Enterococcus faecalis is a common cause of nosocomial infections worldwide. The increasing prevalence of multi-antibiotic resistant E. faecalis strains reinforces this public health concern. Despite numerous studies highlighting several pathology-related genetic traits, the molecular mechanisms of E. faecalis virulence remain poorly understood. In this work, we studied 23 bacterial proteins that could be considered as virulence factors or involved in the Enterococcus interaction with the host. We systematically tested their interactions with human proteins using the Human ORFeome library, a set of 12,212 human ORFs, in yeast. Among the thousands of tested interactions, one involving the E. faecalis virulence factor ElrA and the human protein FHL2 was evidenced by yeast two-hybrid and biochemically confirmed. Further molecular characterizations allowed defining an FHL2-interacting domain (FID) of ElrA. Deletion of the FID led to an attenuated in vivo phenotype of the mutated strain clearly indicating that this interaction is likely to contribute to the multifactorial virulence of this opportunistic pathogen. Altogether, our results show that FHL2 is the first host cellular protein directly targeted by an E. faecalis virulence factor and that this interaction is involved in Enterococcus pathogenicity.The commensal bacterium  Enterococcus faecalis belongs to the human core intestinal microbiota, i.e the limited number of species that are shared by more than 50% of the population1. Although it is a common inhabitant of our intestinal tract, E. faecalis is also a major opportunistic pathogen for critically ill or immuno-compromised patients3. Indeed, E. faecalis is ranked among the most common nosocomial pathogens worldwide infecting the bloodstream and urinary tract. Understanding the molecular mechanisms of E. faecalis pathogenicity is therefore crucial to design new therapeutic approaches to treat enterococcal infections even when strains resistant to antibiotics are involved. E. faecalis does not harbour virulence factors such as effectors and toxins identified in Gram-positive pathogens. Instead, the pathogenicity of E. faecalis relies on a multifactorial process that involves biofilm formation, antibiotic5 and stress resistances8, adhesion to host cells and intracellular survival3.E. faecalis surface exposed proteins involved in different processes are likely to contribute to bacterial pathogenicity e.g., adhesion to host cells, host cell invasion and bacterial protection against host defenses. Except for the enterococcal fibronectin-binding protein A (EfbA)9, most of these surface exposed proteins possess a surface-exposure motif (anchoring or retention motif) such as the WxL domain like of the enterococcal leucine-rich protein A (ElrA)10 or a LPxTG-anchored domain as for the aggregation substance (AS) proteins11, adhesin to collagen (Ace)12, Ebp pilus14, the FSS15, and PrgB16 proteins.Several E. faecalis surface exposed proteins were reported as targeting different host extracellular matrix proteins. Among the members of the AS (aggregation substances) proteins, Asa1 interacts with intestinal epithelial cell lines, macrophages and neutrophils via its binding to CD11b/CD18 integrin, fibronectin and thrombospondin18. Ace is characterized by adjacent immunoglobulin-like folds that define the family of Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs) and it interacts with collagen type I and laminin19. The anchorless EfbA protein binds fibronectin9. Finally, Ebp pili adhere to fibrinogen, collagen, and to platelets20. Overall, these proteins or complexes seem to have multifunctional roles, suggesting that they may interact with different host-cell unknown components through different functional domains.These E. faecalis to interact with its host, we used a systematic high-throughput yeast two-hybrid (HT-Y2H) approach to identify protein interactions between selected surface protein of this bacterium and the human proteome. Such screening approaches were successfully used with pathogenic microbes, including bacteria such as Brucella21 or Pseudomonas syringae22 and viruses such as Epstein-Barr23, Hepatitis C24, HTLVs25, or dengue fever virus26. Here, we report the systematic screening for interactions between 23 selected E. faecalis proteins and the human ORFeome v3.1 (hORFeome), and the identification of a specific interaction between the E. faecalis ElrA and the human FHL2, a member of the Four-and-a-Half LIM Domains Protein family. Disruption of this interaction led to an impairment of the in vivo pathogenic phenotype strongly suggesting a role of this newly identified interaction in the E. faecalis-host interplay.To extend our knowledge on the mechanisms developed by E. faecalis and that could promote interaction with the host namely proteins harbouring a putative surface-exposure motif (anchoring or retention motif) and/or a eukaryotic-like domain and proteins already described as involved in E. faecalis virulence , 1 containing a WxL retention domain (ElrA) and 1 anchorless protein (EfbA). It is well established that cell surface anchored proteins are involved in bacterial-host interactions and that in pathogens, they are often required for the first steps of infection by promoting the interaction with the host targeted cells29. Moreover, these surface exposed virulence factors have either prokaryotic or eukaryotic-like domains, which can be involved in interaction with the host components  the Glucan binding domain and Antigen_C , (ii) a collagen- or fibronectin-binding domain  and (iii) the Leucine rich repeat (LRR) domain (ElrA and EF2250) interaction. Regarding the eukaryotic-associated motif, we found the von Willebrand factor type A (vWA) domain that was originally found in the blood coagulation protein von Willebrand factor (vWF), a signature of adhesion in eukaryotes present in EbsG and EbpA, two Ig-like fold domains (i) the bacterial immunoglobulin-like BIG3 domain detected in EF2505 and (ii) the Cna_B repeat detected in 6 E. faecalis proteins . Note that the Fss proteins 1 to 3 have been shown to bind fibrinogen15. Three proteins exhibiting the ankyrin repeat, fibronectin binding or collagen eukaryotic-like domains were also selected. EF0377 displays 4 ankyrin domains. The ankyrin domains have been shown to promote specific interactions with host proteins leading to the impairment of the targeted molecule activity. Many examples demonstrated the role of ankyrin repeat bacteria effectors in the hijacking of eukaryotic functions by the pathogen during the infection process32. EF0286 and EF1249 have a fibronectin-binding domain  prior to interactomic assays. Each selected E. faecalis ORFs was tested with the human ORFeome v3.1 containing 12,212 human ORFs36. Among the 280,876 tested interactions, a single protein out of the 23 tested gave positive hits. The candidates interacting pairs of proteins were identified, revealing ElrA10, interacted with 3 human ORFs: FHL2 (31042@A08), GOPC (11063@H10) and CDC23 (11088@H06). The interactions were individually validated by mating the yeast strains carrying ElrA fused to the Gal4 DNA binding domain (BD) and the human ORFs flanked by the Gal4 Activating Domain (AD) and growing them on medium selecting for strong interactions. The interaction of ElrA with FHL2 was the only one confirmed. FHL2 is a member of the four-and-a-half-LIM protein family, known to participate in many cellular processes37 and including FHL1, FHL2, FHL3, FHL4 and FHL5/ACT in humans. The LIM domain is a highly conserved double zinc-finger motif38.The interactions between the 23 selected The hORFeome contains 5 different FHL sequences: FHL1 843 ncl (11050@D04), FHL2 840 ncl (31042@A08), FHL3 843 ncl (11031@C01), FHL3 519 ncl (11018@E03) a truncated variant of FHL3 and FHL5 855 ncl (11050@F02). In the same experimental set-up we tested the individual interaction of each clone with the ElrA protein. In stringent selective conditions, the interaction was only detected for FHL2 , the cap domain (aa 61 to 141), the Leucine Rich Repeat domain (aa 142 to 387), the Immunoglobulin-like domain (aa 388 to 473) and the WxL domain (aa 474 to 723). The 21 ElrA-fragments resulting from the amplifications were fused to the Gal4-DB in the pDestDB-GW vector  of ElrA  were amplified Fig.  and clonlrA Fig. . Interes27–723 i.e., the full-length protein without its signal-peptide, (ii) ElrA27–473 i.e., the N-terminal part of ElrA without the FID and the WxL domains and (iii) ElrA474–606 corresponding to the FID were tested with a 3Flag tagged FHL2 in a pull-down assay  ElrAsay Fig. . As expeved Fig. .Figure 427–723  or ElrA27–473 fragments . By confocal microscopy, a clear co-localisation was observed between FHL2 and the wild-type ElrA27–723 fusion protein, but not with the FID-truncated ElrA27–473 fragment. These data are consistent with an interaction of FHL2 with ElrA through the C-terminal part of ElrA in a cellular context  and systemic infection (SI) models. We constructed and tested two independent isogenic elrA mutants deleted in-frame of residues 474 to 606 that encompass the FHL2 interacting domain (elrAΔFID). In the UTI model  in kidney and of 1 log unit decrease (P = 0.00304) in bladder compared to the WT strain, revealing that elrA contributes to pathogenesis of E. faecalis urinary tract infection. Interestingly, the organ bacterial loads upon infection with elrAΔFID were intermediate between the values observed with the WT and those obtained with the ΔelrA-mutated strain, indicating that ElrA-FHL2 interaction is implicated in the ElrA-dependent pathogenesis in the urinary track. We then examined the virulence of the three strains in a systemic infection model post-infection intravenously  and kidneys (1.5 log unit) compared to the WT strain. In contrast, strain elrAΔFID showed an intermediate phenotype and appeared to colonize the liver at a comparable level as the WT while it colonized kidneys at a significant lower level  than the WT, supporting that elrA contributes to E. faecalis systemic infection and partially relies on the interaction of ElrA with FHL2. Altogether, these results indicated that the interaction between ElrA and FHL2 have a role in systemic and urinary infections.Identification of an ElrA domain that interacts with FHL2, led us to hypothesize that deletion of the FID domain may affect virulence as it would impair ElrA interaction with FHL2. To test this hypothesis and considering a potential tissue-specific interaction between ElrA and FHL2, we compared the virulence of a mutant strain deleted for the FID domain to the parental strain (OG1RF) and the Δdel Fig. , the Δelsly Fig. . Strain 39. In Gram-positive bacteria, several bacterial virulence factors are located