on slides Aqua-Mount mounting media (Thermo Scientific). Dendrites were imaged on a Leica SPE confocal microscope using a 63X objective with a 5x digital zoom. Z-stacks were collected at interval of 0.33 μm and maximum intensity projections analyzed. Images were analyzed with the experimenter blinded to the condition and fluorescence intensity was measured using ImageJ along line scans from dendritic protrusions into the adjacent dendritic shaft. Spine enrichment of GFP-tagged FARP1 and mutant constructs was calculated as fluorescence intensity of (GFP/mCherry) spine head/(GFP/mCherry) dendritic shaft and normalized to the wild-type condition.Dissociated hippocampal cultures were prepared from rats at E18 as previously described (Salzberg The atomic coordinates and structure factors for the FERM domains of zfFARP1, mFARP2, and zfFARP2 have been deposited to the protein data bank, with the PDB access codes of 6D2Q, 6D2K, and 6D21, respectively.Supplementary Information


Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight of rice, one of the most devastating bacterial diseases of this staple crop worldwide. Xoo produces a range of virulence-related factors to facilitate its pathogenesis in rice, however, the regulatory mechanisms of Xoo virulence expression have been not fully elucidated. Recent studies have revealed that virulence factor production is regulated via cyclic dimeric guanosine monophosphate (c-di-GMP) signaling pathway that is well-conserved in Xoo and other Xanthomonas species. A set of GGDEF, EAL, HD-GYP, and PilZ domain proteins with diverse signal sensory domains for c-di-GMP synthesis, hydrolysis, and binding is encoded in the Xoo genome. Bioinformatic, genetic, and biochemical analysis has identified an array of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), as well as degenerate GGDEF/EAL, PilZ domain proteins along with a transcription regulator. These signaling components have been characterized to regulate various bacterial cellular processes, such as virulence, exopolysaccharide (EPS) production, biofilm formation, motility, and adaptation at the transcriptional, post-translational, and protein-protein interaction levels. This review summarized the recent progress in understanding the importance and complexity of c-di-GMP signaling in regulating bacterial virulence expression, highlighting the identified key signal elements and orthologs found in Xanthomonads, discussing the diverse functions of GGDEF/EAL/HD-GYP domains, existence of a complicated multifactorial network between DGCs, PDEs, and effectors, and further exploration of the new c-di-GMP receptor domains. These findings and knowledge lay the groundwork for future experimentation to further elucidate c-di-GMP regulatory circuits involved in regulation of bacterial pathogenesis. Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight disease of rice, one of the most devastating bacterial diseases of this staple crop in the world, leading to yield losses of rice production up to 20–50% under disease-favorable conditions  to inject and deliver effectors into rice cells, some of which are critical determinants of pathogenesis during Xoo–rice interaction  is a widespread second messenger in pathogenic bacteria Hengge, . The impc-di-GMP binding promotes changes in the structure, as well as the function of the receptor/effector. This interaction allows changes in gene expression or enzyme activity to occur , X. campestris pv. campestris (Xcc), and X. axonopodis pv. citri (Xac), highlighting the identified key signal elements and orthologs, discussing diverse functions of GGDEF/EAL/HD-GYP domains, existence of a complicated multifactorial network between DGCs, PDEs and effectors, and further exploration of new c-di-GMP receptor domains. These findings and knowledge lay the groundwork for future experimentation to further elucidate c-di-GMP regulatory circuits involved in the regulation of bacterial pathogenesis.Due to the economic importance of bacterial blight of rice and the excellent model of bacterial pathogens in monocot plants, recent studies on Xoo PXO99A, 26 genes were annotated to encode GGDEF, EAL, and HD-GYP domain proteins revealed by bioinformatic analysis, including 11 GGDEF domain standalone proteins (GdpX1-11), 2 EAL domain standalone protein (EdpX1-2), 10 GGDEF/EAL domain proteins (GEdpX1-10), and 3 HD-GYP domain proteins (HGdpX1-3) (pdeR), and PXO_01021 was identified in the genome of Xoo strain PXO99A   (Yang etGdpX1-3) . A 212-kordingly .GGDEF domains are differentiated into three classes: enzymatically functional domains, enzymatically functional domains linked to an EAL domain, and enzymatically non-functional domains based on the domain homology and the signature/motif conservation , GAF, HAMP, Cache_1, CHASE3, Reg_prop- and Y_Y_Y, MHYT, and DUF domains ; Table 2gdpX1 resulted in increased virulence, EPS