Posttraumatic scarring of peripheral nerves produces unwanted adhesions that block axonal growth. In the context of surgical nerve repair, the organization of the scar tissue adjacent to conduits used to span the gap between the stumps of transected nerves is poorly understood. The goal of this study was to elucidate the patterns of distribution of collagen‐rich scar tissue and analyze the spatial organization of cells that produce fibrotic deposits around and within the conduit's lumen.Employing a rabbit model of sciatic nerve transection injury, we studied the formation of collagen‐rich scar tissue both inside and outside conduits used to bridge the injury sites. Utilizing quantitative immunohistology and Fourier‐transform infrared spectroscopy methods, we measured cellular and structural elements present in the extraneural and the intraneural scar of the proximal and distal nerve fragments.Analysis of cells producing collagen‐rich deposits revealed that alpha‐smooth muscle actin‐positive myofibroblasts were only present in the margins of the stumps. In contrast, heat shock protein 47‐positive fibroblasts actively producing collagenous proteins were abundant within the entire scar tissue. The most prominent site of transected sciatic nerves with the highest number of cells actively producing collagen‐rich scar was the proximal stump.Our findings suggest the proximal region of the injury site plays a prominent role in pro‐fibrotic processes associated with the formation of collagen‐rich deposits. Moreover, they show that the role of canonical myofibroblasts in peripheral nerve regeneration is limited to wound contracture and that a distinct population of fibroblastic cells produce the collagenous proteins that form scar tissue. As scarring after nerve injury remains a clinical problem with poor outcomes due to incomplete nerve recovery, further elucidation of the cellular and spatial aspects of neural fibrosis will lead to more targeted treatments in the clinical setting. In this study, we analyzed collagen‐rich deposits formed in response to the injury of the sciatic nerve. Utilizing microscopic and spectroscopic techniques, we determined the distribution and architecture of fibrotic deposits formed in the proximal and the distal stumps of the injured nerve. Among elements of the ECM, collagenous proteins play a crucial role by participating in the formation of neural basement membranes and contributing to the mechanical stability of the nerves. In addition to playing a physiological role, collagenous proteins, most notably fibrillar collagen I and collagen III, participate in the formation of unwanted neural scars , a collagen‐specific protein chaperone that is upregulated during scar formation in a variety of human tissues, including injured joint capsules and nerves, plays a key role. Because of its close association with the collagen production, HSP47 serves as a reliable marker of collagen‐producing cells , 4 weeks (n = 3), 6 weeks (n = 4), and 10 weeks (n = 4) after injury, and then, the corresponding segments of the injured and uninjured nerves were harvested for histological and spectroscopic analyses.Applying a muscle‐sparing approach, a surgeon accessed the proximal sciatic nerve of the right legs of 12‐month‐old White New Zealand female rabbits placed under general anesthesia  for myelin and picrosirius red for collagen fibrils. Subsequently, the samples were counterstained with hematoxylin. A microscope  was used to observe and photograph the nerve specimens. In addition, the samples stained only with picrosirius red were analyzed with a polarized light microscope , as described  mouse monoclonal anti‐HSP47 , (b) mouse monoclonal anti‐αSMA (Abcam), and (c) mouse monoclonal anti‐neurofilament marker (BioLegend Inc.). Subsequently, the primary antibodies were detected with specific secondary anti‐mouse antibodies conjugated to Alexa Fluor 594 or Alexa Fluor 488 (LifeSciences/Thermo Fisher Scientific).Sections of the nerves were double‐stained for both HSP47 and αSMA using mouse monoclonal anti‐HSP47 antibody  and goat polyclonal anti‐αSMA antibody (Invitrogen/Thermo Fisher Scientific). While HSP47 was visualized with a red fluorophore , αSMA was detected with a green fluorophore . Negative controls, in which primary antibodies were omitted, were also prepared and analyzed. These controls were prepared to exclude a possibility of positive staining due to nonspecific binding of the secondary antibodies. Following immunostaining, all samples were treated with 4',6‐diamidino‐2‐phenylindole (DAPI) to stain the nuclei. A fluorescence microscope  was employed to observe immuno‐stained samples. Table Green fluorescence filter was utilized to observe infiltrating eosinophils that fluoresce due to the presence of flavin adenine dinucleotide (FAD) and erythrocytes whose green autofluorescence is caused by peroxidation  the intraluminal compartment of the conduit, (ii) the extraluminal compartment of the conduit, (iii) the proximal stump of the transected nerve, (iv) the distal stump of the transected nerve, and (v) the intraneural area of an uninjured nerve .NIS Elements image analysis software (Nikon Inc.) was used to quantify the αSMA‐positive and the HSP47‐positive cells. In brief, specific areas of the cross‐sections of the nerves  were first selected by outlining regions of interest (ROI). Subsequently, the areas occupied by cells positive for αSMA or HSP47 were measured. Finally, the percent of each ROI occupied by the αSMA‐positive and the HSP47‐positive cells was calculated for each ROI.On average, we analyzed 8 histological sections (4 stained for αSMA and 4 stained for HSP47) per one proximal and one distal stump of a 4‐week‐old rabbit. Those 8 sections covered about 5–8 mm of each stump, measuring from the site of the incision. Please note that obtaining nerve sections of identical dimensions was virtually impossible due to the varying geometries of the sites of nerve injury and surrounding scar tissue formed in different rabbits.p ≤ .05 considered statistically significant . We examined the main effects of specific locations on the relative amounts of the αSMA‐positive and the HSP47‐positive cells. Also, we examined the interaction effect of the luminal localization  and the position of a stump  on the relative content of the αSMA‐positive or the HSP47‐positive cells present in the analyzed regions.The results were evaluated using ANOVA, with 2.8For the fourier‐transform infrared (FTIR) spectroscopy, 5‐μm thick sections of the proximal and distal stumps were prepared from the paraffin‐embedded samples. The sections were placed on MirrIR low‐e microscope slides to allow assays in the reflective mode (Kevley Technologies).−1 wavenumber spectral range, pixel resolution at 25 µm, with 32 scans per pixel, and spectral resolution at 8 cm−1.Following deparaffinization, we scanned the samples using an FTIR spectrometer , as previously described . Applying these settings, however, allowed us to utilize the full‐range of colors to mark areas of the scar tissue with varying collagen content.−1 (attributed to the C=O stretching vibration) provides valuable information about the content of proteins within analyzed regions. Analyzing the ratios of the amide I‐specific and collagen‐specific peaks (AI/C ratio) provides a reliable way to analyze a relevant collagen content directly in a tissue sample  intraluminal area of the proximal injured stumps, (b) intraluminal area of the distal injured stumps, (c) extraluminal area of the injury site of the proximal stump, (d) extraluminal area of the injury site of the distal stump, and (e) intra‐neural area of an uninjured nerve . The results were evaluated using ANOVA, with ≤0.05 considered statistically significant .33.1Collagen‐specific staining of the proximal and the distal sections Figure  collecteMoving 5–8 mm from the edges of the incisions deeper into the proximal and the distal stumps, we observed accumulation of the fibrotic tissue both outside and inside the conduits Figures  and 3. CAt 6 and 10 weeks after injury, analysis of the injury region demonstrated fibrillar deposits, abundantly present both inside and outside the conduit, as well as the original gap region (data not shown). We did not observe any significant presence of cells within the walls of the conduits at any time point of this study.3.2Morphological assays of samples collected 4 weeks after injury demonstrated the deposition of collagen‐rich fibrotic tissue within and around the nerves Figures  and 3. ATwo distinct patterns of organization of collagen fibrils were readily apparent: (a) circumferentially organized fibrils accumulated outside the conduits around the conduit, and (b) transversely organized fibrils accumulated inside the cross‐sectioned conduits Figure . Similar3.3While the picrosirius staining revealed the organization and distribution of the collagen fibrils, FTIR spectroscopy provided more quantitative information on the collagen‐rich deposits Figure . We compF = 20, p < .0001). The main effect of the luminal compartment  was also statistically significant, where the relative collagen content in the extraluminal space was lower than in the intraluminal space  = 4.28, p = .046). There was also a statistically significant interaction between the stump location  and the type of a luminal compartment  on the relative collagen content  = 12.335, p = .001).The main effect of the stump location  was statistically significant where the relative collagen content was higher in the proximal stump than in the distal stump  = 38.113, p < .0001). Table As control uninjured nerves include neither the extraneural scar tissue nor the proximal or distal segments defined by the incision site, we compared the relative collagen content in the intraluminal space of the injured nerves with that of the intrafascicular space in control nerves. We found that the collagen content was higher in the regions encompassing the sites of injury than in healthy control  or its length . Both the perpendicular and the parallel orientations of the vessels were confirmed by observing the longitudinal sections Figure . We obse3.5F = 4.746, p = .033). The main effect of the luminal location  on the percent area occupied by the αSMA‐positive cells was not statistically significant  = 0.114, p = .737). The percent area occupied by the αSMA‐positive cells present in the extraluminal space, however, trended higher compared with the intraluminal space  = 0.311, p = .597).We also performed quantitative assays of the amount of the αSMA‐positive cells as the function of their location in the proximal and the distal stumps, as well as within the intraluminal and the extraluminal spaces of the conduits. The main effect of the stump location  on the area occupied by the αSMA‐positive cells was statistically significant such that the proximal stump contained a higher percentage of the αSMA‐positive cells than the distal stump  = 5.886, p = .019). In contrast to the vessel‐associated distribution of the αSMA‐positive signals, the HSP47‐positive fibroblasts were widely distributed in the scar tissue formed outside and inside the conduits  = 27.1, p < .0001). Similarly, the percentage of the HSP47‐positive cells was significant higher in the extraluminal space than in the intraluminal space  = 15.452, p < .0001). There was no statistically significant interaction between the stump location and the luminal compartment  = 0.072, p = .79).Quantitative assays were also performed to measure the percentage of the HSP47‐positive cells in the proximal and the distal stumps, as well as within the intraluminal and the extraluminal spaces of the conduits. The percentage of the ROI area occupied by the HSP47‐positive cells was significantly higher in the proximal stump than in the distal stump  = 22.934, p < .0001). Finally, in all of the analyzed regions encompassing the sites of injury, the overall percentage of the ROIs occupied by the HSP47‐positive cells was higher than the percentage occupied by the αSMA‐positive cells  = 124.9, p < .0001). Table We also compared the percentage of the HSP47‐positive cells present in the intraluminal space of the injured nerves with that of the intrafascicular space in control nerves. The relative content of HSP47‐positive cells was significantly higher in the analyzed ROIs in the regions encompassing the sites of injury than in the healthy control , neural growth factor (NGF), and insulin growth factor 1  Additional groups of animals must be employed to evaluate the formation of collagen‐rich deposits at earlier and later stages of peripheral nerve regeneration. (b) We have to include assays of noncollagenous elements of the scar tissue to fully understand the processes of nerve repair and the formation of complex neural scars. (c) It will be necessary to introduce a control group in which injured nerves will be repaired without conduits and with conduits fabricated from synthetic materials. One possible option could be to bridge a gap with an autograft or silicone tubes. Introducing these additional groups would make it possible to determine further the role of the conduits in repairing the peripheral nerves. (d) This study does not identify the origin of HSP47‐positive cells. (e) To fully comprehend the role of the scar formation, it will be necessary to perform functional studies of regenerated nerves and compare results as the function of surgical techniques used to bridge the gaps. (f) Although this study was carried out in a relevant animal model, its finding may not fully represent processes occurring in human patients.Based on our results, it appears that the proximal region of the peripheral nerve at the injury site plays an important role in the scarring processes associated with the formation of collagen‐rich deposits. Scarring after peripheral nerve injury and surgery remains a common clinical problem with substantial morbidity and poor outcomes. It is hoped that a better understanding of the mechanisms that caused peripheral nerve fibrosis will lead to novel treatments that may improve outcomes of treatment in the clinical setting.5The results of our study reveal patterns of deposition and the architecture of collagen‐rich scar tissue formed in response to injury of the sciatic nerve. These results indicate significant differences in the scar deposits formed in the proximal and the distal stumps, inside and outside the injured nerves surgically repaired using conduits. Furthermore, our study demonstrated that the role of canonical pro‐fibrotic myofibroblasts in scarring of peripheral nerves might be limited to contracting the wound rather than excessive production of collagen‐rich fibrotic matrices. Further studies will reveal the role and origin of fibroblastic cells responsible for robust scar formation due to the injury of peripheral nerves.The authors declare no conflict of interest.A. Fertala, J. Fertala, A. Steplewski: Substantial contribution to the acquisition, analysis, and interpretation of data. Contributed to drafting the manuscript. M. Wang, M. Rivlin, P. Beredjiklian: Significant contribution to designing an animal model of sciatic nerve injury and surgical procedures. Substantial contribution to the acquisition and interpretation of data form an animal model. Participated in drafting the manuscript. All authors have read and approved the final submitted manuscript.https://publons.com/publon/10.1002/brb3.1802.The peer review history for this article is available at Supplementary MaterialClick here for additional data file.