at the pathogen surface such as the internalins of L. monocytogenes27. In this study, we selected and cloned 23 putative virulence factors of the E. faecalis opportunistic pathogen and tested their ability to interact with host proteins using the hORFeome v3.136. We identified a strong interaction between the virulence factor ElrA of E. faecalis and the human protein FHL2, and determined the minimal interacting domain of ElrA, named FID (FHL2-Interacting Domain). In addition, we showed that FID is partially involved in the ElrA-dependent virulence in vivo. To our knowledge, besides interactions with extracellular matrix proteins, FHL2 is the first intracellular host protein targeted by a virulence factor of E. faecalis.Several bacterial pathogen effectors bind host proteins to hijack the host functions for their own benefits33, only strong interactions are detectable, this explains the low range of positive hits. In the selected surface proteins, ElrA is one of the proteins that is likely to have a transient interaction with the bacterial surface as its WxL domain promotes a non covalent interaction with the peptidoglycan10. Then ElrA could be considered both as a surface and as a secreted protein. For the proteins covalently bound to the bacterial surface (e.g. those with an LPxTG motif), the Y2H have shown limits in the discovery of surface-surface protein interactions. This could be due to the low level of posttranslational modifications in yeast while they are important for many cell surface proteins  or the absence of multimerisation in Y2H experiments while it is required for the activity of many eukaryotic surface receptors. For example, the detection of an interaction between the bacterial flagellin and the TLR5 homodimer receptor is not detectable using Y2H . However, the set of entry vectors encoding E. faecalis ORFs generated in this study, provides an interesting tool of easily transferable inserts to Gateway compatible vectors for other applications, such as the expression of an E. faecalis protein fused with tags that can be used for purification or tandem affinity purification allowing the purification of stable complexes to identify the partners involved41 or with reporters such as the GFP, which can be used to localize the protein42.In our Y2H experimental set-up using stringent conditions10. It was demonstrated that: i) the inactivation of elrA significantly reduced E. faecalis virulence in a peritonitis model and that ElrA contributed to the bacterial intracellular persistence in macrophages and increased the host inflammatory response by stimulating in vivo the production of interleukin-610. Overexpression of ElrA protects from phagocytosis in vitro and increases E. faecalis virulence a mouse peritonitis model43. It was also shown that elrA is the first gene of an operon poorly transcribed under laboratory growth conditions but induced in mice during infection10. However, so far no ElrA ligand was identified. In this study, we identified a physical interaction between ElrA and FHL2, a member of the four-and-a-half-LIM-only family proteins. This interaction happens in vitro and impacts the in vivo infectious process.ElrA is an internalin-like protein that contains a signal-peptide, a leucine-rich repeat domain followed by an Ig-like domain and at the C-terminal end, a WxL domain, which interacts with the bacteria peptidoglycan44. To date in the host-microbe interactions context, a specific interaction has been shown between Tax1 a protein of the human T-cell leukemia virus 1 (HTLV-1) and FHL345. Our study is the first report describing an interaction between a bacterial protein and a specific member of the FHL family. FHL2 has a strong homology with the other members of the four-and-a-half-LIM-only protein family, which includes FHL1, FHL3, FHL4 and FHL5. The human FHL2 amino acid sequence is 48.2% identical with FHL1, 53.4% with FHL3, 48.4% with FHL4, and 59.1% with FHL5. Although all FHLs could promote protein-protein interactions37, we demonstrated that ElrA interacts specifically with FHL2. FHL2 is involved in heart physiology, bone formation, muscular function and placental development. In addition, FHL2 is involved in a wide range of various cellular processes such as cytoarchitecture, cell adhesion, cell survival, cell mobility, signal transduction and other processes through the regulation of gene expression46. FHL2 enhances the transcription of the tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) through the stabilization of NF-κB after activation47. FHL2 is enzymatically inactive but has been shown to interact with more than 100 proteins through its LIM domains48 although, the comparison of various FHL2 partners do not allow the identification of a consensus interacting sequence. The FHL2 partners are components of crucial signaling pathways , proteins known to modulate RNA splicing, DNA replication and repair. The broad range of cellular partners and involvement in cellular processes make FHL2 an interesting target for a pathogenic bacterium to ensure its survival. However, the clear picture of the cellular mechanisms underlying and rising from the FHL2/ElrA interaction remains to be found.The members of the four-and-a-half-LIM-only protein family are involved in many physiological processes probably through their ability to interact specifically with various proteins. Due to their interacting ability, the LIM-only proteins and by extension all proteins containing LIM domain(s), are considered as adaptors or scaffolding proteinsEnterococcus are traditionally considered as commensal extracellular bacteria, during its infectious process it can be found intracellular within urothelial cells shed from the bladder of patients49 or surviving for long periods into macrophages53. Moreover, several laboratories showed that E. faecalis escapes the phagocytic vacuole to avoid lysosomal elimination53. At late stages of infections, intracellular E. faecalis activates phosphatidylinositol 3-kinase signaling to block apoptotic cell death in macrophages53. As FHL2 is detected in the cytoplasm, we could anticipate that FHL2/ElrA interaction occurs during bacterial intracellular survival. However, as ElrA is non-covalently attached to the bacterial peptidoglycan10, we cannot rule out the possible release of ElrA directly into the cytoplasm of the host cell during the early interaction steps. What are the incidences or signalling pathways targeted by the ElrA/FHL2 interaction remain unknown. The interaction with ElrA could even compete with an FHL2 interacting protein or create a new complex with additional partner(s) as it is often the case in the host-pathogens protein-protein interactions54. Interestingly, ElrA is not the only surface-exposed protein that is interacting with host cytoplasmic partners. Indeed, Listeria express at its surface 2 virulence factors implicated in its cytoplasmic survival: ActA (actin assembly-inducing protein) and InlK  that interact with Arp2/3 and the Major Vault Protein (MVP), respectively55. The parallel between ElrA and InlK is interesting, as both proteins are surface-exposed virulence factors and only expressed in vivo55. Like FHL2, MVP is implicated in multiple cellular processes but its precise role remains unknown. MVP is recruited at Listeria surface impairing autophagic recognition and consequently leading to Listeria survival in the cytoplasm56. It is tempting to speculate a similar role for ElrA/FHL2 interaction as E. faecalis has been recently described to resist autophagy52. However the role of ElrA in E. faecalis autophagy resistance remains to be investigated.E. faecalis suggesting a crucial functional role of this domain. Among the 24,788 prokaryotic registered genome57, apart the Enterococcus genus, the FID was detected in an internalin-like protein of Weissella halotolerans exhibiting 35% of identity with the FID of ElrA. This W. halotolerans protein could be an ElrA-like protein as it shares 34% of identity with ElrA and contains in addition to the FID, an LRR repeat domain and a WxL domain. It is noteworthy that W. halotolerans is an opportunistic pathogen for the rainbow trout58.The FHL2 Interacting Domain (FID) of ElrA is 100% conserved in all the sequenced strains of elrAΔFID strain in the peritoneal infection model (data not shown) suggesting that the FID is not involved in the ElrA-dependent virulence via this infectious entry and that probably other domains of the ElrA protein are important for virulence; but it also demonstrated that the FID-deleted protein is stable and functional. On the opposite, in the UTI model, the impairment of the interaction of ElrA with FHL2 led to a systemic infection but with intermediate E. faecalis burdens in mice organs compared to wild-type strain (higher counts) and the fully deleted elrA strain (lower counts), revealing that ElrA contributes to virulence in ascending urinary tract infection. Differences in levels of attenuation of elrAΔFID in the models of UTI and SI suggest that the interaction between ElrA and FHL2 is tissue-specific and that other domains of ElrA may be at least as important as the FID to ensure a complete virulence. This observation may be related to the expression pattern of FHL2. It is expressed at basal level in most of the human and mice tissues (in human:<20 fragments per kilobase of exon per million fragments mapped (FPKM)) but shows a higher level of expression in given tissues such as the heart muscle (516 FPKM), the ovary (456 FPKM), the placenta (74 FPKM) and the urinary bladder (90 FPKM)59).In contrast to the ElrA-deficient strain, we did not find any difference between the wild-type and E. faecalis) and FHL2 (from human) with consequences in 2 infectious models giving new hints to decipher the mode of action of this bacterial effector. This work underlined that the involvement of ElrA in Enterococcus virulence could depend on the route of infection. Understanding the functional role and the significance of the interaction of ElrA of E. faecalis with FHL2 could provide new insights into the mechanisms of virulence of this poorly understood human pathogen.Altogether, our study evidenced a strong interaction between ElrA (from Escherichia coli strains were grown at 37 °C in Luria-Bertani (LB) broth or agar medium supplemented with the required antibiotics: ampicillin (100 µg.mL−1), spectinomycin (100 µg.mL−1), erythromycin (150 µg.mL−1). E. faecalis strains were grown at 37 °C in M17 broth or agar supplemented with 0.5% of glucose (M17G) and the required antibiotics: tetracycline (5 µg.mL−1), erythromycin (30 µg.mL−1). Saccharomyces cerevisiae strains were grown in rich YEPD medium  or in synthetic complete medium (SC) lacking the amino acids  or nucleobase (Ade) corresponding to its auxotrophies. The vectors used for the constructs generated in this work are listed in Table E. faecalis strain V583 and the 39 deletants of the elrA gene in the pDONR and pDestDB-GW vectors are listed in Table The strains used in this work are summarized in Table E. faecalis selected ORFs (EfORFs) were cloned using the Gateway system (Invitrogen) as described in Dobrijevic et al.34. For the Y2H experiments, all the EfORFs were transferred from the pDONR223 into the pDestDB-GW60 using the LR clonase II (Invitrogen) to generate the yeast vectors expressing the DB-EfORF fusion proteins. As we used the Gateway recombination method (Invitrogen), the attB1.1 (GGGGACAACTTTGTACAAAAAAGTTGGC) and attB2.1 (GGGGACAACTTTGTACAAGAAAGTTGGGTC) sequences were added to the 5′ end of each forward and reverse primers, respectively. An amber stop codon in frame with the elrA sequence was incorporated in all reverse primers. The PCR products were then cloned in the pDONR223 as previously described34.The full-length 61 using the LR recombination cloning, generating the p3flag-FHL2, p3flag-FHL3, p3flag-FHL1 and p3flag-FHL5 vectors.The FHL CDS were transferred from pDONR223 to the Gateway destination vector pV189933. Yeast matings were performed with Y8800 and Y8930 strains62. The pDestDB vectors carrying the EfORFs were transformed into the Y8930 MATα yeast and selected on solid synthetic complete (SC) medium lacking leucine (SC-Leu). The autoactivating baits were identified by testing the growth of the yeasts containing the pDESTDB-EfORFs on solid SC medium lacking leucine and histidine (SC-Leu-His) and containing 2 mM of 3-amino-triazole (3-AT); all growing clones were not selected.Yeast two-hybrid screening was performed as previously describedEfORF MATα yeast strain was mated with the 12,212 human AD-ORFs pooled into 130 mini-libraries as described by Rual et al.33 on YEPD agar. After overnight growth at 30 °C, the diploids expressing the GAL1::HIS3 yeast two-hybrid reporter were selected on SC-Leu-Trp-His plates containing 2 mM 3-AT63. De novo autoactivators were eliminated using the counter-selectable marker CYH260. Positive colonies were picked for PCR amplification and identification of the interacting proteins by sequencing of the AD- and DB-ORFs. The Human proteins found to interact with the E. faecalis proteins were then individually tested against each EfORF. For this purpose, we mated MATα Y8930 containing individual DB fused to Ef ORF and MATa Y8800 containing the vector expressing the AD fused to the interacting human candidates. The plates were incubated overnight at 30 °C on appropriate SC minimal media to select for the expression of GAL1::HIS3 or GAL2::ADE2. To eliminate the yeast background due to residual growth on the minimal media, the plates were twice velvet-cleaned as described63. The same procedure was used to test the interaction of ElrA fragments with FHL2. At each step of the interaction screening and validation procedures the following yeast control strains were spotted on the plates: diploids yeasts containing [1] pPC97-CYH (DB-empty) and pPC86 (AD-empty), [2] DB-RB1 and AD-E2F1, [3] DB-fos and AD-jun, [4] pPC97 (DB-empty) and pPC86 (AD-empty), [5] DB-DP1 and AD-E2F1, [6] DB-FRM-3 and AD-PKc_like.Each DB-HEK293T cells (ATCC CRL-3216) and Hela (ATCC CCL-2) were maintained in RPMI 1640 supplemented with 10% heat-inactivated foetal calf serum, 2 mM glutamine, 1X Non essential Amino acid, penicillin (50 U/ml) and streptomycin (50 U/ml) in a humidified 5% CO2 atmosphere at 37 °C. All culture media and supplements were supplied by Life technologies.E. coli ER266 and purified as previously described21. HEK293T cells were transfected with the p3flag-FHL1, p3flag-FHL2, p3flag-FHL3 and p3flag-FHL5 vectors using the jetPrime reagent (Polyplus) following the manufacturer’s instructions. The transfected cells were lysed 10 min on ice in PBS-1% Triton X100-Complete anti-protease cocktail (lysis buffer). Cell debris and nucleus were removed by centrifugation . Then, the cell extracts (200–400 μg of proteins) were incubated overnight at 4 °C with the various GST-ElrA proteins bound to the beads in the lysis buffer. After incubation, the beads were washed six times in the lysis buffer supplemented with 0.5 M NaCl. The original lysates (inputs) and pulled-down proteins were analyzed by Western blotting using the monoclonal mouse anti-FLAG M2 (Sigma-Aldrich 1:2000) or the monoclonal mouse anti-GST  as primary antibodies and the HRP-conjugated mouse anti-human IgG as secondary antibody. The Amersham ECLTM Prime Western blotting detection  was used for the revelation step.GST-ErlA fusion proteins (ElrA(27–473), ElrA(27–723) or ElrA(474–606)) were produced in 21 generating the pCMVHA-FHL2 plasmid. Similarly, the myc-ElrA(27–723) and myc-ElrA(27–473) were produced from the pCMVmyc-ElrA vectors generated by LR with pCMVmyc-GW destination vector64. The transfection in HeLa cells were performed as described21. HeLa cells were grown on coverslips and fixed in 3% paraformaldehyde in PBS, for 15 min. Fixed cells were washed in PBS and permeabilized with 0.5% Triton for 5 min. Coverslips were incubated with primary antibodies in PBS 5% horse serum , or rat anti-HA IgG1 ) for 30 min at room temperature, washed in PBS 5% horse serum and then incubated with the secondary antibodies ), and goat anti-rat IgG conjugated to Alexa Fluor 594 (Invitrogen). Coverslips were mounted onto glass slides using Mowiol (Polysciences). Images were acquired using a Zeiss LSM510 laser scanning confocal microscope and analysed using the Zeiss Zen software .The FHL2 CDS was transferred by LR from the pDONR223 to the pCMVHA-GW vector65. To delete FID domain of the elrA gene, upstream and downstream regions flanking the domain were amplified by PCR using E. faecalis OG1RF genomic DNA as template, and the following primer pairs: (i) FIDup1 (5′-TTAGTCCGTTAGGTCAAGC-3′) and FIDup2 (5′-TTCTTTGACAGGTTGGACG-3′); (ii) FIDdown1 (5′-CGTCCAACCTGTCAAAGAAAGTAGCACGACCCAATATC-3′) and FIDdown2 (5′-AATAGCCACGCCTAAAGTG-3′). The two fragments (803 and 815 pb) were purified and a second PCR using FIDup1 and FIDdown2 allowed the association of the two fragments in a single PCR product that was cloned in pGEM-T Easy (Promega Corporation) to generate pGEM-T-elrAΔFID. After EcoRI restriction of the pGEM-T-elrAΔFID, the ΔFIDelrA fragment was cloned into EcoRI-restricted pG+host966, generating pG+host9-elrAΔFID. The resulting plasmid was then introduced into OG1RF. An in-frame elrAΔFID mutant was selected as previously described10 and the deletion of the FID domain was confirmed by sequencing of the chromosomal locus. The procedure was repeated a second time with another pG+host9-elrAΔFID vector to obtain an independent elrAΔFID mutant. The mutants were named VE18355–3 and VE18355–4  were transurethrally challenged with 1 × 104 colony-forming units (CFUs) of wild-type OG1RF and its derivatives mutants ΔelrA and two independent elrAΔFID mutants (Table The mouse urinary tract infections (UTIs) experiments were performed by following the protocol described by Lebreton ts Table . 48 hour68, the strains were cultured as in the UTI model. A 100 μL aliquot from each strain suspension (1 × 109 bacteria/mL) was injected into the tail vein of each of five female BALB/c mice. Seven days after infection, mice were sacrificed, and their organs (kidneys and livers) were removed aseptically, weighed, and homogenized, to determine the recovered CFUs. The experiments for each animal model were made in triplicate, and comparison among the organ counts was performed by use of an unpaired t test.We used a previously well-established systemic intravenous infection (SI) model2 inhalation. In addition, to minimize animal suffering and distress during the intraurethral catheterization, mice were anesthetized by isoflurane inhalation.Mice were observed twice daily for morbidity and mortality, and any mice that exhibited noticeable morbidity  were euthanized immediately by COAll methods were performed in accordance with the relevant guidelines and regulations.The mouse experiments were approved by the Institutional Animal Use and Care Committee at the Università Cattolica del Sacro Cuore, Rome, Italy , and authorized by the Italian Ministry of Health, according to the Legislative Decree 116/92, which implemented the European Directive 86/609/EEC on laboratory animal protection in Italy. Animal welfare was routinely checked by veterinarians of the Service for Animal Welfare.Supplementary Information


As a collective quasiparticle excitation of the magnetic order in magnetic materials, spin wave, or magnon when quantized, can propagate in both conducting and insulating materials. Like the manipulation of its optical counterpart, the ability to manipulate spin wave polarization is not only important but also fundamental for magnonics. With only one type of magnetic lattice, ferromagnets can only accommodate the right-handed circularly polarized spin wave modes, which leaves no freedom for polarization manipulation. In contrast, antiferromagnets, with two opposite magnetic sublattices, have both left and right-circular polarizations, and all linear and elliptical polarizations. Here we demonstrate theoretically and confirm by micromagnetic simulations that, in the presence of Dzyaloshinskii-Moriya interaction, an antiferromagnetic domain wall acts naturally as a spin wave polarizer or a spin wave retarder (waveplate). Our findings provide extremely simple yet flexible routes toward magnonic information processing by harnessing the polarization degree of freedom of spin wave. Spin waves are promising candidates as carriers for energy-efficient information processing, but they have not yet been fully explored application wise. Here the authors theoretically demonstrate that antiferromagnetic domain walls are naturally spin wave polarizers and retarders, two key components of magnonic devices. As a collective excitation of ordered magnetization in magnetic systems, spin wave (or magnon when quantized) carries spin angular momentum like the spin-polarized conduction electron. Different from the spin transport by conduction electrons, the propagation of spin wave does not involve physical motion of electrons. Therefore, spin wave can propagate in conducting, semiconducting, and even insulating magnetic materials without Joule heating, a troubling issue faced by the present day silicon-based information technologies3. The dissipation in the spin wave system is mainly due to the magnetic damping, which is much weaker than the electronic Joule heating, especially in magnetic insulators4. Magnonics is a discipline of realizing energy-efficient information processing that uses spin wave