production, and flagellar motility, while overexpression of GdpX1 in the wildtype led to attenuated virulence and decreased EPS synthesis. Moreover, GdpX1 suppressed the transcription of genes involved in type III secretion system (T3SS) activation, EPS synthesis, and flagellar motility. These results imply that GdpX1 is a putative DGC and suppresses bacterial virulence traits in this pathogen (Xoo.GdpX1 (PXO_00649) encodes a well-conserved GGDEF domain standalone protein that was predicted as a putative DGC . Its regpathogen  (Yang etpathogen . FurtherXoo strain KACC10331 encodes a well conserved GGDEF domain protein with 99.6% sequence identity to GdpX11 (PXO_04275) from PXO99A. The DgcA's DGC activity was first characterized in the study of a c-di-GMP riboswitch  from Figures 1, 3). Genetic assays indicated that deletion of edpX1 led to increased intracellular c-di-GMP levels, which can be restored to wildtype levels via complementation of edpX1. In vitro colorimetric assays showed EdpX1 displayed PDE activity for c-di-GMP degradation, and a point mutation of E153A at the EAL motif strongly reduced its activity. ΔedpX1 exhibited a severely impaired virulence phenotype in rice, EPS production, and biofilm formation. In trans expression of wildtype edpX1, but not edpX1E153A, was able to restore these phenotypes to near wild type levels. These observations indicate that EdpX1 functions as an active c-di-GMP-specific PDE and promotes virulence, EPS production, and biofilm formation (EdpX1 (PXO_03877), encoding a well conserved EAL domain standalone protein, was annotated as a PDE /RavR (XCC1958), interplayed with the DSF-mediated quorum sensing (QS), and regulated EPS and extracellular protease secretion, and their homologs were well-conserved in other close species and pathovars Xoo PXO99A, PXO_01020, and PXO_01019 (PdeR) were the homologs of RavS and RavR, respectively. PdeR, a GGDEF/EAL/REC-domain containing protein was identified as a RR to form a pair of TCS with the cognate histidine kinase (HK) PXO_01018 (PdeK), but not PXO_01020 , a putative pression   via its CHASE domain. Through a four-step phosphorelay, 2iP binding results in decreased autokinase activity of PcrK and phosphorylation level of PcrR, thereby activating PcrR's PDE activity for c-di-GMP degradation. The TCS PcrK/PcrR affects bacterial tolerance to oxidative stress through regulating the gene transcription. These findings reveal an inter-kingdom signaling by which Xcc intercepts a plant derived hormone signal to promote adaptation to oxidative stress  was identified as a FimX-like, degenerate GGDEF/EAL domain hybrid protein, which showed 96.8 and 93.8% protein sequence identifies to FimXXac2398 and XccFimX, respectively . Deletion of pilZX3 led to the same changes in bacterial virulence and T3SS gene expression as the Δfilp mutant. This indicates that Filp is a novel c-di-GMP receptor to co-regulate bacterial virulence with its interactor PilZX3  containing the cNMP-/HTH-DNA binding domain was identified as a c-di-GMP receptor and transcription factor   screen identified the c-di-GMP receptor MshE, an ATPase associated with the mannose sensitive hemagglutinin (MSHA) T4P in Vibrio cholerae, meanwhile an ATPase of type II secretion system (T2SS) in Pseudomonas aeruginosa, the homolog of MshE was also identified as a signal receptor, indicating both T4P and T2SS are regulated by c-di-GMP signaling , PXO_03396 (a T3SS protein HrcN), and PXO_06188 (a flagellar protein FliI) might be the new c-di-GMP signal receptors. Moreover, diverse proteins and riboswitches were identified as novel c-di-GMP effectors in other pathogenic bacteria, for instance, FleQ, a transcription regulator from P. aeruginosa, and SgmT, a HK from Myxococcus xanthus  characterize the functions of the remaining GGDEF, EAL, and HD-GYP domain proteins, (2) determine the environmental cues that affect the DGC and PDE activities and intracellular c-di-GMP levels through the sensory input domains, (3) characterize the c-di-GMP receptor-mediated regulatory pathways via the transcription and post-translation mechanisms, (4) assess c-di-GMP regulation of bacterial type III secretion system (T3SS), and (5) uncover the specificity in c-di-GMP signaling through protein-protein interaction mechanisms.FY and DX performed experiments. FY, DX, FT, WH, C-HY, and CH analyzed data and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The aim of this study was to evaluate the baking value of rye flours from industrial mills and to indicate which rye flour quality parameters are the most important predictors of wholemeal rye bread quality for commercially milled rye grains. Ten wholemeal rye flours, which were characterized by ash content ranging from 1.43% to 2.42% d.m. (dry mass), were used for the study. The parameters that characterize the flour properties and the baking test were assessed. The study revealed