E. coli, two of the three amidases responsible (AmiA and AmiB) are regulated by a single periplasmic activator (EnvC) that is, itself, controlled by an atypical ABC transporter (FtsEX) tethered to the cytoplasmic septal Z-ring. Here we define the structural basis for the interaction of FtsEX with EnvC and suggest a molecular mechanism for amidase activation where EnvC autoinhibition is relieved by ATP-driven conformational changes transmitted through the FtsEX-EnvC complex.The peptidoglycan layer is a core component of the bacterial cell envelope that provides a barrier to the environment and protection from osmotic shock. During division, bacteria must break and rebuild the peptidoglycan layer to enable separation of daughter cells. In  Escherichia coli, FtsEX is required to separate daughter cells after cell division and for viability in low-osmolarity media. Both the ATPase activity of FtsEX and its periplasmic interaction with EnvC are required for amidase activation, but the process itself is poorly understood. Here we present the 2.1 Å structure of the FtsX periplasmic domain in complex with its periplasmic partner, EnvC. The EnvC-FtsX periplasmic domain complex has a 1-to-2 stoichiometry with two distinct FtsX-binding sites located within an antiparallel coiled coil domain of EnvC. Residues involved in amidase activation map to a previously identified groove in the EnvC LytM domain that is here found to be occluded by a “restraining arm” suggesting a self-inhibition mechanism. Mutational analysis, combined with bacterial two-hybrid screens and in vivo functional assays, verifies the FtsEX residues required for EnvC binding and experimentally test a proposed mechanism for amidase activation. We also define a predicted link between FtsEX and integrity of the outer membrane. Both the ATPase activity of FtsEX and its periplasmic interaction with EnvC are required for resistance to membrane-attacking antibiotics and detergents to which E. coli would usually be considered intrinsically resistant. These structural and functional data provide compelling mechanistic insight into FtsEX-mediated regulation of EnvC and its downstream control of periplasmic peptidoglycan amidases.FtsEX is a bacterial ABC transporter that regulates the activity of periplasmic peptidoglycan amidases via its interaction with the murein hydrolase activator, EnvC. In  FtsEX is a type VII ABC transporter , 2 belon7Streptococcuspneumoniae, FtsEX interacts directly with peptidoglycan hydrolases such as PcsB . Crystals of the EnvC-FtsX periplasmic domain complex belong to space group P212121 with two molecules of the FtsX periplasmic domain and one molecule of EnvC in the asymmetric unit. Analysis of protein contacts using PISA . Occupation of the amidase-binding site by the restraining arm strongly suggests that EnvC has been captured in an autoinhibited state that would necessitate a conformational change in order to bind and activate its cognate amidases.The C-terminal LytM domain of EnvC has an endopeptidase-like fold belonging to the M23 peptidase family . The LytSI Appendix, Fig. S1A). The heptad repeats fall within two stretches on either side of the linker and are clearly important for formation of the coiled coil. We analyzed the composition of the heptad repeats  and their positions within the EnvC structure . The first residue of each heptad is typically a leucine, isoleucine, or valine, and the fourth and fifth positions are most often occupied by either leucine or glutamine . EnvC has 12 heptad repeats within long helix I  and another 9 heptads within long helix II . Consistent with formation of the antiparallel coiled coil, the first, fourth, and fifth positions of each heptad mediate interhelix contacts through classical knobs-into-holes packing  that are joined by a short linker A. The li packing —the firsSI Appendix, Fig. S1C and mapped to the EnvC structure in SI Appendix, Fig. S1D. Heptads 4 to 12 pair with heptads 14 to 21 in the coil, but heptads 1 to 3 and 22 to 25 are unpaired. Heptads 1 to 3 are located at the EnvC N-terminus and face the solvent while heptads 23 to 25 are located within the restraining arm facing toward the interior of the LytM domain . Heptad 22 forms a linker that joins the coiled coil to the restraining arm. The unpaired heptad repeats found within the EnvC N-terminus and the restraining arm are both highly unusual and well conserved, suggesting they are mechanistically important.The 25 heptads are shown schematically in 2. The FtsX monomer bound to the more N-terminal portion of EnvC (long helix I) is well ordered with B-factors that are comparable to those of EnvC. The second monomer, which is bound to long helix II, has higher B-factors, indicating increased mobility—particularly for the first 40 residues. Nonetheless, FtsX residues contacting EnvC are properly resolved in the electron density for both FtsX monomers, giving confidence in the binding site structure and assigned stoichiometry (Movie S1).The two FtsX periplasmic domains bound to EnvC are structurally similar to one another and can be superposed with an rmsd of 1.5 ÅE. coli FtsX fragments are similar to those of Mycobacterium tuberculosis . The FtsX periplasmic domain comprises two βαβ secondary structure motifs that interlock to form a central four-membered β-sheet with two flanking helices. This domain is homologous with the Porter domain found in MacB and LolC and is a conserved feature of all type VII ABC transporters  which partially wraps around each of the two long helices that make up the EnvC coiled coil (2 and 932 Å2) and salt bridges are only found at the periphery of each binding surface . Association between FtsX and EnvC appears to be driven, primarily, by hydrophobic interactions between complementary surfaces formed as EnvC coiled coil helices slot between the X-lobe and central β-sheets of the FtsX periplasmic domains.The two FtsX-EnvC interfaces are shown in led coil D and E. S. pneumoniae FtsX are also perturbed by PcsB binding in NMR experiments  (ftsEX due to insertion of a kanamycin cassette) , do not . We then tested the periplasmic domain variants, knowing that the EnvC-binding function of FtsEX is dispensable for correcting the low-salt growth defect . Similar results were obtained for growth in low-salt broth, although FtsEX variants impaired in EnvC binding grew slower than the wild type in a pattern that broadly matched the degree to which binding was impaired . The results confirm that all 13 FtsEX periplasmic domain variants, including F152E and the X-lobe deletion (Δ145-171), are folded and expressed well enough to rescue growth on low-osmolarity media regardless of whether or not they can bind EnvC.We next characterized our series of interface mutants in the context of full-length FtsEX. We first tested each variants’ ability to support growth on low-osmolarity media since this is an established phenotype associated with FtsEX deficiency  and a usld type)  and MR10assette) , we estah defect . All intE. coli MR2 cells are short and rod shaped while cells lacking ftsEX (E. coli MR10) form long filaments or “chains”  or hydrolysis (E163Q) do not. Failure to complement for the K41A and E163Q variants (both located in the FtsE ABC domain) is not due to poor expression or misfolding since they retain a positive interaction with FtsX—as shown by a bacterial two-hybrid analysis (Minimum inhibitory concentration (MIC) determinations for vancomycin and bacitracin are shown in analysis E. These ftsEX-deficient strain for growth on solid agar containing either vancomycin or bacitracin . MIC determinations for SDS in broth are shown in ftsEX-deficient strains (0.02% SDS). Just as for daughter cell separation and antibiotic resistance phenotypes, detergent resistance can be restored to ftsEX-deficient strains by plasmid-borne ftsEX, but not by variants lacking ATPase activity (K41A) or that are unable to bind EnvC (Δ145-171 and F152E). Side-by-side comparison of each FtsEX variant’s viability on 0.1% SDS-agar confirms these results and additionally flags Y114E as a mildly disrupted interface variant . We have named this structural feature the restraining arm.Having structurally characterized the interaction between FtsEX and EnvC, and probed the interface by mutagenesis, we next considered how FtsEX-EnvC might interact with downstream amidases involved in peptidoglycan remodelling. Peptidoglycan amidases (such as AmiA and AmiB) interact with EnvC via a large groove in its LytM domain and several EnvC variants have been identified that prevent amidase activation without impeding EnvC’s proper localization to the site of division . We mappCenter column). We were also able to purify all three EnvC variants as His-tagged proteins, further confirming their stability (We hypothesized that the EnvC restraining arm has an autoinhibitory role that prevents binding and activation of amidases (AmiA and AmiB) in the absence of stimulation by FtsEX. Using the structure as a guide, we made three different EnvC protein constructs: the first being equivalent to the mature EnvC protein (residues 35 to 419); the second lacking the coiled coil domain, but retaining the restraining arm (residues 222 to 419); and the third comprising only the EnvC LytM domain (residues 278 to 419) D. We thetability F. We theWe next assessed the effect of periplasmic expression of each of our three EnvC constructs on bacterial viability. Cells carrying appropriate expression plasmids were plated-in-dilution on solid agar in which an inducing agent  was either present or absent G. Cells E. coli peptidoglycan amidases  for interaction with the murein hydrolase activators (EnvC and NlpD), or their respective LytM domains (EnvC-LytM and NlpD-LytM). Our prediction was that each amidase should bind its cognate activator’s LytM domain, but not the full-length activators, as they retain autoinhibitory elements. We found that both AmiA and AmiB bind to the EnvC LytM domain, and AmiC binds to the NlpD LytM domain, but none of the amidases interact with full-length EnvC or NlpD .To better understand the location and orientation of EnvC when bound to FtsX, we constructed a molecular model of FtsEX-EnvC based on the crystallographic