as its information carrier7. Besides its low-dissipation feature, the wave nature and the wide working frequency range of spin wave make magnonics a promising candidate of upcoming beyond-CMOS  computing technology.Spintronics extends the physical limit of conventional electronics by harnessing the electronic spin, another intrinsic degree of freedom of an electron besides its charge8, and manipulation of photon polarization is essential for applications in photonics9. The phonon polarization has also been used in encoding acoustic information for realizing phononic logic gates11, and for controlling magnetic domain walls12. In contrast, most magnonic devices proposed or realized so far mainly use the spin wave amplitude18 or phase23 to encode information, and spin wave polarization is rarely used except in very few cases24. The lack of usage of polarization in magnonics has reasons. With only one type of magnetic lattice, ferromagnets can only accommodate the right-circular polarization, hence there is simply no freedom in polarization manipulation. This situation is similar to the case of half-metal, which has only one spin spieces25. In other words, ferromagnet is a half-metal for spin wave. To overcome this disadvantage, a straightforward approach is to use antiferromagnets in manipulating spin wave polarization. In antiferromagnets, due to the two opposite magnetic sublattices, spin wave polarization has complete freedom as the photon polarization. Thus, antiferromagnet is a normal metal for spin wave with all possible polarizations. In comparison with the ferromagnets, besides gaining the full freedom in polarization, antiferromagnets also have numerous other advantages, such as the much higher operating frequency (up to THz) and having no stray field, etc27. Due to these merits, antiferromagnet is regarded as a much better platform for magnonics than ferromagnet30. In order to unleash the full power of antiferromagnets in magnonics, however, it is highly desirable to have a simple yet efficient way in manipulating spin wave polarization, ideally in a similar fashion as in its optical counterpart.Similar to the intrinsic spin property of elementary particles, polarization is an intrinsic property of wave-like (quasi-)particles such as photons, phonons, and magnons. It is more natural to encode information in the polarization degree of freedom than other degrees of freedom such as amplitude or phase. For instance, photon polarization has been widely used in encoding both classical and quantum information31. The former is to create a particular linear polarization, and the latter is to realize the conversion between circular and linear polarizations. The actual realization of these two building blocks relies on the types of the (quasi-)particle in question. For instance, for photon, an array of parallel metallic wires functions as an optical polarizer9, and a block of birefringent material with polarization-dependent refraction indices acts as an optical waveplate31.In general, to be able to manipulate polarization with full flexibility, two basic devices are indispensable, i.e., the polarizer and retarder (waveplate)33 an antiferromagnetic domain wall serves as a spin wave polarizer at low frequencies and a retarder at high frequencies. Due to the extreme simplicity and tunability of a magnetic domain wall structure, the manipulation of spin wave polarization in magnonics becomes not only possible but also simpler and more flexible than its optical counterpart.Here, we show theoretically and confirm by micromagnetic simulations that, utilizing the Dzyaloshinskii-Moriya interaction (DMI) existing in the symmetry-broken systemsred/blue arrows denote the magnetization direction m1,2 for the two magnetic sublattices of an antiferromagnetic domain wall. The domain wall profile is taken as Bloch type, where the magnetization rotation plane (y–z plane) is perpendicular to the domain wall direction 35,i = 1, 2 denote the two sublattices, γ is the gyromagnetic ratio, α is the Gilbert damping constant. Here mi, where K is the easy-axis anisotropy along A and D are the Heisenberg and Dzyaloshinskii-Moriya (DM) exchange coupling constant within each sublattice, and J is the exchange coupling constant between two sublattices. The Heisenberg exchange coupling tends to align neighboring magnetization in parallel. While the DM exchange coupling, existing in magnetic materials or structures lack of spatial/inversion symmetry33, prefers magnetization to rotate counter-clockwise about an axis (x-axis in our case) determined by the symmetry-broken direction.We consider a domain wall structure in an one-dimensional antiferromagnetic wire along mentclass2pt{minimx-axis as θ0(x) and ϕ0(x) are the polar and azimuthal angle of n0 with respect to ϕ0 = const.36, where ϕ0 = π/2) or Néel type (ϕ0 = 0) configuration, or any mixture of the two with 0 < ϕ0 < π/2; in the presence of DMI, the domain wall becomes chiral38 and is pinned to the Bloch type as shown in Fig. We denote the static profile of the antiferromagnetic domain wall along mentclass2pt{minimmentclass2pt{minimmentclass2pt{minimmentclass2pt{minimmentclass2pt{minimmentclass2pt{minimn0(x). Since y–z plane), we call the excitation in VK(x) and VD(x)36: VK(x) = K[1 − 2sech2(x/Δ)] arises due to the easy-axis anisotropy along VD(x) = (D/Δ)sech(x/Δ) is due to the combined action of DMI and the inhomogeneous magnetic texture. Equations (y-polarized (x-polarized) modes.With the static profile mentclass2pt{minimmentclass2pt{minimmentclass2pt{minimVK and VD. In the short wavelength limit (WKB approximation), using Eqs. (x for chosen polarization and wavevector k:VD = 0), the in-plane and out-of-plane modes are degenerate (ωIP = ωOP). This degeneracy is lifted by the introduction of DMI parallel to the magnetization direction40, and in our case the spin wave wavevector The influences of a domain wall on spin wave propagation are all captured by the effective potentials ing Eqs.  and 3),,VK and D = VD = 0), the dispersions and the local spin wave gaps for in-plane and out-of-plane polarization in Eqs. (VK(x) is the well known reflectionless Pöschl–Teller type potential well41. Consequently, regardless of its polarization, the incident spin wave experiences no reflection by the domain wall, and only accumulate a common phase delay. However, when DMI is present , the dispersions and local spin wave gaps for the in-plane and out-of-plane modes are different. Considering that the inter-sublattice exchange coupling J is the dominating energy scale in antiferromagnet, the in-plane gap VDVD > 0 and reaches its maximum value ωD inside the domain wall. This effect of DMI in Eqs. , the y-polarization experiences no (or little) reflection but the x-polarization hits a potential barrier and is strongly reflected; At higher frequencies (ω > ωD), both polarization transmit almost perfectly, but the x- and y-polarization no longer share the same phase delay.In the absence of DMI ( in Eqs.  and 5)  D = VD see Fig. . TherefoTx,y through the domain wall for the linear x- and y-polarization, as well as their relative phase accumulation δϕxy = ϕy − ϕx, as presented in Fig. y-polarization transmit almost perfectly (Ty ≃ 1) for all frequencies above the spin wave gap (ω > ωG). In contrast, because of the effective potential barrier, the x-polarization experiences total reflection (Ty ≃ 0) at low frequencies (ω < ωD). Above the barrier (ω > ωD), however, the x-polarization experiences little reflection (Ty ≃ 1). Because of the different effective potentials experienced at the domain wall, the x- and y-polarizations develop a relative phase accumulation δϕxy, which is frequency-dependent as shown in Fig. The polarization-dependence of the spin wave scattering behaviors by the domain wall are confirmed by micromagnetic simulations based on the LLG Eq.  and GreeωG < ω < ωD), because the x-polarized spin wave is totally reflected, an antiferromagnetic domain wall is naturally a y-polarizer for spin wave. The functionality of the spin wave polarizer is confirmed by a micromagnetic simulation on a straight antiferromagnetic wire shown in Fig. x- and y-polarization of equal amplitude, is injected from the left side of the domain wall, only the y-polarization transmits, indicating that the domain wall is a y-polarizer for spin wave.In the low frequency regime , hence it is an x-polarizer as demonstrated in Fig. x- or y-polarizer depending on the location of the domain wall: an x-polarizer when the wall is at the center segment , both x- and y-polarized spin wave modes transmit almost perfectly through the domain wall, but they accumulate different phases. As a result of this relative phase delay, an antiferromagnetic domain wall functions as a spin wave retarder (or a waveplate). As shown in Fig. ω/2π = ω2/2π = 16.2 GHz, the relative phase delay between the x- and y-polarization δϕxy(ω2) = π/2, hence the antiferromagnetic domain wall is specifically a quarter-wave plate. Figure δϕxy(x) increases by π/2 across each domain wall. By passing through these domain walls, the right-circular polarized spin wave is converted into a 45°-linear mode, a left-circular mode, a −45°-linear mode, and back into a right-circular mode, successively.In the high frequency regime , the working frequency range for the spin wave polarizer and retarder are different, i.e., a polarizer for ω < ωD and a retarder for ω > ωD. However, by modulating the material parameters (such as tuning the DMI strength as in refs. 46), it is possible to have the spin wave polarizer and retarder functioning in the same frequency range. Henceforth, multiple polarizers (of different polarization direction) and retarders (of different phase delay) can be assembled into one magnonic circuit to realize more complex spin wave manipulations.The polarizing and retarding effects sustains at smaller DMI strength 47, where the former is caused by the bulk inversion symmetry breaking, such as in MnSi48 and other B20 compounds49, and the latter is due to the interfacial inversion symmetry breaking, such as in Co/Pt or Co/Ni bilayer51. Here, we adopt the bulk form DMI to demonstrate the working principle of the spin wave polarizer and retarder. However, the materials or structures with interfacial form DMI would work in almost the same manner. The only difference is that interfacial DMI prefers a Néel type domain wall rather than a Bloch type for the bulk DMI38 and the roles of in-plane and out-of-plane modes interchange. Besides the inhomogeneous DMI discussed above, there is also homogenous DMI that makes the antiferromagnet a weak ferromagnet by slightly misaligning the magnetizations in two sublattices33. However, such a misalignment is extremely small because of