that for the analyzed commercial rye flours, the falling number test and the amylograph properties are insufficient parameters for predicting the quality of wholemeal rye bread. The manufacture of good quality wholemeal bread requires the use of rye flour with superior quality, such as fine granulation, low protein content, low total and insoluble pentosans content, and, in particular, a high percentage of water-soluble pentosans content. Breads with a higher volume were obtained from rye flours that were generally characterized by lower protein content, lower total and insoluble pentosans content, and higher water-soluble pentosans content. Flour granulation and the percentage of water-soluble pentosans content especially, had a significant impact on bread’s hardness of crumb and the hardness of crumb’s increase during bread storage.  Secale cereale L.) is a traditional raw material used for the production of bread in Northern and Eastern Europe. Poland, next to Russia, Germany, Ukraine, and Belarus, is a major producer of rye grain [Rye , which were characterized by ash content ranging from 1.43% to 2.42% d.m., were used in this study. The rye flours were produced in different commercial mills located in Poland. The following quality parameters of the tested samples of rye flours were assessed: Protein content (PC) according to ISO 20483:2013 , ash conThe SC test was performed by preparing a suspension from 120 g flour , 364 mL of water, and 46 mL of buffer (sodium phosphate buffer solution pH = 5.0), and that suspension was tested in an amylograph  under constant stirring in the temperature range 30 °C to 42 °C. The slurry was then held at 42 °C for 0.5 h . The det3 of water (temperature in the range of 35–36 °C) to obtain a homogeneous dough with soft consistency. SO fermentation was performed for 20 h at 30 °C and 75% RH. The final step was as follows: The mature SO, 650 g of rye four, 10 g of yeast, 17 g of salt, with the water equivalent to farinograph absorption at 300 FU, decreased in absorption by the water added during preparation of the SO. The dough was prepared in the spiral mixer  at a low speed to obtain a homogeneous mass (for approximately 10 min). Dough temperature after mixing was in the range of 30–32 °C. The dough was placed, for 10 min, in the fermentation cabinet. After fermentation, the dough was divided into pieces  and manually molded. The formed pieces were placed in tins and proofed in at 75% relative humidity and 35 °C until the dough surface reached the top of the tin. Baking was performed in a decking oven Piccolo Wachtel Winkel  with steaming at the beginning of the baking (approximately 10 s). During the first 10 min of baking, the temperature was 260 °C, and the temperature was then decreased to 220 °C. After 40 min of baking, the breads were cooled at room temperature, and stored in polyethylene bags.Rye breads were produced in the laboratory baking trials; bread was made with sourdough (SO). Initially, a part of flour was soured in a one-stage process. SO with a yield of 250% was prepared with 350 g of each rye flour (adjusted to standard moisture of 14%), 3.5 g of starter , and 525Rye bread quality was assessed using the following parameters: Bread yield, bread volume, bread crumb hardness, and crumb moisture. Bread property evaluation was performed 20 ± 4 h after baking. The crumb hardness was also determined four days after baking. The bread yield (YB), (the amount of bread obtained from 100 g of flour) was calculated according to .3/100 g of bread. The bread crumb hardness was determined using the texture analyzer Instron 1140  according to the methodology described by Stępniewska et al. [The loaf volume was measured using the millet seed displacement method, and the specific bread volume was expressed as cma et al. . The moiOne-way analysis of variance (ANOVA) was performed, and the homogenous groups were determined by Tukey’s test. Pearson’s correlation coefficients were calculated (significance level of α was established at 0.05). Principal component analysis (PCA) was also performed. For a better overview, the PCA was performed as in . MoreoveThe rye flour samples significantly differed according to the PC and AC . The PC Granulation is a quality parameter that significantly affects the dough behavior during fermentation. On the one hand, fermentation of dough with a finer flour is more intense, which results from easier access of enzymes to starch. On the other hand, a flour with very fine granulation may have higher buffering properties, which is associated with a higher content of soluble ingredients; that poses a risk of excessive degradation of substrates responsible for creating the dough and bread structure . The ryeThe rye flour samples differed significantly in terms of water absorption, which was in the range of 54.0% (RF1) to 66.6% (RF10) . BecauseThe rye flour