data presented here and preexisting structures of the homologous ABC transporter, MacB . Models ng Phyre  and the ng Phyre A. Interang Phyre  . The model makes three useful predictions relating to function. Firstly, the location of EnvC places the LytM domain a considerable distance from the cytoplasmic membrane, close to the expected location of the peptidoglycan layer. Secondly, the model predicts that interaction with EnvC occurs solely via the FtsX periplasmic domain located between transmembrane segment 1 and 2 and does not require the extracytoplasmic loop between transmembrane helices 3 and 4. Finally, the model suggests a continuous route by which ATP-driven conformational changes in FtsEX can be propagated across the membrane and through the coiled coil domain of EnvC  might be induced to form an extension of the existing coiled coil by packing against the exposed EnvC N-terminus A and B. Movie S6. Three-dimensional (3D) models for the proposed ADP-bound autoinhibited state and the active ATP-bound state are also shown in A hypothetical model showing precisely how a conformational change might be propagated through FtsEX-EnvC is shown in S. pneumoniae . The MacB dimer engages with the MacA hexamer (2:6 stoichiometry)   via an eiometry) . As showIn summary, the structure of EnvC bound to the FtsX periplasmic domains provides valuable structural insight into the bacterial FtsEX-EnvC complex and its roles in peptidoglycan hydrolase activation.SI Appendix. Copurification of the histidine-tagged FtsX periplasmic domain and nontagged EnvC used Ni-IMAC after coexpression in E. coli C43(DE3) from pETDuet1-based vector. The structure of EnvC in complex with the FtsX periplasmic domain was determined by X-ray crystallography using tools from the CCP4 software suite  and MR10 (MG1655 ΔLacI ΔFtsEX::KanR) with wild type or variant ftsEX genes in pETDuet1. Growth in low-salt broth was monitored in LBON50  at 37 °C. Minimum inhibitory concentrations were determined in LB supplemented with 50 μg/mL ampicillin and 1 mM IPTG at 37 °C. Modeling of FtsEX based on structures of MacB used Phyre (Full methods are available in re suite –44. Phasre suite  and M. tc domain  as searcc domain , with E.ed Phyre  and coeved Phyre . Structued Phyre .Supplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary File


The interaction in multisubunit non‐ribosomal peptide synthetases (NRPSs) is mediated by docking domains that ensure the correct subunit‐to‐subunit interaction. We introduced natural docking domains into the three‐module xefoampeptide synthetase (XfpS) to create two to three artificial NRPS XfpS subunits. The enzymatic performance of the split biosynthesis was measured by absolute quantification of the products by HPLC‐ESI‐MS. The connecting role of the docking domains was probed by deleting integral parts of them. The peptide production data was compared to soluble protein amounts of the NRPS using SDS‐PAGE. Reduced peptide synthesis was not a result of reduced soluble NRPS concentration but a consequence of the deletion of vital docking domain parts. Splitting the xefoampeptide biosynthesis polypeptide by introducing docking domains was feasible and resulted in higher amounts of product in one of the two tested split‐module cases compared to the full‐length wild‐type enzyme. A breed apart: The non‐ribosomal peptide synthetase (NRPS) XfpS, which produces the peptides xefoampeptides A and B, can be artificially split without loss of biosynthetic activity by introducing docking domains into XfpS. In fact, in this case, the production of both peptides from the split system increased compared to product levels from the full‐length XfpS. Splitting XfpS into three subunits also led to a functional NRPS system. Several clinically used drugs including antibiotics and anticancer and immunosuppressive drugs are generated by non‐ribosomal peptide synthetases (NRPSs). While we understand very well how these megaenzymes work biochemically Figure , they arNDD from a hybrid system of a polyketide synthase and NRPS was shown to be appended to a Cyc domain.CDD‐NDD interactions for a complete NRPS system including the first structure of a CDD‐NDD complex have been defined for the rhabdopeptide‐synthesizing NRPS, detailing the key amino acids residues of the DD interaction surface.NDD of a C‐domain interacting with short linear motifs (SLiMs) as CDD of a T‐domain] have been observed in the enacyloxin biosynthesis,In contrast to COM domains, the structure of an elucidated X. bovienii. It comprises three modules that produce the main products XFP A (1) and XFP B (2), which are very stable in aqueous solutions and show weak bioactivity against Trypanosoma cruzi, and XFP production was known to be highly reproducible in E. coli. Even more importantly, XfpS, being a relatively small NRPS, encompasses the two typical module domain interfaces connected by DDs in Xenorhabdus, namely, the epimerization domain (E1) to the condensation domain (C2) and the thiolation domain (T2) to condensation domain (C3) interface ‐producing NRPS XfpS from ts XFP A  and XFP xfpS gene encoding the XFP‐producing NRPS XfpS was amplified from genomic DNA of X. bovienii, cloned into an expression vector under control of an l‐arabinose‐inducible PBAD promoter, and expressed in E. coli. Best production of XFP was achieved at 22 °C, which was used for all subsequent experiments also for other xfpS variants (see below). Cultures without the transcriptional activator l‐arabinose were devoid of any 1 and 2. Since we originally wanted to track the amount of soluble XfpS or its derivatives by western blotting, all XfpS variants were N‐ and C‐terminally tagged with a Strep‐Tag II.The X. bovienii biosynthetic gene cluster txlAB coding for the taxlllaid‐producing NRPS was selected,Xenorhabdus connecting E to C domains. The txlA ultimate 53 codons were fused following xfpS codon T1442, thereby introducing the txlACDD region and stop codon . The first 91 txlB codons were cloned in frame prior to xfpS codon Y1476, introducing the translational start codon, the translational start site, and the NDD coding region. The DDs for introduction into the T2‐C3 interface was drawn from the X. bovienii gene cluster paxABC homologue described for X. nematophila HGB081.paxB‐encoded CDD comprising 37 codons was integrated following xfpS L2504 codon insertion into the CDD region and a translational stop. The first 48 paxC codons were cloned in front of the xfpS codon F2526, including the start ATG and a Shine–Dalgarno region . The DD introduction resulted in two XfpS versions of XfpS non‐covalently connected by DDs, namely XfpS‐1 (first module of XtpS) to XfpS‐23 (XtpS module 2 and 3) and XfpS‐12 (XtpS module 1 and 2) to XfpS‐3  pairs known to produce peptides were selected (for more details see the methods section in the Supporting Information). For the E‐ to C‐domain split, a DD pair from the 1 and 2, respectively, when compared to the original wildtype NRPS  by T/C domain‐connecting DDs  NDDCDD. The NDD deletion . Amounts of XfpS‐3 in both experiments are similar and in the same molar range as XfpS‐12 .Finally, the two DD pairs used to create different two‐subunit XfpS variants were combined, leading to an NRPS comprising three individual stand‐alone modules XfpS‐1, ‐2, and ‐3) transcriptionally regulated by one , and ‐3 NDD interaction with the CDD could extent into structural parts of the T domain. Thus, construct n shifted the PaxA/B DD fusion point into α‐helix 4 and construct o exchanged the entire fourth T2‐α‐helix of XfpS . No production is detectable with n but production is restored to above 50 % levels relative to k for o, thus strongly suggesting a role for the T domain in the DD interaction. Furthermore, the product ratio in o shifts slightly back towards that of XfpS (a and b). The product ratio skew in all DD cases linking XfpS‐2 to XfpS‐3 constructs is interpreted as a measure for some sort of impaired movement of domains in the elongation cycle of the NRPS since all constructs dock and produce well above the DD‐deletion control h. Structural information about how the NDD interacts with the T domain will likely to be the way forward to solve this production impediment.Reflecting on the results of the T‐to‐C splits, we examined the recently published LgrA C‐domain/T‐domain interaction structural data.In this work the artificial splitting of a single multienzyme NRPS was achieved through the introduction of DDs whilst maintaining the biosynthetic activity and even increasing it in some cases. The quantification of peptide product in tandem with monitoring the in vivo soluble NRPS protein concentration is was valuable path to gain insight into decoding the reasons for NRPS biosynthetic activity. The chosen method to import the translational coupling capacity from a given gene cluster turned out to be successful. The DD‐deletion strategy in which a crucial translational stop was imported to the translational start junction of the translational initiation process was key to preserving the same number of NRPS subunits. The same numbers of subunits in turn was important for the analysis so that observed effects could be attributed only to the DDs and deletions within the DDs but not to different molar numbers of subunits. Deletion of large parts of the DDs did not terminate NRP biosynthesis entirely, in contrast to the total loss of enzymatic activity in the case of COM domains connecting TycAB.Kd value by more than twelvefold.e and f, in which the CDD‐NDD interaction partsThe DD‐deletion controls shed light on their role in coupling NRPS modules. They seem to be more important when connecting T with C domains and less important for the connection of E and C domains. Deletion of large DD parts never reduced product synthesis below 8 %. Deletion of 6 DD amino acids at their cognate cluster position in the COM‐TycAB caseThe presented data suggest that the artificial DD splitting approach between E‐ and C‐domain modules can be incorporated into the expanding efforts of de novo design of NRPSs.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


