UBE3A in the brain. Due to brain-specific genetic imprinting at this locus, the paternal UBE3A is silenced by a long antisense transcript. Inhibition of the antisense transcript could lead to unsilencing of paternal UBE3A, thus providing a therapeutic approach for AS. However, widespread delivery of gene regulators to the brain remains challenging. Here, we report an engineered zinc finger-based artificial transcription factor (ATF) that, when injected i.p. or s.c., crossed the blood–brain barrier and increased Ube3a expression in the brain of an adult mouse model of AS. The factor displayed widespread distribution throughout the brain. Immunohistochemistry of both the hippocampus and cerebellum revealed an increase in Ube3a upon treatment. An ATF containing an alternative DNA-binding domain did not activate Ube3a. We believe this to be the first report of an injectable engineered zinc finger protein that can cause widespread activation of an endogenous gene in the brain. These observations have important implications for the study and treatment of AS and other neurological disorders.Angelman syndrome (AS) is a neurological genetic disorder caused by loss of expression of the maternal copy of  Other approaches have attempted to reactivate (“unsilence”) the paternal Ube3a allele, with varied success for molecular and phenotypic rescue. Paternal silencing is caused by a long brain-specific RNA transcript that overlaps and is antisense to Ube3a . The initiation, splicing, and function of this transcript are complex. The promoter of the Snurf/Snrpn locus is one start site for the Ube3-ATS, but numerous upstream start sites and exons create transcripts that, in mouse, can be over 1,000 kb.8 Inhibiting the extension of this transcript by insertion of a transcriptional termination sequence,9 topoisomerase inhibitor drugs,10 or antisense oligonucleotides11 results in partial unsilencing of paternal Ube3a, and some phenotypic rescue. These previous studies support that inhibition of Ube3a-ATS is a valid strategy to enhance paternal Ube3a expression.A traditional gene therapy approach using adeno-associated virus and cDNA to restore Ube3a in the brain of an adult mouse model of AS. ATFs are composed of a programmable DNA-binding domain, such as zinc fingers, TALEs or CRISPR/Cas systems, and an effector domain that can activate or repress transcription.13 Zinc finger-based ATFs have been evaluated in phase 2 clinical trials (NCT00476931), which demonstrated that zinc finger ATFs could be well tolerated in human subjects. The lack of similar data for TALE and CRISPR ATFs led us to choose zinc fingers for our initial studies. However, delivery of such ATFs throughout the brain has remained an unmet challenge. In particular, the blood–brain barrier created by the vascular endothelium in the central nervous system limits the method of delivery for most viral vectors, biologics, and drugs to i.c. or i.t. injection, which may be unfavorable for repeated treatments. Since ATFs only provide transient gene regulation they would need to be administered on a regular schedule to be effective for long-term gene regulation. In this study, we explored an alternative delivery approach utilizing an HIV TAT cell-penetrating peptide, which has been shown capable of delivering large proteins to the entire brain from a single i.p. injection.15 We observed that a purified, TAT-linked, zinc finger-based ATF repression protein targeted to the Snurf/Snrpn promoter was able to be injected i.p. or s.c., cross the blood–brain barrier, and activate endogenous Ube3a expression in multiple brain regions of a maternally Ube3a-deficient mouse model of AS.16 Such an approach could eventually be useful for the treatment of AS and other neurological conditions.In this study, we designed artificial transcription factors (ATFs) with the goal of increasing paternal expression of Ube3a can be achieved either by super activation of the Ube3a promoter or by repression of the inhibitory Ube3a-ATS transcript. Eleven zinc finger arrays consisting of six zinc finger modules each were constructed to 18-bp target sites (Supplementary Table S1), including four to a region upstream of the Ube3a locus, three upstream of the Snurf/Snrpn locus (thought to be an initiation site of the Ube3a-ATS17), two to sites common in the approximately nine upstream initiation sites of the Ube3a-ATS,8 and two to a region between the Snord115 cluster and Ube3a . Activating factors contained a VP64 transcriptional activation domain18 and were assayed in HEK293T cells using a reporter plasmid in which luciferase was driven by the Ube3a promoter . Repressing factors contained a KRAB transcriptional repression domain18 and were assayed using luciferase reporter plasmids that harbored the target sites upstream of an SV40 promoter. The factor that provided the most robust activation was A8, and the two factors that produced the most potent repression regulatory effect  were SR71 and S1 .In principle, activation of paternal 19), mCherry red fluorescent protein to aid in protein solubility and visualization, an HA epitope tag for detection, and an SV40 nuclear localization signal to ensure nuclear delivery. The TAT cell-penetrating peptide had been previously used to deliver proteins to the brains of mice following i.p. injection.20 The KRAB/VP64 domain was appended to the C-terminus of the zinc finger protein giving rise to TAT-S1, TAT-SR71, and TAT-A8 . Of these three constructs, only TAT-S1 was able to be purified in sufficient quantity (mg) for multiple mouse injection studies (data not shown). An electromobility shift assay (EMSA) demonstrated that S1 zinc finger array bound its target with an affinity of 5 ± 1.5 nmol/l . The EMSA was repeated with the TAT-S1 and showed similar affinity of 8 ± 1.5 nmol/l (data not shown). The binding preference of the TAT-S1 was investigated using the in vitro Bind-n-seq assay, in which the protein was incubated with oligonucleotides representing all possible 21-bp binding regions, and the preferred targets were identified by next-generation sequencing.21 The observed binding motif indicated a high specificity for the intended target sequence .To evaluate S1 binding to the target locus in vivo, we performed chromatin immunoprecipitation followed by PCR in mouse Neuro2A cells. The S1 zinc finger array bound strongly to its intended chromosomal target site in vivo, while no binding was observed at an unrelated promoter . Thus, the TAT-S1 showed substantial affinity and specificity