samples significantly varied with regard to α-amylase activity and starch properties, such as FN, APV, and FT . These pThe results of pentosans content in the tested rye flour samples are presented in r = 0.893, r = −0.638, r = 0.639, p < 0.05, r = 0.768, r = 0.864, respectively, The total pentosans content (TPC) ranged from 8.3% d.m. (RF9) to 13.4% d.m. (RF10). Generally, rye flours with higher TPC were also characterized by a significantly higher WA. The correlation coefficient between TPC and WA was significant, and equaled 0.868 . Hansen r = 0.673, r = 0.685, respectively, 2 by the dough and has positive results for bread volume. However, excessive depolymerization of pentosans decreases this ability, due to low viscosity and thus has a negative effect on bread properties [r = 0.769, The results of the swelling curve test are presented in operties . The valoperties . It was 3/100 g of bread (bread from flour RF10) to 191 cm3/100 g of bread  were found. No significant correlation was found between parameters that describe the α-amylase activity and the starch properties, such as FN, APV, and FT (The specific bread volume (SBV) ranged from 146 cm= −0.689 . Further, and FT .r = 0.790, r = 0.713, and r = 0.758, respectively), and a significant negative correlation with Gr  ranged from 140% (bread from the flour RF2) to 156% (bread from the flour RF8). The YB was positively correlated with all viscosity parameters of the swelling curve test. Statistical analyses also showed a significant positive correlation between YB and AC, WA and WIP (r = 0.643 (The crumb hardness is an important bread texture parameter. The lowest bread crumb hardness after one (BCH1) and four days’ (BCH4) storage of bread was observed in bread obtained from the flour RF4 , whereas the highest values of these parameters were noted in bread from flour RF8 . The increase in bread crumb hardness during storage of bread (IBCH) ranged from 14.2 N (bread from the flour RF4) to 63.1 N (bread from the flour RF2). No significant correlations were observed between BCH1, BCH4, and IBCH in the parameters that describe the α-amylase activity and starch properties, such as FN, APV, and FT. In the present study, bread from the flour RF3 had significantly higher values of FN, APV, and FT than the bread from the flour RF5 but had similar levels of BCH1, BCH4, and IBCH to the bread from the flour RF5. It was also noted that bread from rye flour RF6, which had optimal values of FN and FT, showed crumb characterized by one of the highest values of BCH1, BCH4, and IBCH. This finding indicates that for commercial rye flour, the FN and the amylograph properties are not sufficient parameters for predicting bread hardness and its increase during storage of bread. Further, it was observed that BCH1, BCH4, and IBCH were significantly and negatively correlated only with Gr and %WSP . General = 0.643 . No corrThe results of PCA shoved that only three out of nine principal components were found to be important, as their values were larger than 1, which is usually considered as the criterion for significance. The first principal component (PC1) accounted for 47.09% of the variation and was the most related to AC, TPC, and WIP; all viscosities obtained from the swelling curve: AV, BV, and CV; and WA. PC1 was negatively correlated with Gr and SBV. The second component (PC2) explained 26% of the variation and was strongly positively related to the %WSP and WSP and negatively related to bread hardness and increase in hardness during storage of bread. The tested industrial wholemeal rye flours were significantly different in terms of all parameters that characterize their baking value. Many significant relationships were found between flour and bread properties.The study revealed that for commercial rye flours, the falling number and the amylograph properties are not sufficient parameters for predicting the quality of wholemeal rye bread. To produce wholemeal rye bread with high volume, low crumb hardness, and a small increase in hardness during storage, it is recommend to use rye flour with fine granulation, low protein content, low total and water-insoluble pentosans content, and a high percentage of water-soluble pentosans content.Bread with a higher volume was generally obtained from rye flour characterized by lower protein content, lower total pentosans content and water-insoluble pentosans content, and a higher percentage of water-soluble pentosans content. Further, flour granulation and the percentage of water-soluble pentosans content had a significant impact on bread crumb hardness, and its increase during storage of bread. Generally, breads with lower crumb hardness were obtained from flours with finer granulation and with a higher percentage of water-soluble pentosans content. Moreover, a positive correlation was found between crumb moisture and water-soluble pentosans content.