The aggregation of hyperphosphorylated tau into amyloid fibrils is closely linked to the progression of Alzheimer’s disease. To gain insight into the link between amyloid structure and disease, the three-dimensional structure of tau fibrils has been studied using solid-state NMR (ssNMR) and cryogenic electron microscopy (cryo-EM). The proline-rich region of tau remains poorly defined in the context of tau amyloid structures, despite the clustering of several phosphorylation sites, which have been associated with Alzheimer’s disease. In order to gain insight into the contribution of the proline-rich region P2 of tau to amyloid fibrils, we studied in vitro aggregated amyloid fibrils of tau constructs, which contain both the proline-rich region P2 and the pseudo-repeats. Using ssNMR we show that the sequence  These diseases see the common event of pathological aggregation of tau into amyloid fibrils, which further accumulate in different forms of aggregates, e.g. Neurofibrillary tangles (NFTs) and Neuropil threads (NTs), in the neurons3. Tau belongs to the family of the microtubule-associated proteins (MAPs)6 and it is mainly expressed in the axons of neuronal cells, where it interacts with microtubule filaments9. Six isoforms, differing in sequence composition, are expressed as the result of alternative splicing of exons 2 and 3 of the MAPT gene10. The longest tau isoform, called htau40, is a protein of 45.9 kDa comprising two insertions of 29 amino acids each (N1 and N2), a proline-rich region, which can be subdivided into a P1 and a P2 domain, five pseudo-repeats  and other neurodegenerative diseases, also known as tauopathies12. Several studies have been directed towards the identification of the protein domains responsible for its aggregation17. Tau is an intrinsically disordered protein with high solubility and a low aggregation propensity in vitro18. In vitro aggregation of tau has been traditionally achieved using polyanions like heparin20 and arachidonic acid21, which, due to their negative charges, promote tau aggregation by neutralization of the positive charges on tau. Biochemical studies could define that the rigid core of the fibrils is composed of the pseudo-repeat region and that the hexapeptide 22. Studies with small tau constructs evidenced a similar role for the hexapeptides bona fide fibrils23. NMR analysis of tau secondary structure in solution reported residual 26: these were hypothesized to provide hydrophobic contributions that in pathological conditions can enhance protein aggregation. The fuzzy coat, composed by the N-terminal domain, the proline-rich region and the C-terminal domain, maintains a higher degree of flexibility in the fibrils28; nevertheless transient contacts between the proline-rich region and the core of the fibrils could be detected using NMR spectroscopy14 supporting its possible roles in tau aggregation29. Solid-state NMR (ssNMR) spectroscopy, electron microscopy (EM), X-ray diffraction, electron paramagnetic resonance (EPR) and other biophysical techniques have been employed to gain insight into the structure of tau fibrils39. One of the challenging aspects that has made the structural characterization difficult is the observation that tau fibrils are often heterogeneous41.Tau fibrils are characterized by a solvent inaccessible rigid core and a fuzzy coat, which can be removed by protein digestion31. Tau fibrils purified from the brain of an AD patient exhibited two morphologies, paired helical filaments (PHFs) and straight filaments (SFs). Both types of fibrils appeared to be composed by two C-shaped protofilaments comprising the R3 and R4 domains31. The hexapeptide 14, the cryo-EM analysis did not provide information about the structural properties of the proline-rich region31.Recently three-dimensional structures for fibrils of tau in AD and Pick’s disease (PiD) have been determined using cryogenic EM (cryo-EM)30. The NPFs are composed of a single protofilament, whose core is composed by the R1, R3 and R4 repeats, and adopts a hairpin-like shape via hydrophobic interactions between the strands30. The WPFs are composed of two NPFs associated together. In PiD, tau isoforms containing three pseudo-repeats are the most expressed1, therefore the R2 domain was not modelled in the cryo-EM structure.Fibrils purified from the brain of a PiD patient where composed of the tau 3R isoform, which lacks the R2 domain. Two morphologies defined as Narrow Pick filaments (NPFs) and Wide Pick filaments (WPFs) could be distinguished42. The resulting structures differed from AD and PiD fibrils structures by inclusion of the R2 domain in the core of the fibrils, which was not observed in the three-dimensional structures reported by Fitzpatrick et al. and by Falcon et al.31. In any of the structures resolved by cryo-EM, only the pseudo-repeat regions could be characterized, but no structural information was accessible for other regions of the tau sequence. In particular, the proline-rich region was not observed, leaving open questions regarding the contribution of this domain to the formation and structure of tau fibrils.Other cryo-EM three-dimensional structures, in this case of heparin-induced in vitro tau fibrils, have also been characterized47. The proline-rich region is also rich in serine and threonine residues, which make it a target for many kinases involved in the pathological hyperphosphorylation of tau48. In addition, the proline-rich region plays an important role for the interaction of tau with tubulin49 and is involved in signaling pathways: the proline-rich motifs  within the sequence are recognized by proteins containing proline-rich motifs binding modules (PRM-binding modules) such as the SH3 domain50.The relevance of the proline-rich region in pathological aspects of tau is supported by the presence of many phosphorylated epitopes for monoclonal antibodies, e.g. AT180, AT100, AT80 and PHF1, which recognize hyperphosphorylated disease-associated forms of tauHere we addressed the involvement of the proline-rich region in the pathological aggregation of tau. We designed simplistic models of tau, namely the two polypeptides P2R2 and P2R3, in order to better understand structural properties of the P2 domain in tau amyloid fibrils. The heparin-induced in vitro fibrils obtained from these two polypeptides, together with those obtained from the tau construct K32, comprising the proline-rich and the pseudo-repeat regions, have been studied using aggregation assays and ssNMR spectroscopy. The combined data provide evidences for the recruitment of the proline-rich region P2 to the core of tau fibrils.7. In order to obtain the backbone and side-chain assignment, NMR 2D and 3D experiments were performed on 25  was added to each reaction mixture to a final concentration of Being an IDP, tau shows little tendency to aggregate in vitro. Polyanions, e.g. heparin, induce in vitro tau aggregation by neutralization of the positive charges and thus allow the establishment of hydrophobic interactions between residues from different tau moleculesmentclass2pt{minimmentclass2pt{minimmentclass2pt{minimant Fig. . The pelmentclass2pt{minimure Fig. . The prementclass2pt{minim51, the residue type could be identified for some cross peaks, e.g. serine, threonine and proline residues, but no specific assignment could be performed  experiments were performed on uniformly The K19 sequence contains the R1, R3 and R4 pseudo-repeats of tau Fig. A, while entclass1pt{minimaentclass1pt{minimaK19 contains a single tyrosine, Y310, which is located in the R3 domain. In the 2D PDSD spectrum of K19, the C13. In the transition from random coil to 14. Thus, their visibility in the INEPT spectrum could be explained by their higher degree of mobility. The analysis of the domains considered to be in the rigid core of the fibrils was complicated by the signal overlap in the ssNMR INEPT spectrum, as well as the clustering of the CApproaching the issue from a different perspective, a 2D INEPT-based In the C13. The peptides were produced recombinantly in E. coli  with one domain of the pseudo-repeat domain  and thus represent two important sequence properties of the full-length htau40 protein  was confirmed using bicinchoninic acid (BCA) assay kit . The plasmids encoding the DNA sequence of either P2R2 or P2R3, which combine the amino acid sequences of the P2 domain (residues S198-Q244) with either the R2 domain (residues V275-S305) or the R3 domain (residues V306-Q336) of htau40, were purchased from GeneArt gene synthesis from Invitrogen, Thermo Fisher Scientific (Germany). The two native cysteine residues C291 and C322 of htau40, located in the R2 and R3 domains respectively, were replaced by alanine in the polypeptides. The DNA sequences of P2R2 and P2R3 were cloned into a pET28a vector, between the BamHI and HindIII restriction sites. Rosetta64. For P2R2 and P2R3, The plasmid carrying the coding sequence of the K32 construct, which comprises the P2 domain (residues S198-Q244) and the complete pseudo-repeats region, including repeats R1, R2, R3, R4 and g, at 37 °C for 1 h in a TLA 100.3 rotor in a bench ultracentrifuge  and resuspended in 65.CD spectra of monomeric K32, P2R2 and P2R3 peptides were acquired on a Chirascan  spectrometer. Samples in sodium phosphate buffer 50 mM, pH 6.8, 50 mM NaCl were diluted in g, at 37 °C for 1 h in a TLA 100.3 rotor in a bench ultracentrifuge . The composition of the supernatant and the pellet was monitored by SDS-PAGE.The fibrillization of unlabeled and 66. The measurements were carried on a 96 well plate in a Cary Eclipse fluorescence spectrophotometer at room temperature, using an excitation and an emission wavelengths of 440 nm and 482 nm, respectively. Fibril formation was monitored performing a Thioflavin T (ThT) fluorescence assayThe morphology of the fibrils was analyzed by negative-stain transmission electron microscopy (TEM). For each sample the fibrils were harvested by ultracentrifugation, discarding the supernatant followed by resuspension in fresh fibrillization buffer  prior to MAS rotor filling and measurement of the ssNMR experiments. DSS  in powder (Sigma-Aldrich) was added to each sample as internal chemical shift reference. PDSD and INEPT experiments were recorded on 68 and ccpNMR67.For the acquisition of ssNMR spectra, Supplementary Information 1.