for its target site.To investigate the potential of an injected ATF to regulate an endogenous gene in the brains of mice, we constructed fusion proteins for the most efficient regulators S1, SR71, and A8. These fusions contained an N-terminal maltose-binding protein for purification, a cell-penetrating peptide consisting of the 10-aa transduction domain of the HIV-transactivator protein . The closest match in the mouse genome contained two mismatches and was >30,000 bp from the nearest gene on chromosome 6. TAT-S1 and TAT-R6 differ by only 26 aa in their DNA-binding domains. The 922-aa TAT-ATFs (S1 and R6) were expressed in Escherichia coli using a cold temperature induction  and purified . Injection of adult wild-type C57BL/6 mice with purified TAT-S1 and TAT-R6  produced a significant peak of mCherry fluorescence in the brain within 4–8 hours . Full-length TAT-ATF protein could be detected by western blot analysis of brain nuclear lysates, in addition to potential lower molecular weight breakdown products . TAT-ATFs also distributed to other parts of the body , although lysates of these organs did not show any detectable full length TAT-ATF (data not shown). The data suggest that TAT-ATFs injected i.p. can cross the blood–brain barrier and enter nuclei in the cells of the brain.To confirm that the upregulation of Ube3a was due specifically to TAT-S1, a negative control construct was made with zinc finger array R6 that was not designed to bind at the et al.16 mouse model of AS that carried a transgene insertion in exon 5 (coding exon 2) at the 5′ end of the maternal Ube3a gene . The insertion causes strong attenuation of Ube3a protein expression from the maternal allele. Adult mice (≥2 months) were used for these experiments since the Ube3a-ATS would be fully expressed from the paternal allele22 and would more accurately reflect an age of human diagnosis (3–4 years old), unlike genetic crosses or early postnatal interventions. Based on the anti-Ube3a-ATS studies described earlier,11 we expected our TAT-S1 to activate Ube3a protein expression from the paternal allele in this mouse model. However, given the half-life of our protein's signal in the brain is between 8 and 24 hours, and the KRAB effector domain is known to provide only transient transcriptional repression,23 we reasoned that multiple administrations are necessary to ensure sufficient presence of the ATF over time to alter gene expression patterns. The ATFs were injected  three times per week for 4 weeks . A final injection was given four hours before harvest to examine distribution of the ATF.To investigate if TAT-S1 could perform its gene regulatory function in the brains of mice, we used the Jiang Figure 4b). Ube3a protein was found to be significantly increased in AS mice treated with TAT-S1, but not TAT-R6, in both hippocampus and cerebellum . The upregulation of Ube3a was confirmed by western blot analysis of brain cytosolic lysates from three different mice that received TAT-S1 treatment . In the western blot, the treatment-dependent appearance of a particular Ube3a isoform (the lower band) can be clearly seen. Both the immunohistochemistry and western blot confirm that the treatment increased Ube3a to a level intermediate between no-treatment AS and wild-type control mice. These results were replicated in an additional experiment in which the treatment was administered by i.p. injection for 7.5 weeks and visualized using a different antibody to Ube3a .We observed no overt signs of toxicity during the 4-week treatment period, based on normal appearance and behaviors of the mice and no visual organ pathology upon dissection (data not shown). We observed widespread distribution of ATFs throughout all regions of the brain . TAT-S1 was able to significantly increase Ube3a expression in both the hippocampus and cerebellum, indicating that the portion of the protein necessary for Ube3a-ATS repression entered the nuclei of cells in the brain. While TAT-S1 must be nuclear to activate Ube3a expression, the Ube3a protein is not necessarily expected to be nuclear. The two anti-Ube3a antibodies used in the current study detected primarily the cytoplasmic form. Previous publications have noted that different isoforms of Ube3a localize to the nucleus, the cytoplasm or both,24 and roles have been proposed for Ube3a at synaptic junctions.25Our study demonstrates that purified TAT-S1 protein can be injected s.c. or i.p. in AS mice, cross the blood–brain barrier, enter neurons throughout the brain, and alter the expression of Ube3a. We believe this to be the first report of an injectable engineered zinc finger repressor that can distribute widely in the brain and regulate the expression of an endogenous gene. Distribution of ATFs was observed by 26 Although formally this study did not demonstrate that delivery was TAT-dependent, adsorptive endocytosis would likely allow the protein to enter many cell types, including both neurons and glia in the brain. However, the Ube3a-ATS is not expressed in glia, in these cells Ube3a is always expressed from both the maternal and paternal alleles. The fact that TAT-S1 upregulated Ube3a strongly suggests that the factors entered neurons, as this is where Ube3a is imprinted by the Ube3a-ATS, the target of TAT-S1. The observation that Ube3a expression was increased by TAT-S1 but not TAT-R6, which differed by only 26 aa in their DNA-binding domains, indicates that the function of TAT-S1 was dependent on the DNA-binding specificity of its zinc finger array. We note that the presence of the maltose-binding protein band in the injected material did not appear to influence the results, as both the TAT-S1 and TAT-R6 samples contained this band but only TAT-S1 increased Ube3a. Therefore, increased Ube3a expression was not caused by potential nonspecific factors such as stress or potential immune response in the mouse, contaminants in the injection solution, or nontargeted actions by other parts of the protein such as detachment of the KRAB domain.The mechanism of TAT-mediated delivery of large proteins was reported to be adsorptive endocytosis.11 TAT-R6 appeared to cause a slight, but not significant, decrease in Ube3a levels in the experiment shown in Figure 3, but a slight, but not significant, increase in the experiment shown in Supplementary Figure S3. These fluctuations are likely due to experimental variation using different cohorts on different days. In the current study, the partial reactivation by TAT-S1 may have been caused by an insufficient