Background: Although domain IV of annexin A5 (anxA5) may be less effective in binding phosphatidylserine (PS), the four domains together may guarantee the maximum binding of anxA5 to the PS membrane. Additionally, previous research has shown that annexin mutants lacking one or more domain(s) have different biological activities compared to the wild-type. The present research mainly aims to study the role of domain IV in the crucial PS-binding function of anxA5. Methods: The domain IV-truncated anxA5 protein was constructed and purified. Isothermal titration calorimetry, flow cytometry and activated partial thromboplastin time were adopted to examine the function of domain IV in anxA5-PS binding directly or indirectly. Results: The domain IV-truncated form of anxA5 is impaired in binding PS liposome and apoptotic cells, and anticoagulation activity. The mutant cannot bind calcium, but binds PS only in the presence of calcium. Conclusions: Truncation of domain IV of anxA5 destroys its calcium-binding ability and impairs its PS-binding activity. Truncation of domain IV may induce conformation change of anxA5 or reduce the hydrophobic interactions between protein and membrane, which may explain the decrease of PS-binding affinity of the mutant. The annexins constitute a multi-member family characterized by the ability to bind acidic phospholipids in a calcium-dependent manner ,2. Each Among all members, human annexin A5 (anxA5) deserves special attention, as it is widely applied in apoptosis detection both in vitro and in vivo, owing to its high affinity in binding phosphatidylserine (PS) exposed on apoptotic cells ,7,8. AddPrevious studies have shown that domain I may play a prominent role in the PS-binding process, while domain III and IV have a limited effect ,21,22. AA mutant of annexin A2 (anxA2) lacking domain IV was observed to show a significant increase in binding of the CARDS (community-acquired respiratory distress syndrome) toxin compared to the wild-type . AnnexinIn this work, we aim to examine whether the deletion of domain IV will affect the functions, especially the central PS-binding function, of anxA5 through the in vitro activity assay of purified proteins. Together with previous research, this work may reveal the role of domain IV in the structure or function of anxA5 from a new viewpoint.Taking advantage of the His-tag in fusion proteins , EwtA5 b. The wtAs shown in As shown in To conclude, the proteins were successfully purified, and could be applied for further investigation.Isothermal titration calorimetry (ITC) is a useful, label-free technique for characterizing the interaction mechanism of binding reactions from the perspective of thermodynamics. Since the interaction between anxA5 and PS is calcium-dependent, the binding between calcium and wtA5 or ctA5 was first measured using ITC.D = 267 μM, while there is no obvious binding reaction between ctA5 and calcium  and wtA5 or ctA5 protein, examining whether truncation of domain IV would affect the binding between them. There was an obvious change in heat during liposome titration in the presence of calcium , but notectively , indicatp ≤ 0.01, n = 3) revealed significant differences between the ratios. However, there was no significant difference (p = 0.1268) between the ratios of PI (propidium iodide)-positive cells. Thus, the differences in the distribution ratios of cells in AnxA5 proteins are widely applied in the field of apoptosis detection, utilizing its high affinity for PS expressed on apoptotic cells. To investigate the role of domain IV, a flow cytometry (FCM)-based apoptosis detection assay was used to estimate and compare the ability of EwtA5 and EctA5 proteins to label apoptotic cells. The EctA5-labeled cells showed a different distribution ratio compared with that of EwtA5 a,b. StatThe above FCM analysis indicates that truncation of domain IV impairs the ability of anxA5 to label apoptotic cells. 