the dominating antiferromagnetic coupling, thus is ignored in our discussions.The DMI has two different forms, the bulk and the interfacial form53. The domain wall structures have been observed in both types of antiferromagnetic materials55, and can be effective controlled by using the exchange bias effects60. Experimentally, it should be more straightforward to realize the spin wave polarizer and retarder proposed here using the synthetic antiferromagnetic wires used for the racetrack memory53.The working principles for the spin wave polarizer and retarder work for both real antiferromagnet and artificial synthetic antiferromagnet, where the latter consists of two antiferromagnetically coupled ferromagnetic wiresIn conclusion, we demonstrated that the antiferromagnetic domain wall with DMI naturally functions as a spin wave polarizer at low frequency, and a spin wave retarder (waveplate) at high frequency. This extremely simple design enables all possible polarization manipulations for antiferromagnetic spin waves. Our findings greatly enrich the possibility of magnonic information processing by harnessing the polarization degree of freedom of spin wave.36 the easy-axis anisotropy K/γ = 3.88 × 104 A m−1, the intra-sublattice Heisenberg exchange constant A/γ = 3.28 × 10−11 A m, the saturation magnetization Ms = 1.94 × 105 A m−1, the gyromagnetic ratio γ = 2.21 × 105 rad m A−1 s−1, and the DMI constant D/γ = 3 × 10−3 A. The inter-sublattice antiferromagnetic exchange coupling between two ferromagnetic wires is J/γ = 5 × 105 A m−1. The dipolar interaction is neglected for this antiferromagnetic environment. To obtain the transmission probability, we first verify that the spin wave perfectly transmits for D = 0, then the transmission probability for a chosen polarization is extracted by taking the ratio between the spin wave amplitude for (i) D = 0 and (ii) D ≠ 0 at the location 500 nm away from the domain wall.The micromagnetic simulations are performed in COMSOL Multiphysics, where the LLG equation is transformed into weak form by using the mathematical module and solved by the generalized-alpha method. The synthetic antiferromagnet wire is composed of two ferromagnetic YIG wires with the following parameters:The data that support the findings of this study are available from the corresponding authors on request.Supplementary InformationPeer Review FileSupplmentary Movie


ECOD relies on a combination of automatic and manual weekly updates to achieve its high accuracy and coverage with a short update cycle. ECOD classifies the approximately 120 000 depositions of the PDB into more than 500 000 domains in ∼3400 homologous groups. We show the performance of the weekly update pipeline since the release of ECOD, describe improvements to the ECOD website and available search options, and discuss novel structures and homologous groups that have been classified in the recent updates. Finally, we discuss the future directions of ECOD and further improvements planned for the hierarchy and update process.Evolutionary Classification Of protein Domains (ECOD) ( Protein three-dimensional structures continue to be determined at an exponential rate, both due to improvements in structure determination techniques and the increase of involved investigators –3. AdditWe developed the Evolutionary Classification Of protein Domains (or ECOD)  as a hieFollowing the initial release of ECOD (v22), we implemented a weekly update pipeline for ECOD coupled to the release of new structures from the PDB. By quickly and efficiently classifying known structures, and through dedicated manual curation, we are able to classify all depositions in the PDB without overburdening manual curators or relying on solely classifying a reduced set of representative structures. Briefly, the ECOD update pipeline separates a weekly PDB release into a set of individual protein queries based on peptide chains within the PDB depositions (putative fragments or peptides are removed early in the process and either placed into the peptide/fragment categories or reincorporated into ECOD as segments of multi-chain domains). Each member of this set of peptide chains is then individually queried against ECOD reference libraries using a combination of sequence  and structural (DALI) aligners –12. BLASWe initiated weekly updates in February 2014 with ECOD version 33. In the following 123 weekly PDB releases, 187 ± 51 structures were classified each week on average Figure . These cThose chains that cannot be partitioned into domains and assigned automatically are manually curated (8). The result of manual curation is either to partition a peptide chain into domains and to assign those domains to H-groups within the ECOD hierarchy, or to assign a chain, in part or wholly, to one of the ECOD special architectures. In either case, the manual assignment of these domains or special architecture regions helps to automate subsequent assignments. Over time, our manual curation workload has remained steady in the face of the gradually increasing rate of structure releases, and the ratio of domains to non-domain  regions has decreased. We attribute these shifts to the change in focus from small, structurally novel, globular domains, to larger complexes wherein associated domains may contain more extended regions in the deposited coordinates increasing numbers of extended structures in recent PDB releases likely result from their physical ordering by protein–protein interactions within large complexes, or from a shift from X-ray crystallography refinement techniques, where disordered regions are more likely to be excluded from modeled regions, to microscopy refinement techniques, where large disordered regions are more likely to be included in the deposited structural coordinates.http://prodata.swmed.edu/complete/distributions).ECOD is highly redundant with respect to sequence and structural similarity. Since the release of ECOD we have departed from our F-group model of Pfam HMM-based families mixed with HHsearch-based single-linkage clusters. Now we use families described by Hidden Markov Models (HMMs), some of our own creation. With the increased week-to-week stability and performance that this change provided, we were able to recalculate ECOD representative sets on a weekly basis and provide them as a standard distribution. Domain clusters are generated using BLASTCLUST at 99%, 70% and 40% sequence filtering over 90% alignment coverage of both the query and the reference sequences. Because H-groups can contain multiple T- and F-groups, filtering only occurs between members of the same F-group. This rule ensures that each F-group is represented at least once, although representatives may be sequence-similar above the level of filtering to members of other F-groups. Representatives are selected for each version from clusters preferring (i) existing F40 representatives (i.e. from previous versions), (ii) manually curated domains, (iii) provisional manual domains, (iv) domains from structures determined by X-ray crystallography, (v) domains from structures with higher X-ray crystal resolution, (vi) and domains from more recently released structures. These representative sets are made available, along with their PDB structure sets, at our webpage  and potentially homologous groups (X-groups). In addition, we partition groups of homologous domains into sequence families of closer relatives, frequently with similar functions. Because family classification in itself is to a large degree reliant on manual identification of functional motifs and interaction partners, we rely on the well-established classification provided by the Pfam sequence database to the extent possible . Putativhttp://hmmer.org) searches against the UniRef90 non-redundant protein database  must be created through manual curation and populated with at least one representative. Although not all curation results in the creation of new groups, on average 2.5 new X-groups (no clear homology to any known domain) and 1.9 new H-groups (some inconclusive evidence of homology to known domains) were created and populated in each weekly update. Proteins assigned to these new groups were generally partitioned as single-domain (60%), with a smaller fraction partitioned into more than one domain (40%). Domains in newly created X-groups are not homologous to any other ECOD domain; however, they may gain new homologs in subsequent versions. Of the 273 X-groups and 220 H-groups defined by manual curation during the weekly updates, 75% of both have received additional domains subsequent to their inception. Many of these additions are redundant, considering only the addition of new F40 domains, only 14% and 12% have received new additions, respectively. These novel PDB chains are generally not classified by other structural domain classifications. Only proteins observed in 19 and 27 of the new ECOD X- and H-groups are observed in SCOPe v2.06. Similarly, only proteins within 31 of the new ECOD X-groups and 34 of the new ECOD H-groups are observed in CATH v4.1.Plasmodium falciparium. The structure of RH5 from Plasmodium was determined and found to have a novel alpha-helical fold that may form an obligate multimer  protein family is necessary for invasion of host erthyrocytes in multimer ,21. The multimer . A subsel domain .New groups in ECOD can contain domains from viral proteins. The fast evolution of viral proteins can be responsible for significant structural variation and elaboration. The initial 2.3 A X-ray structure of the hantavirus glycoprotein Gn (5fxu) ectodomain  was manuThe mamallian mitoribosome, although descendant from alpha-proteobacteria, contains many unique structural features . The strThe mission of ECOD is to provide timely and consistent domain classification of proteins released by the PDB. We maintain a unique database uniquely focused on the full classification of the available structures, and by full coverage of the residues in those structures. Here, we have presented some of the improvements to website search and the result of more than 18 months of consistent weekly updates. In the future we intend to focus on making quality-control data available through the same portal, formalizing our description of sequence F-groups, and describing the inter-domain interfaces present in the protein complexes in which ECOD domains participate. We anticipate that the types of structures classified by ECOD will continue to shift toward large complexes determined by microscopy methods, and that the fraction of single-domain X-ray crystal structures will continue to decrease. This shift will require a continued focus on the automated treatment of these structures, and the curation tools used to classify them manually.