concentration of TAT-S1 at the target site. Future experiments could increase either the dose or the timing of administrations. The partial response might also be due to additional Ube3a-ATS transcripts initiating from upstream promoters.8 Future experiments could examine additional ATFs that would inhibit these promoters and perhaps be delivered as a combinational therapy.In this study, S1 treatment only partially restored Ube3a expression to wild-type levels. This intermediate level was expected since previous therapeutic approaches using topoisomerase inhibitors, antisense oligonucleotides, and stop codon insertions had resulted in partial restoration of Ube3a from the paternal allele.27 In principle, the use of ATF biologics with short-term effects similar to drugs may have advantages over approaches such as permanent gene engineering, for which distribution, dosage and reversibility are problematic.12 The Ube3a-ATS transcript is only expressed in mature neurons of the brain , dominant (Huntington), or haploinsufficient .The prospect for long-term treatment with proteins has been demonstrated by insulin and numerous other biologics.transcription factor assays.In vitro  Zinc finger coding regions were designed by modular assembly methods33 and synthesized by BioBasic . The arrays were cloned using XhoI and NotI into the PGK promoter-driven mammalian expression vector pPGK-VP64,34 which appended an N-terminal HA epitope tag and SV40 nuclear localization sequence, and either a C-terminal VP64 transcriptional activation domain18 or a KRAB transcriptional repression domain, cloned at the NotI and PstI sites.18 Recognition helices of the zinc finger arrays are provided in Supplemental Table S1. Target sites for the ATFs were cloned between NotI and XhoI sites upstream of the SV40 promoter in pGL3-control plasmids , as listed in Supplementary Figure S1. In 24-well plates, HEK293T cells at 80% confluency in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 1 U/ml of penicillin, and 1 µg/ml of streptomycin were cotransfected with 100 ng of ATF expression plasmid, 25 ng of modified pGL3-control firefly luciferase reporter plasmid containing ATF target sites, and 25 ng of pRL-TK-Renilla Luciferase plasmid , using Lipofectamine 2000 . Cells were harvested 48 hours posttransfection by removing media, washing with 500 µl of 1× Dulbecco's phosphate-buffered saline , followed by lysis in 100 µl of 1× Passive Lysis Buffer (Promega) with 1× Complete Protease Inhibitors . Clarified cell lysates (20 µl) were used to determine luciferase activity using DualGlo reagents  in a Veritas microplate luminometer . The firefly luciferase signal was normalized to renilla luciferase controls. All experiments were performed in duplicate and repeated on 3 different days. Expression of TAT-S1 and TAT-R6 protein. TAT-ATF coding regions were cloned into the pMAL-c2X vector  between EcoRI and HindIII. The full sequence of the TAT-S1 protein is provided in Supplemental Figure S1, and the recognition helices of TAT-R6 are provided in SupplementalTable S1. Proteins were expressed in NEB5α E. coli (New England Biolabs) by inoculating 5 ml overnight cultures into 800 ml of Luria Broth medium  with 50 µg/ml Carbenicillin. Cultures were shaken overnight at 37 °C, then moved to 4 °C, and induced with isopropyl β-d-1-thiogalactopyranoside (0.5 mmol/l) and gently shaken for 48–72 hours. The cold temperature induction was critical to obtaining high yields; induction at room temperature or 37 °C was far less efficient. The culture was pelleted and resuspended in 30 ml of Zinc Buffer A , 90 mmol/l KCl, 1 mmol/l MgCl2, 100 µmol/l ZnCl2). The resuspended pellet was microfluidized, which was critical for obtaining full-length protein. Microfluidized lysates were run over columns of amylose resin  that had been prepared by washing twice with deionized water, twice with Column Buffer (20 mmol/l Tris–HCl (pH 7.4), 0.2 mol/l NaCl, 1 mmol/l ethylenediaminetetraacetic acid (EDTA)) and twice with ZBA. Protein was eluted in Elution Buffer  and concentrated to ~16 mg/ml using a Centricon Plus-70 . Proteins were sterile filtered to remove any residual bacteria and routinely checked to assure no detectable endotoxins were present. Protein integrity was evaluated using Coomassie-stained gels and concentration established via Nanodrop A280 UV absorption. Protein concentrations for injections refer to the full-length + maltose-binding protein band intensities, of which only half was considered to be the 100-kD full-length protein . Proteins were stored at −80 °C in ATF Injection Buffer . A dose of 160–200 mg/kg consisted of a 0.25 ml injection i.p. or s.c. containing 4 mg of total protein in a 20–25 kg mouse.Electromobility shift assays. Biotin-labeled DNA targets were generated by PCR-amplification using a 5′ biotinylated forward primer of 69-mer oligonucleotides containing 18-base pair zinc finger target site (Supplementary Table S2). PCR reactions contained unlabeled reverse primer in a 4:1 ratio over the biotinylated primer. Amplified targets were column purified . Protein–DNA complexes were mixed on ice and then incubated in the dark for 1.5 hours at room temperature in ZBA, 0.05% NP-40, 0.1 mg/ml bovine serum albumin, 10% glycerol, and 35–75 pmol/l target oligo. Protein–DNA complexes were separated from the unbound probe using 7% polyacrylamide Tris/boric acid/EDTA gels run in 0.5× Tris/boric acid/EDTA  and then transferred onto Biodyne B nylon membranes. Complexes were UV cross-linked to the membranes for 4 minutes . The biotinylated DNA was visualized using the LightShift Chemiluminescent EMSA Kit  according to the manufacturer's protocol. Equilibrium binding constants (apparent KD) were calculated from protein titration experiments. Gel images on X-ray film  were scanned and then quantitated using ImageJ. All reported EMSA measurements were averages of at least three experiments performed with independent protein dilutions. Representative data are shown in Figure 1b.Chromatin immunoprecipitation assay. The mouse neuroblastoma cell line Neuro2a (ATCC #CCL-131) was grown in Dulbecco's modified Eagle's medium supplemented with 10% bovine calf serum. Neuro2a cells were grown to 70% confluency and transfected with 15 µg S1-KRAB-expressing plasmid per 10-cm dish using Lipofectamine 2000 (Life Technologies). Cells were cross-linked 48 hours posttransfection by incubation with 1% formaldehyde solution for 10 minutes at room temperature. Chromatin immunoprecipitation assays were performed as