Bacillus/Clostridium group, as well as in the Fusobacteria and Thermotogales lineages. The NiaR structure consists of two domains: an N-terminal DNA-binding domain, and a C-terminal regulation domain containing a metal-binding site. In this paper, we report the crystal structures of apo and niacin-bound forms of NiaR from Bacillus halodurans (BhNiaR). The analysis of metal-binding and niacin-binding sites through the apo and niacin-bound structures is described. Each N- and C-terminal domain structure of BhNiaR is almost identical with NiaR from Thermotoga maritima, but the overall domain arrangement is quite different. A zinc ion is fully occupied in each subunit with well-conserved residues in the C-terminal domain. Niacin is also located at a hydrophobic pocket near the zinc ion in the C-terminal domain.The niacin-responsive repressor, NiaR, is transcriptional repressor of certain nicotinamide adenine dinucleotide (NAD) biosynthetic genes in response to an increase in niacin levels. NAD is a vital molecule involved in various cellular redox reactions as an electron donor or electron acceptor. The NiaR family is conserved broadly in the  The NAD molecule participates in many biochemical transformations, including several reactive centers. Nearly 88% of enzymes that rely on NAD are involved in catabolic pathways. Approximately half of these enzymes are related to the use of carbohydrates, and the remaining enzymes contributes to the oxidation of other substrates such as carboxylic acid, amino acid and fatty acid4. In many bacteria, NAD+ is synthesized through two metabolic processes, the de novo and salvage pathways. In the de novo pathway, NAD+ is synthesized from l-aspartate or l-tryptophan by the consecutive action of NadBACDE 5. The salvage pathway is a recycling system producing NAD+ from exogenous sources such as niacin , nicotinamide, nicotinamide riboside or endogenous breakdown products of NAD+ by NAD+-consuming enzymes6. Despite some variations in the early steps of the two pathways, the final step of NAD+ synthesis from nicotinic acid adenine dinucleotide to NAD+ is highly conserved7.Nicotinamide adenine dinucleotide (NAD) is an essential molecule that plays an important role as both electron donor and electron acceptor in cellular metabolism+ biosynthesis are regulated by several transcription factors, the NAD+-dependent repressor (NadR), the Nudix-related transcriptional regulators (NrtR), and the niacin-responsive repressor (NiaR). NadR in Enterobacteria acts as a repressor of certain NAD+ biosynthetic genes responding to NAD+, nicotinic acid phosphoribosyltransferase (pncB) in the salvage pathway and nadBA involved in the de novo pathway4. In addition, NadR has enzymatic activities as nicotinamide riboside kinase and nicotinamide mononucleotide adenylyltransferase, which function in NAD+ synthesis11. The NrtR acts as a regulator of NAD+ biosynthesis genes and salvage genes such as, nadBACDE, nicotinamidase (pncA), pncB, and nicotinamide riboside transporter pnuC13. NiaR, previously called YrxA, was found mainly in Bacillus/Clostridium species, as well as Fusobacteria and Thermotogales14. NiaR represses transcription of certain NAD+ biosynthetic genes in response to elevated niacin levels. NiaR also represses the expression of the transporter for niacin uptake , pncA and pncB in the salvage pathway, and two operons, cysteine desulfurases (nifS) and nadBCA, sharing a promoter region, in the de novo pathway15. The transcriptomic analysis of Streptococcus pneumoniae NiaR showed that NiaR regulated gene expressions, including niaX, pnuC, and nadC, in response to niacin16. The biochemical and bioinformatic analyses provided the mechanism of NiaR and the metabolism of NAD+ synthesis in Bacillus subtilis17. The NiaR regulon constitutes a transcriptional regulation system of NAD+ synthesis in several groups of Gram-positive bacteria6.Both de novo and salvage pathways for NAD+ synthesis pathway regulators14. The NiaR from Thermotoga maritima (TmNiaR) displayed metal binding and dimeric structure. The NiaR protein is composed of two domains. The N-terminal domain is a DNA-binding domain containing a helix-turn-helix motif and the C-terminal domain is a regulatory 3H domain in which three histidines are well conserved. A nickel ion is occupied in the C-terminal domain of TmNiaR and the function of this domain was assumed to involve binding to small molecules18.Previous studies suggested that niacin binds to the 3-histidine domain (3H domain) of NiaR and NiaR belongs to a family of de novo NADBacillus halodurans  is composed of 179 amino acids, with 54% sequence identity with NiaR in B. subtilis (BsNiaR). Further sequence comparisons of BhNiaR show that it is 37% identical to TmNiaR, 38% identical to S. pneumonia NiaR, and 42% identical to Clostridium symbiosum NiaR . The structural data of BhNiaR provides details of metal binding and ligand interaction. DNA binding affinity of BhNiaR depending on niacin or metal ions was assessed by electrophoretic mobility shift assay (EMSA).We determined the crystal structures of BhNiaR was refined to 2.0 Å resolution with crystallographic Rwork and Rfree values of 20.18% and 22.78%, respectively. Although the refined model contained a subunit in an asymmetric unit composed of 173 residues with a zinc ion and 129 water molecules, it could be generated to a homo-dimeric structure by a symmetric subunit. The N-terminal domain is composed of residues 7–70 and C-terminal domain is composed of residues 71–179. The N-terminal region (residues 1–6), the linker loop region (residues 68–74) connecting the N- and C-terminal domains, and the internal region (residues 137–147) in the C-terminal domain were poorly ordered due to lack of electron-density maps. The niacin-bound BhNiaR crystals were grown by co-crystallization with 2 mM niacin. The structure of the niacin-bound form was determined at 1.8 Å resolution and refined to crystallographic Rwork and Rfree of 19.43% and 24.26%, respectively. The refined model of niacin-bound BhNiaR also contained a subunit with interpretable residues 7–179 in an asymmetric unit. The N-terminus (residues 1–6), the linker loop region (residues 68–74), and the internal region (residues 137–145) in the C-terminal domain were poorly ordered. A niacin molecule was located near a zinc ion and surrounded by a hydrophobic pocket close to the dimeric interface. All refined models for BhNiaR indicated favored or allowed regions of the Ramachandran plot. The detailed refinement statistics are summarized in Table The apo crystal structure of BhNiaR structure was composed of two domains, an N-terminal DNA-binding domain (residues 1–70) and a C-terminal dimerization/substrate-binding 3H domain (residues 71–179), connected by a flexible loop (residues 66–75) . The dimeric structure is stabilized by the hydrogen bonds and hydrophobic interactions centered around a faced β5 strand forming an anti-parallel sheet; 22 residues are involved in hydrophobic interactions and 13 residues are involved in hydrogen bonds (PDBsum: http://www.ebi.ac.uk/thornton-srv/databases/pdbsum/Generate.html). The dimer interface is mainly produced by hydrophobic residues, which are highly conserved through NiaR family proteins, such as Val42, Ser48, Leu49, Ala59, Thr60, Ala61, Val78, Val111, Tyr112, Gly113, Ile115, Thr116, Ala117, Ser118, Leu119, Phe130, Met134, Leu141, Val149, Met151, Leu176, and Phe179. Thirteen hydrogen bonds were formed between Glu10 Oε2 and Ser178 Oγ, between Gln41 Nε2 and Thr146 O, between Lys51 Nz and Asp114 Oδ2, between Ala59 N and Asp114 Oδ2, between Ala59 O and Asp114 N, between Tyr64 Oη and Asp108 Oδ2, between Val111 O and Arg133 Nη2, between Asp114 O and Ser118 N, and between Thr116 N and Thr116 O. This finding demonstrated that BhNiaR exists as a functional dimer in solution. A dimer is also consistent with results from size-exclusion chromatography with multi-angle light scattering (SEC-MALS)  value of 0.2 Å. The most noticeable structural difference was the loop region between the N-terminal and C-terminal domains  Fig. . The niains Fig. .2+, Cu2+, or Zn2+ as the most probable metals in the TmNiaR structure based on coordination geometry, anomalous difference Fourier peaks, refinement results, and similar ligating residues in the Metalloprotein Database19. A zinc ion fully occupied the metal-binding site in the apo and niacin-bound forms of BhNiaR, which was confirmed by ICP-MS and also was verified by a peak on an omit map at the counter levels even at 9σ . The zinc ion is located between layered α-helices and a β-sheet of the C-terminal domain. The metal-binding site appeared to be fully occupied, with the temperature factors for a zinc ion being 43.95 Å2 and 22.81 Å2 in the apo and niacin-bound forms, respectively.Previous work suggested Ni 9σ Fig. . In addiTmNiaR, it has been reported that four conserved residues  and a water molecule coordinated with a nickel ion in octahedral geometry14. However, in the apo structure of BhNiaR, three residues , corresponding to TmNiaR residues , and a water molecule coordinated with a zinc ion in tetrahedral geometry: Glu91 Oε1, His150 Nε1, His152 Nε2, and Wat108 O. The His83 residue was not involved directly in metal coordination, with a distance of 3.8 Å. In addition, in the niacin-bound structure, the coordinated water molecule was replaced by a carboxyl oxygen (O2) of the niacin molecule and the His83 residue formed a hydrogen bond with a second carboxyl oxygen (O1) of the niacin molecule   containing the nifS-nadB promoter region. The DNA was mixed with various amount of BhNiaR either in the absence or presence of niacin. BhNiaR bound to its cognate DNA with a dissociation constant (Kd) of ~ 0.5 µM in the presence of niacin, whereas the DNA-binding ability was decreased in the absence of niacin  in T. maritima . Although their N- and C-terminal domains were well matched structurally , the domain arrangements were different. When the C-terminal domains were superimposed, the N-terminal domain of BhNiaR was rotated approximately 170° at the center of the flexible linker region (residues 64–72), compared with that of TmNiaR  in the TmNiaR dimer was 40 Å, whereas in BhNiaR (at residue Arg40) the distance was 57 Å  conserved in HPr-like proteins with a water molecule in tetrahedral geometry. Another conserved residue, His83, is not involved in zinc ion coordination but is involved in niacin binding. The niacin bound to the C-terminal domain of BhNiaR is located near a zinc ion, His83, and a well-conserved hydrophobic pocket. Both the niacin and the metal ion cooperatively affect DNA-binding affinity of BhNiaR, and the EMSA results show that the absence of a metal ion causes a significant defect in the DNA-binding affinity of BhNiaR.The crystal structures of the niacin-responsive repressor, niaR genes were amplified by polymerase chain reaction (PCR) using genomic DNA of B. halodurans as a template. The amplified niaR genes were inserted into the NheI/XhoI-digested expression vector pET-28b(+) (Novagen), resulting in a hexahistidine-containing tag (His-tag) at its N-terminus with a PreScission protease cleavage site. The recombinant niaR genes were transformed in Escherichia coli BL21 (DE3) star pLysS cells (Invitrogen). The transformed cells were grown at 310 K to an OD600 of ~ 0.5 in Luria–Bertani medium and overexpression of BhNiaR protein was induced continuously with 1.0 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) for 4 h at 303 K. After the cells were