VP9071–415 encompasses the conserved N-terminal domain of VP90 but lacks the hypervariable domain, which forms the capsid surface spikes. The structure of VP9071–415 is comprised of two domains: an S domain, which adopts the typical jelly-roll β-barrel fold, and a P1 domain, which forms a squashed β-barrel consisting of six antiparallel β-strands similar to what was observed in the hepatitis E virus (HEV) capsid structure. Fitting of the VP9071–415 structure into the cryo-electron microscopy (EM) maps of HAstV produced an atomic model for a continuous, T=3 icosahedral capsid shell. Our pseudoatomic model of the human HAstV capsid shell provides valuable insights into intermolecular interactions required for capsid assembly and trypsin-mediated proteolytic maturation needed for virus infectivity. Such information has potential applications in the development of a virus-like particle (VLP) vaccine as well as small-molecule drugs targeting astrovirus assembly/maturation.Human astrovirus (HAstV) is a leading cause of viral diarrhea in infants and young children worldwide. HAstV is a nonenveloped virus with a T=3 capsid and a positive-sense RNA genome. The capsid protein (CP) of HAstV is synthesized as a 90-kDa precursor (VP90) that can be divided into three linear domains: a conserved N-terminal domain, a hypervariable domain, and an acidic C-terminal domain. Maturation of HAstV requires proteolytic processing of the astrovirus CP both inside and outside the host cell, resulting in the removal of the C-terminal domain and the breakdown of the rest of the CP into three predominant protein species with molecular masses of ∼34, 27/29, and 25/26 kDa, respectively. We have now solved the crystal structure of VP90IMPORTANCE Human astrovirus (HAstV) is a leading cause of viral diarrhea in infants and young children worldwide. As a nonenveloped virus, HAstV exhibits an intriguing feature in that its maturation requires extensive proteolytic processing of the astrovirus capsid protein (CP) both inside and outside the host cell. Mature HAstV contains three predominant protein species, but the mechanism for acquired infectivity upon maturation is unclear. We have solved the crystal structure of VP9071–415 of human astrovirus serotype 8. VP9071–415 encompasses the conserved N-terminal domain of the viral CP. Fitting of the VP9071–415 structure into the cryo-EM maps of HAstV produced an atomic model for the T=3 icosahedral capsid. Our model of the HAstV capsid provides valuable insights into intermolecular interactions required for capsid assembly and trypsin-mediated proteolytic maturation. Such information has potential applications in the development of a VLP vaccine as well as small-molecule drugs targeting astrovirus assembly/maturation. Astroviridae family possess a nonsegmented, positive-sense, single-stranded RNA (ssRNA) genome with a nonenveloped icosahedral capsid  (Members of the l capsid . Astroviopy (EM) , 3. Humaopy (EM) . Human aopy (EM) , 7.1–415]), a hypervariable central region (VP90416–646), and an acidic C-terminal domain (VP90647–782) (–The ∼7-kb genomic RNA of human astrovirus is polyadenylated and contains three open reading frames (ORFs) , 9. ORF18–ion VP904–646, and647–782) . The aci647–782) , 18. Upo647–782) , 20  . When ov647–782) –23.1–415), which comprises the capsid shell , whereas both VP27/29 and VP25/26 are from the hypervariable region with a different N terminus, which forms the capsid surface spike , was crystallized as monomers with 1 molecule per crystallographic asymmetric unit (CAU). The structure of VP9071–415 shows two domains: an S domain that adopts the typical jelly-roll β-barrel fold and a P1 domain that has the appearance of a squashed β-barrel consisting of six antiparallel β-strands. A Dali search indicated that VP9071–415 is a close structural homolog of the HEV CP in spite of the lack of any detectable sequence similarity  (GenBank accession number AF260508) was cloned between the NcoI and HindIII sites of the vector pET28a (Novagen) by using forward primer 5′-GGCGCCCATGGAAAAACAAGGTGTCACAGGACCAAAACCT-3′ and backward primer 5′-GCGCCAAGCTTTTAGTGATGATGATGATGATGACCACCACCATGACCTAAACTAGGCTGATTCATC-3′.The coding sequence for HAstV-8 CPEscherichia coli by using the Rosetta 2(DE3) strain. Cells were first grown to an optical density at 600 nm (OD600) of 0.8 to 1.0 at 37°C and then induced with 1 mM IPTG  for 19 h at 30°C. Cells were harvested and lysed by using a sonicator (Branson 250 Sonifier) for 15 min at 4°C. The lysis buffer consisted of 300 mM NaCl, 50 mM Tris (pH 7.5), 5 mM 2-mercaptoethanol, 5 mM imidazole, 1 mM PMSF (phenylmethylsulfonyl fluoride), 0.5 μg/ml pepstatin, and 0.5 μg/ml leupeptin. The lysate was clarified by centrifugation at 12,000 × g at 4°C for 45 min. The supernatant was subjected to affinity purification using Ni-nitrilotriacetic acid (NTA) resin (Thermo Scientific). Bound protein was eluted by using an elution buffer containing 500 mM NaCl, 50 mM Tris (pH 7.5), 5 mM 2-mercaptoethanol, and 300 mM imidazole. Recombinant protein was further purified by ion exchange chromatography using a HiTrap Q HP column  and by size exclusion chromatography using a HiLoad Superdex 200 16/60 gel filtration column . The final protein was at least 95% pure as judged by SDS-PAGE. The protein concentration was determined by the A280 reading from a NanoDrop 2000/2000c spectrophotometer (Thermo Scientific). The molar extinction coefficient (ε) of CP71–415 was calculated to be 1.58 by the ExPASy ProtParam tool. Approximately 5 mg of purified protein could be obtained from 6 liters of cell culture.A 6×His tag and a GGG linker sequence were engineered at the C terminus of the construct to facilitate protein purification. Recombinant protein was expressed in 71–415 was overexpressed by using M9 minimal medium containing SeMet and a mixture of six other amino acids to prevent methionine synthesis (To obtain selenomethionine (SeMet)-labeled proteins, VP90ynthesis . SeMet-l71–415 proteins, both native and SeMet-labeled forms, were concentrated to 10 mg/ml and subjected to crystallization screening. The best crystals were obtained by the hanging-drop vapor diffusion method at 25°C by mixing 2 μl of the protein solution with an equal volume of reservoir solution containing 0.2 M ammonium phosphate and 22% polyethylene glycol 4000 (PEG 4000). For data collection, crystals were briefly soaked in a cryoprotectant made from mother liquor supplemented with 23% (vol/vol) glycerol and flash-cooled in a nitrogen cryostream. Diffraction data were collected at the Advanced Photon Source (APS)  Life Sciences Collaborative Access Team (LS-CAT) F line. For SeMet-labeled crystals, a single-wavelength anomalous dispersion (SAD) data set of 720 frames was collected at the peak wavelength for Se (0.97872 Å) by using a detector-to-crystal distance of 200 mm, an exposure time of 2 s, and an oscillation angle of 1°. All of the data were processed by using the HKL2000 program  and UCSF Chimera programs, respectively.Cartoon and surface representations were generated with the PyMOL (5IBV.Atomic coordinates and structure factors have been deposited in the RCSB Protein Data Bank (PDB) under accession number 71–415 , VP9071–313 , and VP9071–283 , were cloned and expressed in E. coli  and the P1 domain (residues 257 to 415) (10 helices from the GH loop). The P1 domain has an antiparallel β-barrel structure composed of seven β-strands  and three α-helices . The P1 domain is connected to the S domain through an asparagine-rich linker loop . A substantial amount of surface area  is buried between the S and P1 domains. This interaction, which is mostly hydrophobic in nature, is mediated by β(C)2(C) and the CD loop, EF loop, and GH loop from the S domain and the domain linker loop, β9, β12, β13, β15, α4, and α5 from the P1 domain. The C terminus of the P1 domain is located externally near the S-P1 domain interface. In the astrovirus capsid, the C terminus of P1 is expected to connect to the P2 domain, which forms the dimeric spikes on the outer surface of the viral capsid (The structure of VP90 to 415)  and c. T to 415) . The sur71–415 was best aligned to the jelly-roll domain of carnation mottle virus (CMV)  (Z score = 16.0) (Z score = 15.8) (Z score = 15.7) (Z score = 15.6) (Z score was HEV (Z score = 7.0). When both the S and P1 domains of VP9071–415 were used as the search model, HEV came up with the highest Z score, 27.0, which was followed by CMV (Z score = 16.0), TBSV (Z score = 14.4), RMV (Z score = 14.0), and OV (Z score = 13.8)  , TBSV (Z = 16.0) , ryegras = 15.7) , and HEV = 15.6) , 32. Whe = 13.8) . The fin = 13.8) .71–415 allowed us to build an atomic model of the astrovirus capsid. Together with the crystal structure of the capsid spike (Structures of both immature human astrovirus (HAstV-8) (EMD-5414) and mature human astrovirus (HAstV-1) (EMD-5413) have been established by cryo-EM reconstruction to a 25-Å resolution . Therefo71–415 fits well into the EM maps of the immature and mature astrovirus capsids, producing nearly identical models. Due to the lack of a meaningful difference, only the mature capsid model is presented . Thresented  and b. Aresented  and e. S71–415 dimer buries the smallest amount of surface areas among all types of capsomeres found in the T=3 capsid. There are two types of dimers: one that is relatively flat, sitting on 2-fold axes , and another that has an inwardly bent conformation, located on quasi-2-fold axes . Due to the different bending angles, we observed dramatic differences in the gap distances between the two P1 domains in the two types of dimers, with ∼5 Å for the C-C dimer and ∼25 Å for the A-B dimer  between adjacent subunits  facing the surface depression on 5-fold and quasi-6-fold symmetry axes  that closely interacts with the S domain from the opposing side. Therefore, in principle, VP27 could remain tethered to the capsid through the β-sheet interaction mediated by β15. In comparison, VP25 starts its N terminus at residue 424, which is beyond the P1 domain, and therefore would lose its grip on the capsid after its cleavage  through a number of intermediates, including VP38.5 . Why is 71–415. In the context of the capsid, the most susceptible sites are R359 and R361, which are located in a structurally flexible loop hovering over the P1 domain  that are potentially susceptible to trypsin. These six residues are all surface exposed in the crystal structure of VP901 domain  and 5. T1 domain , suggest1 domain .71–415 will provide a valuable structural framework for further biochemical and genetic analyses to pinpoint cleavage sites essential for astrovirus infectivity and the associated mechanisms.The proteolytically induced maturation of the capsid results in a >100-fold increase in infectivity , 24. TheE. coli formed predominantly dimers (data not shown). Comparison of the crystal structure of the HEV VLP and the HAstV capsid model shows that the HAstV CP-CP interaction around the 3-fold symmetry axis is substantially weaker, with ∼2,000 Å2 of buried surface , compared to ∼3,000 Å2  in HEV. Therefore, the lack of efficient assembly of astrovirus VLPs may be due partly to a weak interaction around the 3-fold symmetry axis.Recombinant human astrovirus proteins, in the form of either VP70 or VP90, with or without the first 70 amino acid residues, have been found to self-assemble into VLPs when overexpressed in mammalian cells –23. It i–71–415 capsid model should be able to identify additional mutations that favor trimer formation. The ability to obtain large quantities of VLPs would greatly facilitate detailed characterization of the astrovirus capsid function and the maturation mechanism. Furthermore, development of VLPs that can encapsidate exogenous nucleic acids would allow them to be used as delivery agents of RNA or DNA for different purposes.Several approaches may help to enhance the efficiency of astrovirus VLP assembly. For instance, peptide sequences can be either an insertion or conjugated to the N/C terminus of the astrovirus CP to promote trimer formation. Disulfide bond engineering at the trimer interface may also help to stabilize astrovirus CP interactions. Furthermore, molecular modeling based on our VP9071–415 was reported (5EWN contains two independent HAstV-1 VP9071–415 molecules. Superimposition of HAstV-8 VP9071–415 onto each of the two HAstV-1 VP9071–415 molecules gives average root mean square deviations (RMSDs) of 0.92 Å and 0.90 Å for 324 common Cα atoms, which are slightly higher than the average RMSD of 0.83 Å from superimposing the two HAstV-1 VP9071–415 molecules. Major variations between the two VP9071–415 structures from the two different serotypes are mapped to the following four regions: the S-P1 domain linker, the loop connecting α3 and β12, the loop connecting β13 and β14, and the loop connecting α5 and β15 (Near the end of manuscript preparation, the crystal structure of HAstV-1 VP90reported . The str and β15 . Additio and β15 .71–415 that are highly variable among the eight HAstV serotypes, HAstV-8 VP9071–415 is colored according to the level of sequence conservation (To highlight sequence regions of VP90ervation  and c. T