previously described with minor modifications.35 About 30 µg of sonicated chromatin was incubated with 3 µg of monoclonal anti-HA antibody , monoclonal anti-RNA Polymerase II antibody (Covance 8WG16), or control mouse IgG (Sigma). Immunoprecipitates were captured using 3 µg rabbit anti-mouse serum and StaphA cells (Sigma). After washes and reversal of DNA–RNA–protein cross-links, standard and quantitative PCR were performed to confirm specific binding to the S1 target sequence in chromatin immunoprecipitation samples as compared to 0.1% input control or relative to binding at an unrelated promoter on chromosome 4. All primer sequences are listed in Supplementary Table S2. Primers Snurf-F and Snurf-R to the mouse Snurf gene promoter target site were used as positive control primers, while mmchr4-F and mmchr4-R to a region on mouse chromosome 4 served as a negative control. Specific fragments were amplified using GoTaq (Promega) DNA polymerase . PCR products were separated on a 1.5% agarose gel and visualized using the Gel Doc XR+ System (BioRad). Quantitative real-time PCR was used to calculate binding enrichment using the ΔΔCt method.Bind-n-Seq assay. Bind-n-Seq was performed essentially as previously described.21 Briefly, we used barcoded 93-mer double-stranded oligonucleotide targets containing Illumina  primer binding sites and a 21-nt random region. Various protein concentrations were incubated with 3 µmol/l oligo target, 0.12 µg/µl herring sperm DNA, 100 µmol/l ZnCl2, 5 mmol/l dithiothreitol, 1% bovine serum albumin, 5% glycerol, and 1, 50, or 100 mmol/l KCl. Bound complexes were precipitated using amylose resin and then washed with a corresponding buffer of 10 mmol/l Tris pH 8.5, 100 µmol/l ZnCl2, 1 mmol/l MgCl2, 5 mmol/l dithiothreitol, and 1, 50, or 100 mmol/l KCl. Eluted and amplified DNA was sequenced on an Illumina HiSeq2000 or MiSeq. Motifs were determined using MERMADE, available at korflab.ucdavis.edu/Datasets/BindNSeq/.Mice. All mice were maintained and experiments conducted according to University of California, Davis approved Institutional Animal Care and Use Committee (IACUC) guidelines and protocols. The AS mouse model16 was obtained from Jackson Laboratories  and maintained on a mixed background of SV129 and C57BL/6. Wild-type C57BL/6 mice (Jackson Laboratories) and SV129 mice  were used for matings with Ube3a-deficient female mice. Genotyping was performed according to the supplier's protocol, with the modification that the annealing temperature was changed to 60 °C. Primers are listed in Supplementary Table S2. All animals receiving injections were older than 2 months of age at the time of the first injection.Maestro in vivo imaging of mice. All mice used in the initial blood–brain barrier study were C57BL/6 male mice. All mouse images were performed on a Maestro 2 Imager , use and training provided by the Center for Molecular and Genomic Imaging at UC Davis. The green filter was used with acquisition settings of 550–800 nm in 10-nm steps. Before injections, an image was taken of the purified protein using the auto-expose option. Timepoints used were 15 minutes, 4 hours, 8 hours, and 24 hours postinjection. At each timepoint, fluorescent images were taken of the injection site, the head and the back. Exposure times for the injection site, head, and back images were established from a previous pilot experiment at 229, 1,959, and 1,986 ms, respectively. On all images, a mock-treated animal served as a control, mock was the ATF/protein elution buffer of ZBA, 500 mmol/l maltose. All images were based on the same raw inputs for mCherry signal and background mouse signal. Analysis of mCherry signal was performed by measuring the mCherry by using a consistent sized area in the brain region using Maestro software. The measurement area was the same for each mouse across all treatments. Additional later images were taken of the Ube3a-deficient mice when treated, to ensure brain localization of the TAT-ATFs.Immunohistochemistry. For the images in Figure 4b,c (50-µm-thick sections), one brain hemisphere was washed in 1× TBS , fixed in 10% buffered paraformaldehyde overnight, and then placed into 30% sucrose for 3 days. Following brain saturation, the tissue was frozen in Tissue-Tek CRYO-OCT Compound  and then sectioned on a Leica  cryotome. Blocking was performed using Superblock (Life Technologies) for 1 hour, followed by a 10% goat serum, 0.3% Triton X in 1× TBS for 2 hours. Following aspiration, slides were stained with primary anti-HA 1:150 (Roche 12013819001) that was directly labeled with an Apex Antibody labeling kit , or anti-Ube3a 1:100 (Sigma E8655) labeled with the Apex Antibody labeling kit, in 5% goat serum and 0.15% Triton X and incubated at 4 °C overnight. Slides were washed three times with 1× TBS, stained with 4′,6-diamidino-2-phenylindole for 10 minutes, washed three times with 1× TBS, and then mounted with coverslip using Prolong Gold (Life Technologies). Images were acquired using a Leica DM6000B epifluorescent microscope. A 10% linear reduction in brightness was applied equally to the green channel of all images to reduce autofluorescence and clarify features. All fluorescence intensity measurements were performed on unaltered images using ImageJ software 1.48v. Fluorescence intensities were measured in the hilus region of the dentate gyrus in the hippocampus and the Purkinje cell layer in the cerebellum.Supplementary Figure S3 (5-µm sections), paraffin embedding was utilized. One hemisphere of the brain was placed in 1× TBS and then fixed in 4% paraformaldehyde overnight. Brains were fixed in an additional 3 hours in 10% paraformaldehyde solution and then placed into wax for sectioning. Slides of these sectioned tissues were incubated overnight at 56 °C, washed with xylene twice for 10 minutes, and then soaked in 100% ethanol twice for 10 minutes. Antigen retrieval was performed using 1× Target Retrieval Solution  at 95 °C for 1 hour and then washed in 1× SSC  at room temperature for 5 minutes while rocking gently. Blocking was performed using Superblock for 1 hour, followed by a 10% goat serum, and 0.3% Triton X in 1× TBS for 2 hours. Following aspiration, slides were stained with primary anti-Ube3a 1:1,000  with coverslip and incubated at 4 °C overnight. Coverslip were removed and the slide was washed three times with 1× TBS. The secondary antibody, goat-anti-rat Alexa Fluor 647 1:1,000  or goat-anti-rabbit Alexa Fluor 488 1:1,000 (Abcam 150077) in 1× TBS, 5% goat serum, and 0.15% Triton, was applied