8 and (1.48 ± 0.04) × 108 U/mole respectively  lower than that of wtA5, indicating that domain IV is important for the anticoagulation activity of anxA5.AnxA5 protein has potent anticoagulation activity resulting from its PS-binding ability, blocking the availability of PS for coagulation reactions . An APTTectively . The antThe experiments above reveal the role of domain IV of anxA5 from different perspectives, including the binding of PS liposome and apoptotic cells, and its anticoagulation activity. The apoptosis detection application and anticoagulation activity of anxA5 are basically reflections of its PS-binding activity. Interestingly, ITC analysis showed that the domain IV-truncated form of the anxA5 protein does not bind calcium, but binds to PS liposome only in the presence of calcium. Together with the ITC experiments, the FCM analysis and APTT assay illustrate that, in nature, domain IV of anxA5 is essential for achieving its maximum PS binding. Additionally, domain IV is critical for anxA5 binding of calcium. Previous study has shown that a natural variant of anxA4, losing the majority of domain III and all of domain IV, binds to PS liposome in a calcium-independent way, which is distinct from the full length of anxA4 . FunctioIndividual domains and integral proteins may exhibit a broad range of diversity and specificity in both structure and function , which mConsidering the research above, the reasons why truncation of domain IV led to the decrease of anxA5-PS binding affinity may be explained in terms of two aspects. Firstly, the anxA5 mutant lacking domain IV may undergo conformation change, leading to a loss of calcium-binding ability and a decrease in PS-binding affinity. Secondly, domain IV may contribute to anxA5-PS binding via hydrophobic interaction with the membrane, which could explain why there is an obvious decrease in entropy change (TΔS) during the ctA5-PS interaction.Subsequent research could be conducted to study similar mutants lacking one or more domains, as well as investigating the domains individually, to further reveal the four domains from the perspective of their uniqueness, not just being repeats of annexin. 6-EGFP-(GS)4 (pHEGS). The fragments of wild-type anxA5 (wtA5) and its mutant (ctA5) lacking domain IV (the last 66 residues) were amplified using the primers indicated in The EGFP fragments were amplified with primers e-F and e-R1, the product of which was again amplified using primer pairs e-F and e-R2 . The rec6-CBD-Intein (pHI). Subsequently, the wtA5 and ctA5 fragments were inserted into pHI to construct intein-wtA5 (IwtA5) and intein-ctA5 (IctA5) expression vectors. The intein fused vectors were designed to obtain non-fusion proteins wtA5 and ctA5 after self-cleavage of intein, as described below. The non-fusion proteins were designed for isothermal titration calorimetry analysis and anticoagulation assay.The CBD-SspDnaB intein , wtA5 and ctA5 fragments were amplified using the primers indicated in The recognition sites of restriction enzymes used are shown in bold and underlined. All primers listed were ordered from GenScript , and plasmids were sent to GenScript for sequencing.E. coli BL21 (DE3) and induced to express protein as previously reported [The above expression vectors were transformed into reported . The EGFreported . BrieflyAfter quantification by bicinchoninic acid (BCA) staining, the purities of the proteins were analyzed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and stained with Coomassie brilliant blue.2 in HBS) was injected to the protein solution (60 μM protein in HBS) at 15 °C [The isothermal titration calorimetry (ITC) analyses of non-fusion wtA5 and ctA5 proteins were performed on a MicroCal iTC200 . For calcium titration, calcium chloride solution (7 mM CaClat 15 °C . For lipat 15 °C . The proat 15 °C . After tJurkat cells were induced to undergo apoptosis by addition of etoposide (25 μM), and apoptosis detection was carried out as previously described . The apo2  was added to the cup, after which the clotting time was recorded. APTT results are expressed as U = (sample clotting time-buffer clotting time)/(buffer clotting time × protein molar number).Activated partial thromboplastin time (APTT) was determined using a Thrombotimer  with an assay kit from Sunbio  according to the manufacturer’s instructions. The goat plasma (50 μL) was mixed with purified non-fusion proteins or buffer (50 μL) and APTT reagent (50 μL) in a coagulation cup, followed by incubation for 5 min at 37 °C. Then, CaClBased on previous investigations on anxA5 and other annexins, we successfully designed and purified domain IV-truncated anxA5 proteins. Through ITC, FCM, and APTT assays, we estimated the role of domain IV in anxA5-PS binding, either directly or indirectly. The results showed that domain IV is critical for anxA5 in binding calcium, and maximizes the binding between anxA5 and the PS membrane. Considering previous reports, domain IV may contribute to the interaction between anxA5 and PS through the influence of protein conformation, or a hydrophobic interaction between them. This work characterizes the role of domain IV in anxA5-PS binding, which is expected to help further define the relationship between the structures and functions of annexin members.