Through interaction with various other proteins, DnaA also controls the frequency and/or timing of chromosomal replication at the initiation step. Escherichia coli DnaA also recruits DnaB helicase, which is present in unwound single-stranded DNA and in turn recruits other protein machinery for replication. Additionally, DnaA regulates the expression of certain genes in E. coli and a few other species. Acting as a multifunctional factor, DnaA is composed of four domains that have distinct, mutually dependent roles. For example, C-terminal domain IV interacts with double-stranded DnaA boxes. Domain III drives ATP-dependent oligomerization, allowing the protein to form a filament that unwinds DNA and subsequently binds to and stabilizes single-stranded DNA in the initial replication bubble; this domain also interacts with multiple proteins that control oligomerization. Domain II constitutes a flexible linker between C-terminal domains III–IV and N-terminal domain I, which mediates intermolecular interactions between DnaA and binds to other proteins that affect DnaA activity and/or formation of the initiation complex. Of these four domains, the role of the N-terminus (domains I–II) in the assembly of the initiation complex is the least understood and appears to be the most species-dependent region of the protein. Thus, in this review, we focus on the function of the N-terminus of DnaA in orisome formation and the regulation of its activity in the initiation complex in different bacteria.The primary role of the bacterial protein DnaA is to initiate chromosomal replication. The DnaA protein binds to DNA at the origin of chromosomal replication ( N-. N-oriC oriC (E. coli consensus sequence: 5′-TTATNCACA-3′). Domain III belongs to the ATPases Associated with diverse cellular Activities (AAA+) class of proteins; upon interaction with adenosine triphosphate (ATP), but not adenosine diphosphate (ADP), domain III changes conformation to enable the protein to properly oligomerize into a filament. The structure of such a filament bound to dsDNA and the means by which DnaA melts oriC is not fully understood. Nonetheless, the interaction between DnaA monomers within the filament introduce a conformational change in the bound DNA to melt its double-stranded structure at the DUE [E. coli DnaA domain III, together with domain I, recruits DnaB helicase to an open complex and helps position the helicase onto the ssDNA [E. coli and possibly in Caulobacter crescentus or the sporulation initiation inhibitor protein Soj and the initiation-control protein YabA in B. subtilis [E. coli DnaA, domains III and IV are sufficient in vitro for opening the oriC region; i.e., proteins that lack domains I and II unwind oriC in vitro in a manner similar to that of the full-length protein [E. coli DnaA is required to maintain its function in bacterial cells. Indeed, it has been shown that DnaA domain I, similar to domain III, mediates interactions between DnaA monomers and interacts with other proteins, including the helicase DnaB (see below).Domain IV is responsible for DNA binding via a helix-turn-helix motif . The dom the DUE ,19,20. S the DUE ,22,23,24he ssDNA ,26; howehe ssDNA ,28,29,30subtilis ,33,34,35 protein . HoweverE. coli through mutually exclusive interactions with regulatory proteins and DnaB. Altogether, the findings indicate that domain I is important for the activity of DnaA at the orisome.Although the N-terminal domain is crucial for DnaA activity in vivo, its role in orisome formation is the least understood of the four domains. The reason for that is, in part, related to the lack of structure of full-length DnaA. The structure of the N-terminal portion of DnaA ,38, whicE. coli, B. subtilis, H. pylori, and Mycoplasma genitalium DnaA domain I have been solved; for the last, however, no functional analyses have been performed to date. Despite high sequence diversity  [The structures of iversity , domain iversity . E. coliβ2-α3-β3 ,38. H. p1 and α2 , and B. 3 and β3 ; M. genimonomers ,42,43. Ndimerize . The α2 and DnaB ,44, the A (HobA) , and theA (HobA) , respectA (HobA) ,44.E. coli DnaA weakly interacts with ssDNA [It has been reported that the structure of DnaA domain I is similar to the K homology domain (KH domain) ,40. KH dth ssDNA , though th ssDNA ,36,46. TDomain II is unstructured and the most variable in sequence . AccordiE. coli is a gram-negative, non-sporulating, facultatively anaerobic bacterium. Although E. coli constitutes a natural microflora in the lower intestine of warm-blooded organisms, including humans, some strains are pathogenic. This bacterium can survive and multiply outside of its host despite a decline in growth over time. The genomes of natural isolates of E. coli range from 4.5 to 6.0 Mb and encode approx. 4200–6500 genes. The bacterium has been used as a model organism for studying bacterial processes, including chromosomal replication and the cell cycle. Therefore, E. coli DnaA is one of the best characterized initiator proteins, especially within the context of structure-function relationships. In fact, studies on E. coli DnaA pioneered work on other initiators, including those in Archaea and Eukaryota. The resolved structure of E. coli DnaA domain I (1–86 aa) complements comprehensive biochemical data collected to date. It has been shown that domain I is engaged in numerous protein-protein interactions that include other DnaA monomers, as well as proteins that regulate DnaA activity at the orisome . Domain I of E. coli DnaA also participates in recruiting the replisome protein DnaB helicase; thus, it is important for the transition between the initiation and DNA synthesis (elongation) phases of replication.E. coli DnaA, possibly due to dimerization of domain I, mediate long-distance interactions between DnaA monomers (S. coelicolor (see below), and that this interaction facilitates or stabilizes DnaA binding to distantly located DnaA binding sites [oriC, possibly by bringing DnaA monomers into a closer contact so they can make a filament via domain III . Hda consists of an N-terminal β-clamp-binding consensus sequence and the AAA+ domain, which shares homology with DnaA domain III. Hda-ADP in a complex with a β-clamp of DNA polymerase III interacts with DnaA domains I, III, and IV shortly after initiation ,58, and  to DnaA ,59. DnaA to DnaA ,60,61. I to DnaA . Intereslication . Such anlication ,63. It wlication .oriC [E. coli during logarithmic growth.The molecular mechanisms of DnaA domain I interactions with HU, Dps, L2, and YfdR, and their roles in the initiation of chromosomal replication are much less understood than those described above. The HU protein is a DNA-binding protein that functions in compaction of the bacterial chromosome (by inducing DNA bends) and regulates DNA-related processes, including replication and transcription . HU is coriC . In partoriC . MoreoveIn contrast to HU, proteins Dps, L2, and YfdR inhibit initiation ,69,70. DoriC and thus inhibits DnaA-dependent DUE unwinding. Thus, L2 interferes with prepriming complex formation because it precludes DnaB loading, which is required for further replisome assembly. It has been suggested that L2 coordinates replication with transcription under specific, yet unknown, conditions.L2 is a ribosomal protein that has recently been shown to interact with the N-terminus of DnaA . In vitrE. coli DnaA in a Phe46-dependent manner [YfdR, a protein encoded by a set of genes of the cryptic phage CPS-53, binds to domain I of t manner . ConsistB. subtilis is a gram-positive soil bacterium that sporulates under suboptimal growth conditions [B. subtilis range from 4.0 to 4.3 Mb and encode approx. 4000–4500 genes. Many B. subtilis cellular processes, including chromosomal replication, adjust to environmental conditions to promote vegetative growth, sporulation, or spore germination. Accordingly, a master Spo0A regulator, which is responsible for entry into sporulation, directly controls the activity of oriC [B. subtilis oriC is bipartite, i.e., it contains two clusters of DnaA boxes separated by a dnaA gene; both clusters are required for the initiation of chromosomal replication in vivo [dnaA gene [B. subtilis DnaA-ATP has been shown to interact with oriC in a manner characteristic of AAA+ proteins; upon orisome assembly, DnaA-ATP forms a helix-like structure that unwinds DNA and binds to ssDNA [B. subtilis DnaA has a predominant role in DnaA filament assembly and is thus a target for binding numerous regulatory proteins, such as Soj, YabA, and the primosomal protein DnaD, none of which is found in E. coli [B. subtilis DnaA domain III is the best characterized domain of the entire DnaA protein, whereas the roles of the other domains in the formation and activity of the initiation complex are much less understood. Knowledge of the role of the B. subtilis N-terminal domains  in orisome assembly is particularly scarce. It is known that the N-terminal domains are not required for filament formation and ssDNA binding by B. subtilis DnaA in vitro [B. subtilis DnaA domain I dimerizes. Most residues involved in the dimerization of E. coli DnaA domain I are conserved in B. subtilis DnaA  have been reported [B. subtilis helicase is loaded onto ssDNA via a “ring-making” mechanism, which is different from the “ring-breaking” mechanism in E. coli [Unlike in reported ,85. More E. coli ,87. ThusH. pylori is a gram-negative pathogenic bacterium that resides in the human stomach, a relatively stable, albeit hostile, ecological niche [H. pylori range from 1.5 to 1.7 Mb and encode approx. 