for 2 hours, the slides were then washed three times with 1× TBS, and then dried before placing coverslips. Coverslips were applied with 4′,6-diamidino-2-phenylindole-containing Vectashield . The images in Supplementary Figure S3 received no alterations. All fluorescence intensity measurements were performed on unaltered images as above.For the images in Western blot. For the experiments in Figure 2d, one hemisphere from each brain was flash frozen in liquid nitrogen. Tissues were treated first with hypotonic buffer (2 mmol/l 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 5), 1 mmol/l KCl, 0.1 mmol/l EDTA, 10% Glycerol, 1× protease inhibitor cocktail , 50 µmol/l phenylmethylsulfonyl fluoride, 50 µl of 100 µmol/l dithiothreitol, in diethylpyrocarbonate-treated water) and then ground with motorized pestle for 35 seconds. The remaining nuclear pellet after centrifugation was lysed with radioimmunoprecipitation assay buffer , 1 mmol/l EDTA, 1× Protease Inhibitor Cocktail). The nuclear lysate was quantified using a BCA assay (Life Technologies). Polyacrylamide gels  were loaded with 250 µg of protein per sample and run at 115 V for 1.5 hours and then transferred to polyvinylidene difluoride membrane using a semi-dry blotting apparatus at 75 V for 1.5 hours. The membrane was blocked in 5% dry milk in TBST  and then probed for 48 hours at 4 °C with primary a rat anti-HA antibody conjugated to horseradish peroxidase at 1:100–250 . The western was incubated with the ECL Plus Reagent  for 15 minutes and imaged on a Storm 860 imager . Images were not altered.Figure 4e, mouse brains were harvested and immediately snap frozen at −80 °C. Proteins were extracted using the CelLytic NuCLEAR Extraction Kit (Sigma) according to the manufacturer's instructions. Protein concentration was determined utilizing the BCA Protein Assay kit (Pierce). About 20 µg of protein were separated on a Novex 4–12% Bis-Tris gel (Life Technologies) using 3-(N-morpholino)propanesulfonic acid buffer and BOLT Antioxidant as described by the manufacturer. Using the Mini Trans-Blot Cell (BioRad), proteins were transferred onto nitrocellulose membranes, in transfer buffer  methanol), overnight at 30 V in the cold room. After transfer, protein loading was evaluated using Ponceau S staining. Membranes were then blocked with 5% dry milk in TBST  and probed with anti-Ube3a antibody . After incubation with goat anti-mouse horseradish peroxidase , chemiluminescence was detected using the ECL Prime Western Blotting Detection Reagent .For the experiments in Figure S1. Sequence of proteins and reporters used in this study.Figure S2. TAT-ATFs protein purification.Figure S3. High-resolution images of Ube3a activation by TAT-S1 but not TAT-R6.Table S1. DNA binding sequence and recognition helices of the zinc finger arrays used in this study.Table S2. Primers used in this study.


Bifidobacterium bifidum lacto-N-biosidase was used as a model protein to evaluate the minimal functional unit due to its interest and structural complexity. By expressing different truncated forms of this enzyme, we show that Model A architectures cannot be reduced to Model B. In particular, there are two structural requirements general to GH20 enzymes with Model A architecture. First, the non-catalytic domain GH20b at the N-terminus of the catalytic GH20 domain is required for expression and seems to stabilize it. Second, the substrate-binding cavity at the GH20 domain always involves a remote element provided by a long loop from the catalytic domain itself or, when this loop is short, by an element from another domain of the multidomain structure or from the dimeric partner. Particularly, the lacto-N-biosidase requires GH20b and the lectin-like domain at the N- and C-termini of the catalytic GH20 domain to be fully soluble and functional. The lectin domain provides this remote element to the active site. We demonstrate restoration of activity of the inactive GH20b-GH20-α construct (model A architecture) by a complementation assay with the lectin-like domain. The engineering of minimal functional units of multidomain GH20 enzymes must consider these structural requirements.Hexosaminidases are involved in important biological processes catalyzing the hydrolysis of N-acetyl-hexosaminyl residues in glycosaminoglycans and glycoconjugates. The GH20 enzymes present diverse domain organizations for which we propose two minimal model architectures: Model A containing at least a non-catalytic GH20b domain and the catalytic one (GH20) always accompanied with an extra α-helix (GH20b-GH20-α), and Model B with only the catalytic GH20 domain. The large  They cazy.org ). GH20 hcazy.org ,5. In adBifidobacterium bifidum metabolizes type 1 human milk oligosaccharides by the action of extracellular sialidases, fucosidases and lacto-N-biosidase.GH20 enzymes comprise not only β-N-acetylhexosaminidases (EC.3.2.1.52), catalyzing the removal of terminal non-reducing N-acetylhexosamine residues, but also lacto-N-biosidases (EC.3.2.1.140), hydrolyzing the terminal lacto-N-biosyl residues from the non-reducing end of oligosaccharides such as human milk oligosaccharides (HMOs). HMOs play an important role in the development of intestinal flora and in the modulation of the immune system of breast-fed infants –8. HMOs B. bifidum lacto-N-biosidase (LnbB) as a model protein because of its complex domain organization among GH20 enzymes and its significance in the metabolism of human milk oligosaccharides. The LnbB gene encodes for a membrane-anchored extracellular enzyme of 1112 amino acid residues. Recently, it was cloned and the recombinant full length protein was characterized confirming that this enzyme catalyzes the hydrolysis of the tetrasaccharide Galβ1,3GlcNAcβ1,3Galβ1,4Glc to lacto-N-biose  and lactose , codon-optimized for the expression in Escherichia coli were produced by GenScript . The LnbB gene (37–1064) was cloned into the pET24b plasmid  using the restriction sites NdeI and XhoI, and the construct A gene (40–528) into the pET28a+ vector using NdeI and Bpu1102. Constructs B (176–497) and Bα (176–528) genes were obtained by PCR reaction using construct A as template, and constructs D (40–776) and F (546–776) genes using the full length LnbB gene. The following forward and reverse primers were used (restriction site are underlined): P1 (CATATGAAGCCGAAATATAAAGAACGT-3’5’-GGAATTC) and P2 (GCTCAGCGGCGTCAAC-3’5’-TAATA) for construct B; P1 and P3 (GCTCAGCACGGGAGTCACTCCA-3’5’-TAATA) for construct Bα; P4 (CCATGGCTGATGACTCCGCAGCAGGCTA-3’5’-GCGTAT) and