collected by centrifugation at 4200g for 10 min at 277 K, the pellets were immediately frozen at 193 K.The v/v) glycerol) and 1 mM phenylmethylsulfonyl fluoride, homogenized by an ultrasonic processor  and separated from the insoluble fraction by centrifugation at 31,000g for 60 min at 277 K. The recombinant BhNiaR protein in soluble fraction was loaded on a nickel-charged His-trap immobilized metal affinity chromatography (IMAC) column  and eluted with buffer B  glycerol, and 300 mM imidazole). Enzymatic removal of the His-tag from the BhNiaR proteins was achieved by overnight incubation with PreScission protease. The uncleaved His-tagged proteins were removed from the native proteins by applying to a nickel-charged His-trap IMAC. The next purification step was size-exclusion chromatography on a Superdex-75 column  with elution buffer  glycerol, 1 mM dithiothreitol and 1 mM MgCl2). The purified BhNiaR protein was concentrated to 15 mg ml−1 using Centricon YM-10 (Millipore).The frozen pellets were placed in a lysis solution containing buffer A  was replaced by GCA-encoded alanine . PCR products were treated with DpnI for digestion of preexisting recombinant plasmids containing the wild-type niaR gene. PCR products were introduced into E. coli DH5α, and then the mutant plasmids were isolated and purified from the cells. The mutated niaR sequence was confirmed by DNA sequencing .Site-directed mutagenesis of BhNiaR protein was performed by the sitting-drop vapor diffusion method using a 96-well crystallization plate  and commercial screening solution from Hampton Research, Qiagen and Emerald Biosystems at 296 K. Each sitting-drop was prepared by mixing 0.75 μl of the concentrated protein and reservoir solution, respectively. The BhNiaR protein grew as two forms of crystals, apo form and niacin-bound form. Crystals of apo BhNiaR were obtained in 2% (v/v) tacsimate, pH 6.0, 0.1 M Bis–Tris, pH 6.5, and 20% (v/v) PEG 3350 solution and were grown to 0.05 × 0.1 × 0.05 mm within 2 weeks. Crystals of niacin-bound BhNiaR were obtained by co-crystallization with 2 mM niacin and were grown in 0.1 M Tris–HCl, pH 8.5, and 15% (v/v) PEG 6000 solution.Initial crystallization of the v/v) glycerol in the reservoir solution and then flash-cooled in liquid nitrogen. X-ray diffraction data of apo and niacin-bound crystals of BhNiaR were collected at 100 K and at the wavelength, 0.97941 Å, using synchrotron radiation of the Pohang Accelerator Laboratory in Korea. Diffraction data were collected with a Pilatus 6 M detector on 11C Micro-MX beamline for the apo form and with an ADSC Q315r CCD detector on 5C-SBII beamline for the niacin-bound form. The crystals were exposed to X-rays for 1.0 s per image, and 270 frames (apo) and 100 frames (niacin-bound) were obtained with each 1.0° oscillation. The data were processed and scaled with DENZO and SCALEPACK of HKL2000 software22.Each crystal was transferred into a cryo-protectant solution containing 20% (BhNiaR were solved by molecular replacement using the program PHASER MR from the CCP4 program suite23 using the TmNiaR structure (PDB code 1J5Y) as a search model14. The structures of apo and niacin-bound forms built by COOT24 and were refined by PHENIX. All refined structures of BhNiaR were evaluated by MolProbity25 and have been deposited in the Protein Data Bank. The figure was generated using the computer program PyMol 26. The refinement statistics of BhNiaR are shown in Table The crystal structures of both forms of PDB code J5Y as a BhNiaR, 34-bp double-stranded DNA (Forward : 5′–CTCATTTACATATGTCTTGACATCTATATTTACA–3′ and Reverse : 5′–TGTAAATA TAGATGTCAAGACATATGTAAATGAG–3′) containing own promoter region (nifS-nadB) was prepared by Oligo Synthesis . The reaction mixture was made by mixing the native BhNiaR (0–3 µM) and the reaction buffer  glycerol with or without 1 mM niacin) prior to mixing the double-stranded DNA (80 nM). All reaction mixtures were incubated for 30 min at room temperature, after which each mixture was loaded on a pre-chilled 6% native polyacrylamide gel in 0.5 X Tris–Borate, pH 8.3. Electrophoresis was performed at 277 K and the gel was visualized using SYBR Green (ThermoFisher Scientific).To confirm the DNA-binding affinity of BhNiaR protein was injected  at a flow rate of 0.5 ml min−1. The data were evaluated using the Zimm model for static light-scattering data fitting and represented using an EASI graph with a UV peak in the ASTRA V software (Wyatt).SEC-MALS experiments were performed using a fast protein liquid chromatography system  connected to a Wyatt DAWN Heleos II instrument and a Wyatt Optilab T-Rex differential refractometer. A Superdex-200 10/300 GL  gel filtration column pre-equilibrated with 20 mM Tris–HCl (pH 8.0), 200 mM NaCl, and 1 mM DTT was normalized using ovalbumin (40 kDA) as the protein standard. The BhNiaR, PDB ID, 7CV0; Niacin-bound BhNiaR, PDB ID, 7CV2.Coordinate and structure factors have been deposited in the Protein Data Bank (PDB): apo Supplementary Figures.


Ly6/uPAR/α-neurotoxin domain (LU-domain) is characterized by the presence of 4-5 disulfide bonds and three flexible loops that extend from a core stacked by several conversed disulfide bonds . This highly structurally stable protein domain is typically a protein-binder at extracellular space. Most LU proteins contain only single LU-domain as represented by Ly6 proteins in immunology and α-neurotoxins in snake venom. For Ly6 proteins, many are expressed in specific cell lineages and in differentiation stages, and are used as markers. In this study, we report the crystal structures of the two LU-domains of human C4.4A alone and its complex with a Fab fragment of a monoclonal anti-C4.4A antibody. Interestingly, both structures showed that C4.4A forms a very compact globule with two LU-domain packed face to face. This is in contrast to the flexible nature of most LU-domain-containing proteins in mammals. The Fab combining site of C4.4A involves both LU-domains, and appears to be the binding site for AGR2, a reported ligand of C4.4A. This work reports the first structure that contain two LU-domains and provides insights on how LU-domains fold into a compact protein and interacts with ligands. Ly6/uPAR/α-neurotoxin domain (LU-domain), also named three-fingered protein domain or TFPD, are widespread in the animal kingdom and mainly comprises either secreted or glycosyl-phosphatidylinositol (GPI) anchored single domain proteins with diverse biological functions e.g., α-neurotoxins), acetylcholine esterases , L-type calcium channels  or targeting cell membranes  Proteins with via homodimeric assembly using either non-covalent interactions  e.g., iriditoxin and α-cobratoxin) 2+ response e.g., uPAR, C4.4A, Haldisin, TEX101, CD177, and PINLYP) Notwithstanding the prevalence of single LU-domain-containing proteins in the animal kingdom, there are only a few examples where two or more LU-domains forming the functional unit. In venomous snakes, certain neurotoxins evolved unique functions via β-sheet interactions creates a large central hydrophobic ligand-binding cavity that mediates the high-affinity binding of its primary ligand, the serine protease urokinase-type plasminogen activator. Biophysical studies have shown that this interdomain assembly in uPAR is highly flexible and that this has biological relevance Despite the clear functional importance of these multiple LU-domains proteins, their three-dimensional structures remain largely unexplored with a single exception. The urokinase-type plasminogen activator receptor (uPAR) is a GPI-anchored membrane protein containing three LU-domains  and several crystal structures have been solved for this founder of the LU-domain protein family LYPD3), which contains two LU-domains followed by a mucin-type region rich in serine, threonine and proline (STP-rich region) and a C-terminal GPI-anchor stratum spinosum in stratified squamous epithelia of the skin and for squamous differentiation of epithelia in other organs such as esophagus, vagina, oral cavity, and rectum e.g., breast Prompted by the close relationship between LU-domain flexibility and function of uPAR, we decided to solve the crystal structure of another protein containing multiple LU-domains to gain further insight into the structural versatility of this fold. We chose to focus on C4.4A  to facilitate the capture and purification of the protein e.g., the two LU-domains in human C4.4A share only 30% and 28% sequence identity with the DII of uPAR). Single-wavelength anomalous dispersion phasing using a biosynthetically selenomethionyl labeled C4.4A (yielding ~70% Se-incorporation in Met) was unsuccessful due to poorly diffracting SeMET crystals (>4 Å). Traditional multiple isomorphous replacements (MIR) or phasing with sodium bromide Recombinant human C4.4A was expressed in The complex between C4.4A and the Fab fragment of 11H10 was purified by size exclusion chromatography and yielded well-diffracting crystals of C4.4A:Fab (11H10). The Fab fragment was positioned into the crystal by MR, and the Fo-Fc map now revealed electron density for C4.4A. Extensive manual building, together with iterative refinement, finally yielded crystal structures of both the C4.4A:Fab complex and C4.4A with good statistics. The structure of C4.4A was refined to 2.59 Å with R-factor and R-free of 0.2063 and 0.2503, respectively; 92.8% residues in favored Ramachandran region . Notably, the DII-Lk1 connecting βIIB and βIIC of C4.4A is quite long (9 amino acids vs only 6 in the DI-Lk1). The six β-strands in the DI form a large continuous β-sheet (61% β-sheet content), whereas the DII has a small β-sheet (49% β-sheet content)  cause protein misfolding and loss-of-function There are two C4.4A molecules in the asymmetric unit of the crystal. Superposition of the two molecules shows that their structures are highly similar with each other with RMSD of 0.72 Å for all Cα, further supporting the low flexibility of the structure under these conditions. Another notable key difference between the two LU-domains in C4.4A is the arrangement and the number of disulfide bonds. The DI has four disulfide bonds , while the DII has five    . These hfor DII) , Fig. 2CThe structure of C4.4A in the complex with the Fab fragment of mAb 11H10 is highly similar to the C4.4A alone Fig. A, with alabeled as H/Arg103 in Fig. ) inserts into the groove of C4.4A-DI and C4.4A-DII and forms hydrogen bonds with Asp65 and Gln165 of C4.4A  in DI and the linkers between β-strands in DII (Lk1 and Lk2) Fig. . The Fabof C4.4A . On the Usually, the conformation of loop is susceptible to ligand binding and/or environment due to its flexibility. However, despite containing a long loop in DII-Lk1, the binding of Fab doesn't induce the conformational change of this loop. Further structural analysis shows Tyr132 and Tyr139 located in DII-Lk1 form hydrogen bonds with DI to stable the conformation of DII-Lk1 .The structure of the complex demonstrates that the mAb 11H10 recognizes a conformational epitope on intact C4.4A-DIDII containing both LU-domains. This observation is excellently aligned with biochemical results showing by Western Blot Fig. D, E thatC4.4A was reported to interact with both α6β4 integrin and MMP14, promoting wound healing and metastasis Based on our crystal structure of C4.4A:Fab complex, we studied its molecular interaction of C4.4A with AGR2 by carrying out the protein-protein docking between C4.4A and AGR2 (PDB ID: 2lnt) LU-domains contain three to six highly conserved disulfide bonds with a unique