1400–1800 genes, with only a few regulatory proteins controlling cellular processes [H. pylori oriC resembles B. subtilis oriC, i.e., it is bipartite and consists of two clusters of DnaA boxes, oriC1 and oriC2, separated by a dnaA gene [H. pylori oriC and DnaA-DNA interactions have recently been well characterized [H. pylori DnaA, particularly concerning domain III. For instance, it is not known whether H. pylori is regulated by ATP binding and hydrolysis, and no protein homologous to Hda has been found in H. pylori. Moreover, no proteins interacting with domain III of H. pylori DnaA have been identified thus far. As domain III is highly homologous among species, it likely forms a filament that is typical of DnaA. The N-terminus of H. pylori DnaA has been relatively well characterized. It comprises 110 amino acids  and does not self-associate [H. pylori DnaA domain I interacts with HobA, a protein essential for H. pylori survival. To date, HobA is the only known protein that interacts with DnaA, and it influences DnaA assembly at oriC [oriC1 [E. coli DiaA [E. coli DnaA and HobA–H. pylori DnaA interaction surfaces co-evolved [H. pylori DnaA binding to oriC, whereas DiaA increases but decelerates E. coli DnaA binding to oriC. Interestingly, the kinetics of responses involving domains III–IV do not depend on the stimulating protein (DiaA or HobA). In a hybrid system in which E. coli domain I was fused to domains II–IV of H. pylori DnaA (EcIHpII-IVDnaA), DiaA stimulated EcIHpII-IVDnaA in a manner similar to that of HobA stimulation of H. pylori DnaA, though with a sensitivity characteristic of DiaA [al niche ,89. The rocesses ,91. H. pnaA gene . The strcterized ,93,94,95 at oriC ,97. IndeC [oriC1 . Despiteoli DiaA ,98. Simi-evolved . Despite of DiaA . This suH. pylori DnaA or any domain of the DnaA protein participates in helicase loading onto an open complex because no DnaA-DnaB interactions, either between isolated proteins or within an orisome, have been shown thus far. Glu21, which is important for interactions of E. coli DnaA with E. coli DnaB, is present in H. pylori (Glu 25), but Phe46 is missing. It should be noted that H. pylori DnaB helicase is atypical, and unlike bacterial hexameric helicases, it forms a dodecamer that dissociates into hexamers upon interaction with DnaG primase [It is not known whether the N-terminus of  primase ,100. RegS. coelicolor is a gram-positive soil bacterium. It possesses a large, 9 Mb chromosome encoding approx. 8300 genes, which is almost twice as large as the E. coli or B. subtilis chromosome. S. coelicolor grows as substrate mycelia, which differentiate into an aerial mycelium and spores upon nutrient depletion. The key elements of the initiation of S. coelicolor chromosomal replication, DnaA and oriC, have been identified, and their interactions have been characterized [S. coelicolororiC contains two clusters of DnaA boxes separated by a short spacer DNA [B. subtilis and H. pylori, the number of distinct nucleoprotein complexes is higher in S. coelicolor (up to 4 complexes per hairpin) than in the other two bacteria (1 complex per loop), as visualized by electron microscopy [S. coelicolor DnaA is one of the largest known DnaA proteins (656 amino acids) due to the presence of a long domain II, which comprises an additional stretch (approx. 150 amino acids) of predominantly acidic amino acids. Such an exceptionally large domain II should enable DnaA dimers or oligomers to interact with distantly located DnaA boxes to establish a functional nucleoprotein complex. Domain I of the S. coelicolor DnaA protein dimerises [S. coelicolor DnaA. Thus, further studies are required to gain insight into protein-protein interactions that lead to assembly or regulation of a functional S. coelicolor orisome.cterized ,105,106.acer DNA ; in totacroscopy ,92,106. E. coli, whereby green fluorescent protein (GFP) of 238 amino acids was inserted into domain II or into the C-terminal region of domain I (right after β3), without the loss of DnaA functionality in vivo [E. coli protein. In addition, comprehensive deletion analysis within domain II of E. coli DnaA showed that at least 21–27 residues are required to sustain the correct conformation of the entire protein, possibly because they properly align domain I with domains III–IV [E. coli domain II resulted in an under-initiation phenotype [oriC, it is tempting to speculate that the length of domain II is adjusted according to the spacing between DnaA boxes. Regarding this hypothesis, the S. coelicolor DnaA protein can bind to widely spaced DnaA boxes due to the presence of a long domain II, whereas the H. pylori DnaA protein, with a relatively short domain II, binds to closely spaced H. pylori DnaA boxes [H. pylori DnaA does not dimerise  and the transition from the initiation to the DNA synthesis step. The N-terminal domains are also engaged in coordinating chromosomal replication with the cell cycle  and other cellular processes  or environmental conditions .The N-terminal domains of bacterial DnaAs are essential for full protein activity upon initiation of chromosomal replication, ensuring cooperativity of the protein in DNA binding and correct spatial assembly at oriCs, the mechanisms of replisome assembly and the strategies of regulating DnaA activity. However, there are relatively few experimental data that assert the general features of the N-terminal domains with respect to the structure-function relationship of orisomes in different species. Nonetheless, dimerization and interaction with other proteins are the most conservative features of domain I. Domain II serves as a linker that coordinates the function of largely independent domains I, III, and IV.It should be noted that the N-terminal domains exhibit the least conserved sequence , and accE. coli and S. coelicolor DnaAs dimerize. Helix α1 is crucial for dimerization in E. coli, but amino acids and interaction surfaces involved in S. coelicolor DnaA dimerization are unknown. In contrast, H. pylori DnaA domain I was shown not to interact, and there are no data regarding the dimerization of B. subtilis DnaA domain I. It was proposed that domain I dimerization and a sufficiently long, flexible domain II help to establish long-distance interactions. Thus, it was suggested that for some orisomes, domain I dimerization is not important when DnaA boxes are closely spaced at oriC, such as for H. pylori oriC [H. pylori DnaA participates in long-distance interactions between DnaA-oriC1 and DnaA-oriC2 subcomplexes [H. pylori, B. subtilis, and other bipartite orisomes  [It was experimentally shown that domain I in ori oriC ,95,113. omplexes , raisingacteria) ,114.E. coli DiaA, H. pylori HobA, and B. subtilis SirA) is mediated by helices α2 and α3, which likely comprise a common interface for protein-protein interactions  or H. pylori (BsIHpII-IVDnaA)), in which domain I is swapped for B. subtilis domain I. Such proteins should be able to interact with SirA, and this interaction could possibly destabilize orisomes formed by chimeric DnaAs.Interaction of DnaA domain I with other proteins , the loading proteins , or other species-specific factors.Interaction between DnaA domain I and the helicase has only been demonstrated for cus DnaA , and Dnalis DnaA ,79). It red DciA ), the orE. coli DnaA and initiators from other species is especially puzzling. Within this context, the N-terminus of E. coli DnaA appears to be an omnipotent domain. However, within the context of environmental challenges, physiology, and genetics, E. coli is not that different from other species, particularly B. subtilis or S. coelicolor. This makes it difficult to justify such an increase or decrease in the properties or interaction partners , the nuteolysis .E. coli DnaA-DiaA and H. pylori DnaA-HobA heterocomplexes have revealed relatively high specificity of interactions between initiation proteins [Functional and structural studies on proteins . This fiproteins ,118, amo


A transcription factor that regulates skeleton formation in sea urchin embryos has evolved a new domain that is essential for this process. Related research article Khor JM, Ettensohn CA. 2017. Functional divergence of paralogous transcription factors supported the evolution of biomineralization in echinoderms. eLife6:e32728. doi: 10.7554/eLife.32728As an embryo develops, complex regulatory networks control where and when genes are activated, resulting in tissues and organs forming at the right time and place. Changes to these networks, in particular to DNA sequences that bind transcription factors, can affect how an organism develops and looks .Transcription factors are proteins that help turn specific genes on or off by binding to nearby DNA, and a somewhat controversial theory suggests that changes affecting the strength of transcription factor binding may modify regulatory networks . Such chSea urchins are a popular model organism in developmental biology and like most other echinoderms, the larvae of sea urchins are very different to their adult form. Sea urchins develop from an egg into a planktonic larva, before transforming into a bottom-dwelling juvenile, and unlike most echinoderms, they form a larval skeleton in the early embryonic stages .A well-studied group of cells called the micromeres are key to this process, and are the first cells internalized as the embryo acquires its form . The molOne key transcription factor in this regulatory network is Alx1, which is exclusively activated or expressed in the micromeres soon after they form . PreviouKhor and Ettensohn blocked Alx1 with a compound called a morpholino, and at the same time, injected the embryo with a version of Alx1 that is insensitive to this substance and restores skeleton formation. To identify the roles of the Alx1 protein, they deleted or mutated various parts of the morpholino-insensitive Alx1 and tested if the embryos were still able to build a skeleton. Most parts of the protein were dispensable, but a small domain unique to Alx1, called Domain 2, turned out to be essential for skeleton formation. Furthermore, Khor and Ettensohn discovered that when Domain 2 was inserted into Alx4, which is an adjacent copy of Alx1, sea urchins were able to form a normal skeleton.Khor and Ettensohn then compared the genomes of other echinoderms and discovered that Alx4 was highly conserved within all members of this group, whereas Alx1 varied greatly. The Alx1 proteins of close relatives of the sea urchin were functionally interchangeable, while the Alx1 proteins of more distantly related echinoderms were not. Khor and Ettensohn suggest that this is due to differences in the regions outside Domain 2. Alx1 and Alx4 are thought to be the result of an ancient gene duplication, and the acquisition of Domain 2 may have determined their different roles .This study is a sterling example of a transcription factor altering its protein sequence and presumably its affinities, leading to the functional differences of Alx1 and Alx4. Khor and Ettensohn emphasize that to fully understand these evolutionary changes more research is needed to clarify how Alx4 contributes to the formation of the skeleton in adults. We also need to examine the protein structure of Alx1 more deeply to discover what Domain 2 interacts with, and how it initiates skeleton formation.The transcription factor Alx has the potential to become an informative model for transcription factor evolution because of its detailed gene regulatory network and the comparisons that can be made between species that diverged at different times. This will deepen our knowledge of how mechanisms beyond mutations in DNA sequences have shaped the evolution of gene regulatory networks.