P5 (CTCGAGCAGGCTGCCGGTCAGC-3’5’-GCGTAT) for construct D, and P6 (CCATGGTGGATGCGGGTATC-3’5’-GCGTAT) and P5 for construct F. All constructs were cloned into the pET28a+ vector. E. coli DH5 cells were transformed and positive transformants were verified by DNA sequencing.Two synthetic genes of Lacto-N-biosidase from E. coli BL21 (DE3) star cells were grown following an autoinduction protocol [-1 kanamycin, 25mM Na2HPO4, 25mM K2HPO4, 50mM NH4Cl, 5mM Na2SO4, 2mM MgSO4, 0.5% glycerol, 0.05% glucose and 0.2% lactose at 250 rpm and 30°C for 24 h. The full length protein and constructs A, B and Bα were also obtained using 1 mM IPTG induction after 16 h of growth at 37°C in LB medium with kanamycin achieving similar results. Cells were harvested by centrifugation , resuspended in buffer A  and disrupted in a cell disrupter (20000 psi). After centrifugation , the supernatant was loaded into a 1mL Ni2+-charged HiTrap chelating column chromatography. Proteins were purified to homogeneity by a linear gradient of Buffer B . Collected fractions were analyzed by SDS-PAGE and fractions containing the proteins were combined and dialyzed against buffer A with a 30 kDa cut-off membrane. All proteins were purified further using an XK16/60 Superdex 200 column , preequilibrated with buffer A. Fractions were pooled, concentrated with a Centricon YM-10 unit (Merk Millipore) and stored at 4°C. Protein concentrations were determined spectrophotometrically by the BCA method measuring A590 nm using BSA as standard [Expression and purification of the full length protein and constructs were carried out as follows: recombinant cultures of protocol  in LB mestandard .Protein size was determined using dynamic light scattering in a Nano ZS Nanosizer  with a laser light wavelength of 632.8 nm and a scattering angle of 173 degrees. Temperature was set at 25°C. Protein solutions in buffer A were measured without previous dilution. Full length protein showed a single peak for monodisperse particles of 10 nm and PDI 0.30, which is consistent with monomeric form.p-nitrophenyl β-lacto-N-bioside  were performed at 30°C in a volume of 0.1 mL using 96-wells microtiter plates measuring p-nitrophenol concentration at 405 nm. Enzymatic reactions were performed in a reaction mixture containing 0.25 mM substrate and 50mM of citrate-phosphate buffer (pH 4.5). After pre-incubation for 5 min, reactions were initiated by addition of the enzyme . At regular time intervals, reactions were quenched by the addition of 0.15 mL of quenching buffer  and p-nitrophenol release was measured by absorbance at 400 nm with a microplate-reader MRX (Cultek). Triplicate measurements at each concentration were performed.Lacto-N-biosidase activity assays with Enzymatic reactions with constructs A and F were performed using standard conditions in a Bravo liquid handling Robot (Agilent). After pre-incubation of construct A (50 nM) and F (25 nM-500 nM) for 5 minutes, addition of the substrate initiated the reaction. Triplicate measurements at each concentration were performed. The complex of constructs A and F was also characterized using gel filtration chromatography. The mixture of construct A (42 μM) with excess of construct F (245 μM) was loaded to the Superdex 200 column pre-equilibrated with buffer A and calibrated with protein standards. Fractions were further analyzed by SDS-PAGE and their hydrolase activity was determined. This purification was performed by duplicate.Enzymatic activities of construct A (50 nM) in the presence of BSA at 1:1, 1:4 and 1:10 ratio were analyzed using standard conditions.http://www.cazy.org, [http://www.pdb.org). Protein sequences were retrieved from UniProt (http://www.uniprot.org). Assignment of protein domains were obtained from different databases  and the consensus domain organizations are reported in The list of currently available 3D structures of GH20 enzymes was retrieved from CAZY database  and Bacteroides fragilis (Bf3009) enzymes remains unknown.To date, eleven of the thirteen GH20 enzymes structures fit into Model A . As summFor the monomeric proteins, SpHex and ScHex structures, which share 94% sequence identity, comprise the GH20b domain followed by the catalytic GH20 domain ,27. Hex1For the dimeric proteins, each subunit of HexA, HexB ,29 and OAggregatibacter actinomycetemcomitans (DspB) and the β-hexosaminidases from Streptococcus pneumoniae (StrH). Both are monomeric proteins in solution where DspB presents only one domain, the unique GH20 domain [S. pneumoniae were crystallized [The remaining two GH20 enzymes present structures that are assigned to Model B : dispers0 domain , and Strtallized . The GH2The full length protein was truncated according to the two different models of structure organization we propose, and three different truncated proteins were designed: construct A, B and Bα .et al. (2013) in which different LnbB truncated forms were designed, revealed that a truncated protein (37–520) equivalent to construct A exhibits no catalytic activity. A longer truncated protein (37–663) composed by GH20b-GH20-α and a part of the lectin-like domain was reported to be as active as the full length. These data indicate that the GH20b-GH20-α architecture is not the minimal functional unit for LnbB, as it is for the rest of the model A GH20 enzymes. Further studies are presented to determine the details of the minimum requirement for LnbB to be catalytically active.Construct A (residues 40–528) follows the architecture organization of Model A enzymes and is composed of the GH20b and GH20 domains always followed by the extra α-helix. This construct would be expected to be active since several enzymes of model A exhibit just these two domains alone in the wild-type protein . Furthermore, a truncated form of Hex1T  is reported to be active . HoweverConstruct B (residues 176–497) mimics Model B enzymes and is formed by the single catalytic GH20 domain alone. Lastly, construct Bα (residues 176–528) was designed to tackle the open question whether the accompanying GH20b domain can be removed in Model A enzymes. This construct encodes the catalytic GH20 domain with the extra α-helix at the C-terminus. Both constructs, B and Bα, were designed for the study of the behaviour of the GH20 catalytic domain in model A proteins.E. coli. The full length protein (37–1064), without the signal peptide and membrane anchor regions, was expressed as C-terminal His6-Tagged protein. Constructs A, B and Bα were expressed as N-terminal His6-Tagged proteins. Cultures