signature motif: CCxxxxCN (x is arbitrary amino acid), which is tightly packed at the palm region In many cases, the palm surface of LU-domain is important in interacting with the ligand, as shown by the structure of the multiple-LU-domains protein (uPAR) and single LU-domain protein (CD59 and some three-fingered snake venom toxins) via intermolecular disulfide bonds A novel mode of homodimerization of LU-domains was revealed in our C4.4A structure. All of known three-fingered snake venom toxins contains only one LU-domain. A few toxins exist nevertheless as non-covalent homodimers in solution e.g. κ-bungarotoxin and haditoxin LYPD5) is extracellular protein predominantly expressed in stratum granulosum of human skin under homeostatic condition, and was predicted to contain two LU-domains with disulfide bonding pattern similar to C4.4A Haldisin  was generated by conventional mouse hybridoma technology after immunizing FVB mice with purified recombinant human C4.4A produced in TM II kit (Life Technologies) and the following primers:Hybridomas producing 11H10 mAb (IgG1κ) were cultured and used to generate cDNA for the corresponding light and heavy chains using the Cells-to-cDNAVκ light-chain 5`-primer:GAATTCGACATTCTGATGACCCAGTCT;TATCκ light-chain 3`-primer:GCGGCCGCACACTCATTCCTGTTGAAGCTCTTGAC;AH heavy-chain 5`-primer:VGAATTCCAGGTTACTCTGAAAGAGTCTGG;TATH heavy-chain 3`-primer:CGCGGCCGCACAATCCCTGGGCACAATTTTCTTGTC.AThe cDNAs were amplified with conventional PCR using the Platinum Pfx DNA Polymerase (Invitrogen) and products with the proper size (app. 700 bp) were purified from a 1% agarose gel with the QiaQuick Gel Extraction Kit (Qiagen). The cDNAs were cloned into pBlueScript KS+ using the introduced EcoRI and NotI restriction enzyme sites (underlined in the primer sequences) and the Rapid DNA ligation kit (Roche). Subsequently, DH5α Competent cells were transformed and DNA was isolated from individual clones and analyzed by restriction enzyme digestion before sequencing. Five-six clones for each chain were sequenced and revealed 100% identical sequences. The amino acid sequences are shown below:11H10 Fab light chain:DILMTQSPAILSVSPGEGVSFSCWANQNIGTSIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNSWPIFTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.11H10 Fab heavy chain:QVTLKESGPGILQPSQTLSLTCSFSGFSLNSFGTGVGWIRQPSGKGLEWLAHIWWNDYKYYNAALESRLTISKDTSNNQVFLKIASVDTADTATYYCARLRLRYFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC.Drosophila S2 cells and purified by immunoaffinity chromatography as described Recombinant C4.4A-ent-uPAR-DIII fusion protein was produced in Drosophila S2 cells and purified by immunoaffinity chromatography as described The generation of various C4.4A domain constructs was produced in The Fab peak was pooled and mixed with C4.4A-DIDII and the resultant complexes purified by gel filtration on a Superdex 200 column (GE Life Sciences) with 20 mM Tris, 150 mM NaCl and pH 7.4 as the running buffer. The eluted fragments contained the target complex were mixed and concentrated to 10 mg/mL using an Amicon Ultra Centrifugal Filter Device  with a molecular mass cutoff of 10,000 Da, the aliquots were stored frozen at -80°C.w/v) polyethylene glycol 4000, 0.1 M citric acid in pH 3.6, as described previously The C4.4A crystals were obtained at 293K by the sitting-drop vapor-diffusion method in a concentration of 10 mg/mL. The precipitant condition is 22.5% (v/v) glycerol and flash-frozen in liquid nitrogen. Diffraction data for both C4.4A and C4.4A:Fab complex were collected on beamline X29 at Brookhaven National Synchrotron Light Source (NSLS) and were processed using the automated data-processing pipeline xia2 Prior to X-ray data collection, the crystals were transferred to the precipitant solution containing 25% . No MR solution can be found with all these models alone. Importantly, with the Fab positioned, we obtained the MR solution of the model of DI, giving a z-score of translational function higher than 12.0. The log-likelihood gain (LLG) from Phaser was also positive, indicative of a successful MR solution. The model was adjusted according to electron density map in Coot.MR method was used to solve C4.4A:Fab structure. The Fab fragment was positioned into the crystal lattice using After several rounds of refinement of the DI in C4.4A:Fab crystal, the well-fit model of DI was used as the search model for C4.4A crystal dataset, which had higher resolution (2.6 Å) than C4.4A:Fab complex (2.8 Å). The MR using Phaser produced the successful solution, and identified both molecules in the asymmetric unit. Phenix Autobuild module was used to perform iterative model building, refinement and density modification, leading to an improved electron density map. Iterative cycles of model building and refinement were performed until the model cannot be improved.The model from the C4.4A crystal was then used as the searching model to successfully place into the C4.4A:Fab crystal by MR method with the positioned Fab as fixed model. The completed model of the complex was further improved by several cycles of refinement and manual adjustment until converged. All relevant data collection and refinement statistics were summarized in Table Ka values calculated by using the H++ webserver + ions were added to counterbalance the charge of the protein. The system contained 8,630 water molecules and 28,786 atoms in total. It was underwent MD simulations with AMBER ff99SB-ILDN force field -1·Å-2 harmonic position restraints on the protein, followed by 2,500 steps of steepest-descent and 2,500 steps of conjugate-gradient minimization without restraints. The system was then gradually heated from 0 K up to 298 K in 20 steps of 2 ns. After that, 2000 ns-long productive MD simulations were carried out in the NPT ensemble. The most representative structure was identified by the cluster analysis The homology model of Haldisin was built automatically by the SWISSmodel web server based on the crystal structure of C4.4A Supplementary figures and tables.Click here for additional data file.


Drosophila kinesin-II, interacts with Arm for Wg signaling. Molecular basis for this interaction has remained unknown. Here we identify a critical Arm repeat (AR) required for binding Klp64D and Wg signaling. Arm/Armadillo (Arm) is crucial for transducing Wingless (Wg) signaling. Previously, we have shown that Klp64D, a motor subunit of  Wnt signaling is evolutionarily conserved, and hence Drosophila has been extensively utilized for genetic dissection of the Wnt signal transduction pathway. Secreted Wingless  activates a cascade of signal transduction events. Armadillo  plays a key role in transducing the canonical Wnt/Wg signaling by acting as a transcriptional regulator4. Arm is also an essential component of adherens junctions (AJs) together with E-cadherins (E-cad) and 6. In the absence of Wg signal, Arm is accumulated at the adherens junctions (AJs) where it forms a complex with E-cad or is rapidly degraded 7. Expression of Wg target genes is activated when stabilized Arm enters the nucleus and recruits other co-activators8.The Wnt signaling pathway controls diverse biological processes such as growth, patterning and differentiation. Misregulation of the Wnt pathway can lead to human diseases including cancer and metabolic disorders9. Klp64D, the Drosophila homolog of Kif3A, is a subunit of the kinesin-II microtubule-based heterotrimeric motor protein complex10. Recently, we have reported that Klp64D is also required for Wg signaling as a binding partner of Arm11. Klp64D recruits Arm and its interacting partner Dishevelled (Dsh) for Wg signal transduction. In wing development, Wg signaling is induced from the dorso-ventral (DV) boundary of wing imaginal disc. In the absence of kinesin-II function, Arm is abnormally accumulated with intracellular vesicles in the DV boundary region. Thus, it has been suggested that Klp64D might be involved in intracellular trafficking of Arm for Wg signaling11. However, specific regions of Arm and Klp64D proteins that are critical for their binding and Wg signaling have not been determined.Arm is required for development of fly organs at different stages. It has been shown that loss of Klp64D causes mislocalization of Arm in differentiating photoreceptor cells of pupal retina12. Arm contains a long repeat region called the Arm repeat (AR) domain in the middle portion of the protein. Arm repeats (ARs) are evolutionarily conserved structures found in many proteins14. Metazoan AR-containing proteins have different numbers of AR units, ranging from 1 (ARMC1) to 13 (ARMC4)15. The AR regions of Arm and mammalian 18. APC and Tcf compete for binding Arm because the ARM domain is the main binding site that typically interacts with one partner of the destructive complex at a time20. In contrast, the BCL9 homolog Legless (Lgs), which also interacts with Arm for Wg target gene expression, can bind to a different region of Arm repeats without competition with Tcf23. Thus, although the central groove region of Arm is a major site for binding to partners, other individual Arm repeats may also be involved in interaction with partners. In our earlier study, the AR region of Arm was shown to interact with the coiled-coil domain and/or the C-terminal cargo domain of Klp64D11. It is an intriguing question whether Klp64D interacts with a specific AR region(s) to regulate Wg signaling.It has been shown that distinct roles of Arm at AJs and Wg signaling are genetically separable. The N-terminal region of Arm plays a role in AJs while the C-terminal domain is essential for Wg signaling functionIn this study, we analyzed the interactions between different ARs of Arm and Klp64D kinesin-II motor subunit. We demonstrate that AR2 specifically interacts with the cargo domain of Klp64D. Our data indicate that AR2 and the Klp64D cargo domain are essential for the function of Arm and Klp64D in vivo, respectively. Overexpression of these interacting domains are sufficient to antagonize Wnt signaling. This study suggests a role of AR2 and Klp64D cargo domain in Wg signaling and provides an insight into a new tool for inhibiting Wnt signaling through dominant-negative interactions.11. The N-terminal region of Klp64D contains the motor domain whereas the C-terminal region consists of a coiled-coil domain and the tail domain for cargo binding   and Klp64D mutant protein deleted in the C-terminal tail domain (Klp64DΔtail). Pull-down analysis indicated that ArmΔAR2 cannot bind the Klp64D tail while Klp64DΔtail fails to bind AR2  Fig. b. These AR2 Fig. . These rUAS-AR transgenic flies. To detect the expression of these transgenes, AR constructs were designed to be expressed as AR-HA fusion proteins. We could detect the expression of AR-HA constructs for AR3, 7, 8, and 10 in wing disc, but AR2-HA expression was not detectable. Hence, we generated untagged UAS-AR transgenes inserted at the same chromosomal site (59D3 on 2R) using phiC31 integrase 24 to induce similar expression levels. Ubiquitous overexpression of AR2, 3, 7, or 8 using act-Gal4 caused larval lethality while AR10 did not affect development. Thus, overexpression of AR2, 3, 7 and 8 can dominantly interfere with normal development.We have identified 5 AR regions that bind to Klp64D Fig. b, c. To C96-Gal4. Overexpression of AR3, 7, 8 or 10 that fails to bind the tail domain of Klp64D did not cause any effect on the wing   was not significantly rescued by p35 overexpression . While p35 efficiently suppressed cleaved caspase 3 (Cas3) staining