were grown at 30°C for 24 h with autoinduction medium. The full length protein and construct A were obtained as soluble intracellular proteins with yields of 16 and 27 mg/L of culture respectively. SDS-PAGE analysis and MALDI-TOF mass spectrometry confirmed the theoretical 112 kDa of the full length protein and 57 kDa of Construct A. Further characterization by gel filtration chromatography showed that the full length protein is monomeric with particle size of about 10 nm by dynamic light scattering, whereas Construct A is mainly dimeric in solution .Engineered proteins of LnbB enzyme were cloned and expressed in solution . The fulOn the other hand, constructs B and Bα were only present in the insoluble pellet and no soluble protein was achieved after different expression conditions such as autoinduction or IPTG induction at different temperatures (data not shown). This seems to indicate that the single GH20 catalytic domain of LnbB, with or without the extra α-helix, is not stable enough and yields insoluble aggregates. Thus, GH20b domain at the N-terminus of GH20 is required for LnbB expression and stability. This domain is present in all model A enzymes being the relative orientation of GH20b and GH20 catalytic domain the same in all structures . The num-1, but construct A was inactive, as expected according to previous studies [The hydrolytic activity of the soluble constructs was assayed using p-nitrophenyl β-lacto-N-bioside as substrate at pH 4.5 and 30°C. Full length protein showed a specific activity of 27.2 ± 3.5 s studies . The GH2While this minimal GH20b-GH20-α architecture assures the activity of SpHex and Hex1T ,36 and aInterestingly, we have detected that this pattern is reproduced in all proteins of model A: there is always a remote element assisting in the definition of the substrate binding cavity by means of direct interactions with conserved amino acids directly involved in protein—substrate interactions. By contrast, when loop 2 is short, this remote element comes from the accompanying domains, as in LnbB, or from the C-terminus domains of the other monomer in dimeric structures, such as, GcnA, HexA, HexB and OfHex1. For example, in the homodimeric structure of GcnA, domain III forms a significant part of the dimer interface . The C-tS. gordonii hexosaminidase or in subsite -2 of the LnbB, or participate in the second shell of this cavity behind the conserved arginine and aspartate residues in subsite -1 of hexosaminidases.Therefore, all GH20 structures of model A clearly show the presence of a remote element folding on the substrate-binding cavity providing amino acid residues that either complete the active site, as in the extended subsite -1 of the 6-Tagged proteins were purified and analyzed by SDS-PAGE and MALDI-TOF, confirming the corresponding theoretical molecular masses of 83 and 28 kDa for constructs D and F, respectively. Gel filtration chromatography showed that both proteins were monomers in solution (-1). Therefore, both C-terminal domains in LnbB, the CBM32 and Ig like domains are not important for the catalytic activity, and construct D is the minimal functional unit of LnbB. Next, a complementation experiment was assayed by adding Construct F (the single lectin domain) on the inactive Construct A. Activity was restored in a concentration-dependent manner when increasing the concentrations of Construct F  is inactive because of the lack of the remote element, would addition of the lectin domain (as a single protein) reconstitute the active site and restore activity? Toward this end, Construct F (the single lectin domain, solution . Construstruct F . At a mostruct F . Fractiostruct F  and the struct F  demonstrThis structural-functional analysis allows us to come to different conclusions. First, we identified two levels of domain organization in GH20 enzymes: model A with GH20b-GH20-α architecture and model B with the single catalytic GH20 domain alone. As LnbB constructs show, model A enzymes cannot be reduced to model B, since GH20b domain is a structural requirement in the N-terminus of GH20 to assure protein stability. Secondly, GH20b-GH20-α architecture is not always the minimal functional unit. For the LnbB enzyme here studied, the isolated GH20b-GH20-α domain is inactive and requires the lectin domain which provides an essential loop to shape the active site. Based on these results and analysis of the structures of GH20 enzymes, we propose a broader mechanism by which important interactions in the substrate binding cavity of the catalytic GH20 domain are provided by a remote element in all structures, which must be preserved to ensure a proper definition of the active site. The remote element can directly be provided by a long loop 2 of the GH20 domain itself. In this case, GH20b-GH20-α can act as a minimal functional unit, and large multidomain enzymes can be engineered to reduced forms with a minimal model A architecture. In other circumstances, when loop 2 is short, proteins dimerize or present an accompanying domain in the C-terminus of GH20 that provide this remote element. In the latter cases, protein engineering of these enzymes must be designed maintaining the accompanying domains. The proposed model can stimulate further studies on other members of the superfamily.


Background: Csd6 is one of the cell shape-determining proteins in H. pylori.Results: The active site of Csd6 is tailored to function as an l,d-carboxypeptidase in the peptidoglycan-trimming process.Conclusion: Csd6 constitutes a new family of l,d-carboxypeptidase.Significance: The substrate limitation of Csd6 is a strategy that H. pylori uses to regulate its helical cell shape and motility. Helicobacter pylori causes gastrointestinal diseases, including gastric cancer. Its high motility in the viscous gastric mucosa facilitates colonization of the human stomach and depends on the helical cell shape and the flagella. In H. pylori, Csd6 is one of the cell shape-determining proteins that play key roles in alteration of cross-linking or by trimming of peptidoglycan muropeptides. Csd6 is also involved in deglycosylation of the flagellar protein FlaA. To better understand its function, biochemical, biophysical, and structural characterizations were carried out. We show that Csd6 has a three-domain architecture and exists as a dimer in solution. The N-terminal domain plays a key role in dimerization. The middle catalytic domain resembles those of l,d-transpeptidases, but its pocket-shaped active site is uniquely defined