induced by overexpressing proapoptotic gene hid . AR2 overexpression did not induce ectopic Cas3 staining , and the notched wing phenotype of AR2 overexpression was not noticeably suppressed by p35 . These data suggest that wing notching caused by Arm knockdown or AR2 overexpression is unlikely due to cell death.Next, to test whether overexpression of AR proteins can impair Klp64D function in vivo, each of AR transgenes was overexpressed along the DV boundary region of wing disc using ing Fig. a–e. In cing Fig. g. For quing Fig. j. To deting Fig. f, g, j. ing Fig. h, j. SinC96-Gal4  also inhibited the expression of Sens, thus showing partial disruption of Sens staining along the DV boundary , a downstream target of Wg signaling, is reduced by Klp64D knockdown using al4 Fig. l 11 whenrol Fig. k. AR2 ovUAS-HA were used as control in the absence or presence of exogenous Wg secreted in the medium. Wg-containing medium was added 4 days after transfection to induce the downstream signaling. In the absence of Wg, the luciferase ratio was near zero in the presence or absence of exogenous Arm . However, these arm2 mutant clones grew large when wild-type Arm was provided by UAS-arm within the clones . These data indicate that AR2 is essential for Arm function during wing development.Our data above suggested that AR2 overexpression is sufficient to interfere with the interaction between Arm and Klp64D, thus inhibiting Wg signaling. To determine whether the AR2 domain is necessary for the Arm function, we tested whether Arm protein lacking the AR2 domain can rescue the defects of loss-of-function UAS-GFP  . Furthermore, the wing notching phenotype by AR2   partially suppressed the notching phenotype of Klp64Dtail overexpression, showing only 7 ± 3% (n = 30) reduction in the margin  resulted in slightly stronger suppression to 3 ± 2% margin loss (n = 30), respectively  were very small compared with the size of wild-type control clones , arm2 mutant clones were small regardless of whether they are located close to the DV boundary or away from it . This is expected because Arm is required cell-autonomously for Wg signal transduction. Similarly, Klp64Dk1 mutant clones were small independent of their positions in wing pouch . Hence, the tail domain of Klp64D is required for the Klp64D function in wing growth, while its overexpression causes dominant-negative effects.We then tested whether the Klp64D tail domain is required for the Klp64D function, using mutant clones generated by MARCM. 11. Because AR2 overexpression causes dominant-negative effects on the Klp64D-Arm interaction, we examined whether AR2 overexpression also leads to increased overlap of Arm stain with Golgi. In wild-type control (C96 > +), basolateral region of wing cells showed relatively low levels of GM130 vesicles that overlap with Arm puncta  did not show significant effects on junctional Arm stains , and the notched wing phenotypes are not suppressed by p35. These data suggest that the effects of AR2 and Klp64Dtail are probably not due to non-specific cell death. We also found that AR2 overexpression did not significantly affect the junctional pattern of Arm staining in wing disc . To further overcome the limitation of our overexpression experiments, we also examined the loss of function analysis. Our data from MARCM clonal analysis suggest that the AR2 domain is essential for the function of Arm in wing development. Similar genetic tests also show that the tail domain is essential for the function of Klp64D in wing disc. Taken together, our data suggest that AR2 and Klp64D tail regions are important for the interaction between Arm and Klp64D and these domains are required for their normal function in wing margin development.AR2 overexpression strongly inhibits the Arm effects on Wg signaling activity in the TOP-Flash reporter assay, suggesting that AR2 overexpression antagonizes Arm function. Further, overexpression of the tail domain of Klp64D causes similar dominant notching effect along the wing margin. This suggests that Klp64Dail Fig. d. It shoisc Fig. c’, althoDrosophila embryogenesis, the central most Arm repeats such as AR3-6 and 8 of Arm are important for Wg signal transduction27. However, other individual Arm repeats may also be involved in interaction with binding partners29. For example, AR1 region of β-catenin interacts with specific proteins such as the BCL9 transcriptional regulator22. AR12 region of Arm/30. Biochemical and genetic interactions between AR2 and Klp64D also suggest a possible role of individual AR in interacting with specific partner proteins. The entire Arm repeat domain (aa 141–670) binds in vitro to two domains of Klp64D, the coiled-coil domain and the tail domain11. AR2 is the only repeat that interacts with the tail domain. Overexpressed AR2 may specifically interfere with the interaction between Arm and the cargo domain of Klp64D, leading to dominant-negative effects in the wing margin. In contrast, AR3, 7, 8, and 10 repeats can bind to Klp64D but not to its tail domain 32. Since Arm and Klp64D are co-localized with Rab5 endosome marker11, endosomes may be involved in Arm localization to Golgi. We also found that some Arm puncta overlap with DE-Cad , suggesting that Arm may co-localize with DE-Cad on the way to the plasma membrane from ER. All three of DE-Cad, Arm and GM130 punctate stain were also found to overlap at a low level . Precise mechanisms for the increased Arm localization with Golgi by Klp64D RNAi or AR2 overexpression needs further studies.It has been shown that Klp64D knockdown in the DV boundary region of wing disc results in increased overlapping localization of Arm with GM130 Golgi marker11. Hence, it would be interesting to see whether the interaction between AR2 region of Arm and Klp64D are conserved orthologs of mammalian Drosophila strains were grown and maintained at room temperature (22 °C). Klp64Dk1 allele causes a premature stop codon10 , USA). UAS lines used were UAS-Klp64D RNAi , UAS-Klp64D33, UAS-arm RNAi (8515 BDSC), UAS-armS1034, UAS-arm, arm2 mutant  and C96-Gal4 (BDSC).All arm mutant clones, males of hs-FLP, FRT 19A tub-Gal80; nub-Gal4, UAS-GFP/UAS-GFP were crossed with females FRT19A arm2/FM7c flies. Control clones were generated by crossing males of hs-FLP, FRT19A, Tub-Gal80; nub-Gal4, UAS-GFP/UAS-GFP with females of FRT19A/FRT19A. The same method was used to generate Klp64Dk1 clones. Mutant clones were induced at 60 h after egg laying (AEL) by an hour heat shock at 37 °C. Larvae were aged at room temperature until 3rd instar (about 120 h AEL) and wing imaginal discs were dissected for immunostaining.To generate 40. Mounted wings were photographed under AxioImager Zeiss optics with 5 × and 10 × lenses. Alternatively, whole flies or portions of flies were photographed through a dissection microscope. Quantification of wing notching was performed using Image J software by selecting all the margin area and measuring the margin length that was lost by notching using the Measuring and Counting tool.Wings from adult flies were dissected in isopropanol and mounted in Canadian Balsam mounting medium (Gary’s magic mount) following the protocol of Roberts and LawrenceWing imaginal discs were dissected from third instar larvae. Wing discs were fixed in PBS/0.2% saponin on a rotator for at least 20 min at RT. Anti-Dlg antibody (gift from Kyung-Ok Cho), anti-Sens antibody (gift from Hugo Bellen), anti-Dll antibody and anti-GFP were diluted 1:500, 1:1,000, 1:500, and 1:200 respectively, in PBS/0.2% saponin/0.5% goat serum and incubated at 4ºC overnight (O/N), rinsed three times in PBS/0.2% saponin, incubated in anti-rabbit FITC (1:200), anti-mouse Cy3 (1:200) and in anti-pig Cy5 (1:200), anti-sheep FITC (1:200) for 2 h at RT, and rinsed again four times in PBS/0.2% saponin. Secondary antibodies conjugated with Cy3, Cy5 or FITC were from Alexa Flour (Molecular Probes). Fluorescent images were acquired using Zeiss confocal microscope.∆AR2) and Klp64D tail domain (Klp64Dtail) were amplified by PCR using DGRC LD23131 cDNA (for Arm) and genomic fly extract (for Klp64D) and cloned into a pUAS-attB vector 35 using NotI and XbaI sites. The following primers were used to make Arm repeats constructs:To generate transgenic flies, Arm repeat domains , Arm-HA, Arm deleted in the AR2 domain (ArmAR2: 5′-GCGGCCGCCAAAATGGGAGATGGAGGGAGATCCACT-3′ and 5′-GCTCTAGACATACCGGTGTCCAGGTCGAA-3’.AR3: 5′-GCGGCCGCCAAAATGTTCCCGCAGAATTTCACACAA-3′ and 5′-GC TCTAGACATTTCATATCGTCCACTTGGTCT-3’.AR7: 5′-GCGGCCGCCAAAATGGGAACCGTCACAAATGCTAAA-3′ and 5′-GC TCTAGACATTTGACCACCGCGTGCTTTAGC-3’.AR8: 5′-GCGGCCGCCAAAATGTCTGATCAACTACCAGGACGA-3′ and 5′-GC TCTAGACATTGGCCCTGGTTGCCAGCTCAG-3’.AR10: 5′-GCGGCCGCCAAAATGTTCCCAGGCCGCCATGATGGT-3′ and 5′-GC TCTAGACATTGTTCATAATGGCATGTCGCG-3’.S2R + cells were grown in M3 media (Sigma) supplemented with 10% 10X insect medium supplement (Sigma) and 1% penicillin–streptomycin solution. Cultured cells were transfected with 1–3 µg DNA using Cellfectin II Reagent following a protocol from the manufacturer (Thermo Fisher Scientific).Drosophila S2R + cells express all of the Wg signaling components necessary to respond to exogenously added Wg36. For Arm, UAS-ArmAR2 and USA-HA control transfections, 1 µg DNA, 1 µg WISER (luciferase reporter plasmid) (gift from J.P Vincent) and 1 µg act-Gal4 (in the well of UAS-HA) in 98 µl EC buffer were combined with 16 µl Enhancer (Qiagen), 38 µl Effectene (Qiagen) in 3.5 × 106 cells/1.6 mL of growth media in a 6-well plate. To assess the effect of Arm and Arm AR2 on Wg signaling, 400 µl of media with or without Wg protein was added 2 days after transfection. 1 day later, the cells were lysed to assess luciferase levels using the Dual-Luciferase Reporter Assay System (Promega) containing Tcf reporter (firefly luciferase) and a constitutively active control reporter (renilla luciferase). Wg activity was calculated as the ratio of firefly to renilla luciferase activities with SEM (Standard error of the mean) of three independent experiments with the HA control. Statistical significance was based on a two-tailed t test. To obtain Wg-containing media, S2 tub-wg cells (DGRC) and S2 cells were grown in M3 media (Sigma) as described37. Cells were pelleted by centrifugation. The presence of Wg protein was confirmed by Western blotting by mouse anti-Wg antibody (DSHB).FL, GST-Klp64DC, GST-Klp64DN, GST-Klp64DTail, MBP-ArmFL, MBP-Arm repeats, MBP-ArmC, MBP-ArmN or MBP-AR domains38. Arm repeats used were: AR1 (aa 149–188), AR2 (aa 189–231), AR3 (aa 232–272), AR4 (aa 273–314), AR5 (aa 316–357), AR6 (aa 358–398), AR7 (aa 399–437), AR8 (aa 438–481), AR9 (aa 482–527), AR10 (aa 528–595), AR11 (aa 596–636), and AR12 (aa 637–677). Bacterial cell lysates were prepared as described39. An equal amount of blocked glutathione Sepharose 4B beads (Bioprogen) with GST, GST fusion protein or beads alone were incubated with lysates containing MBP-fusion proteins O/N at 4ºC. After several washes with pull-down buffer , sample buffer was added, beads were boiled and protein were resolved on SDS-PAGE. For western blotting, proteins were electrophoretically transferred onto nitrocellulose membranes, blocked in 5% skim milk (Biorad) and incubated with primary goat anti-GST (Santa Cruz) or rabbit anti-MBP antibody (Santa Cruz), and secondary goat anti rabbit or anti goat (Molecular Probes) antibody. Protein bands were visualized using ECL kit (GenDepot).For GST pull down, the following recombinant proteins were expressed and purified: GST-Klp64DSupplementary Information.