by the four loops I to IV, among which loops I and III show the most distinct variations from the known l,d-transpeptidases. Mass analyses confirm that Csd6 functions only as an l,d-carboxypeptidase and not as an l,d-transpeptidase. The d-Ala-complexed structure suggests possible binding modes of both the substrate and product to the catalytic domain. The C-terminal nuclear transport factor 2-like domain possesses a deep pocket for possible binding of pseudaminic acid, and in silico docking supports its role in deglycosylation of flagellin. On the basis of these findings, it is proposed that H. pylori Csd6 and its homologs constitute a new family of l,d-carboxypeptidase. This work provides insights into the function of Csd6 in regulating the helical cell shape and motility of H. pylori. Helicobacter pylori is a spiral-shaped Gram-negative bacterium that colonizes the upper gastrointestinal tract in approximately half of the world's human population. Its infection of the gastric mucosa is associated with various gastric diseases, including chronic gastritis, peptic ulcer, mucosa-associated lymphoid tissue lymphoma, and gastric adenocarcinoma  and the antibiotics such as clarithromycin and amoxicillin (or metronidazole). However, increasing drug resistance in H. pylori requires the discovery of new antibiotics , where mDAPmeso-2,6-diaminopimelate, and the neighboring peptides are cross-linked exclusively by the 4→3 linkage between the main chain of d-Ala4 from one strand and the side chain of mDAP3 from another strand  of the M14 metallopeptidase family and cleaves the γ-d-Glu2-mDAP3 bond of the muramyl tripeptide to produce the muramyl dipeptide  activities (hp0518 gene in H. pylori 26695 strain) was identified as another member of the peptidoglycan trimming pathway and also a cell-shape determinant of H. pylori  was suggested to effect the Nod1 activation and ultimately the NF-κB transcriptional activity  that can possibly catalyze nonclassical 3→3 cross-linking of peptidoglycan. The YkuD domain-containing l,d-transpeptidases  have been identified in a range of bacteria, including Enterococcus faecium, Bacillus subtilis, and Mycobacterium tuberculosis  , a sialic acid resembling sugar  , which ang sugar , 38. Theng sugar , 39. Intnization . It suggH. pylori Csd6, its biochemical, biophysical, and structural characterizations have been performed in this study. Mass analyses using the synthetic muramyl peptides indicate that Csd6 functions only as the l,d-carboxypeptidase  and not as the l,d-transpeptidase . Analytical ultracentrifugation and single molecule fluorescence resonance energy transfer (FRET) analyses indicate that Csd6 exists as a dimer in solution. A Csd6 monomer has a three-domain architecture consisting of the N-terminal domain , the middle l,d-CPase domain (Lys-67–Glu-202), and the C-terminal nuclear transport factor 2-like domain . The NTD shows very remote structural similarity to other known protein structures and plays a key role in dimerization. The middle catalytic domain has an overall fold of the l,d-TPase domain, validating the bioinformatics prediction. However, here this catalytic domain is referred to as the “l,d-CPase” domain, because we as well as others show that Csd6 functions only as an l,d-CPase. The Csd6 l,d-CPase domain resembles those of well characterized l,d-TPases, but its pocket-shaped active site is uniquely defined by the four loops I–IV, among which loops I and III show the most distinct variations in sequence length and conformation from known l,d-TPases. The d-Ala-complexed structure suggests possible binding modes of both the substrate and product to the l,d-CPase domain. The NTF2-like domain possesses a deep pocket for possible binding of a hydrophobic ligand such as Pse, and an in silico docking study supports its role in the control of the glycosylation level of flagellin. This work provides further insights into the strategy of H. pylori for regulating its helical cell shape and motility, which are crucial for its virulence. The reported structural information would serve as the foundation in a search for new drug targets to fight infections by H. pylori.To better understand the molecular function of csd6 gene from H. pylori 26695 strain, encompassing residues Val-13–Lys-330 of the gene product (HP0518), was cloned into the expression vector pET-28b(+) (Novagen) to express the recombinant Csd6 protein fused with a His6-containing tag at its N terminus. It was overexpressed in Escherichia coli Rosetta2(DE3)pLysS cells using a Luria Broth culture medium. Protein expression was induced by 0.5 mm isopropyl β-d-thiogalactopyranoside, and the cells were incubated for an additional 20 h at 18 °C following growth to mid-log phase at 37 °C. The cells were lysed by sonication in buffer A  containing 10% (v/v) glycerol and 1 mm phenylmethylsulfonyl fluoride. The crude lysate was centrifuged at 36,000 × g for 1 h. The recombinant Csd6 protein showed a tendency to aggregate at 4 °C; therefore, it was purified at room temperature in two column chromatography steps. The supernatant was applied to an affinity chromatography column of HiTrap Chelating HP , which was previously equilibrated with buffer A. Upon eluting with a gradient of imidazole in the same buffer, the Csd6 protein was eluted at 120–150 mm imidazole concentration. The eluted protein was applied to a HiLoad XK-16 Superdex 200TM column , which was previously equilibrated with 20 mm Tris-HCl, pH 7.9, and 150 mm sodium chloride. Fractions containing the Csd6 protein were pooled and concentrated to 5.8 mg ml−1 for crystallization using a YM10 ultrafiltration membrane (Amicon). This construct gave the best crystals in crystallization experiments. Other constructs were also tried for expression and crystallization. The Val-13–Lys-330 construct fused with a C-terminal His6-containing tag was not expressed in E. coli Rosetta2(DE3)pLysS cells. The constructs covering residues 4–330 or 17–330 were expressed at very low levels and were insoluble. The construct covering residues 1–330 (full-length) was expressed in a soluble form, but its crystals diffracted poorly to 7 Å only.The PCR-amplified l,d-TPase assay, LdtMt2, an l,d-TPase encoded by the rv2518c gene of M. tuberculosis H37Rv strain, was used. The N-terminal region (Leu-20–Ala-42) of LdtMt2 is predicted to form a putative transmembrane helix; the gene covering residues Ala-55–Ala-408, fused with a His6-containing tag at its C terminus, was cloned into pET-21a(+) (Novagen). The recombinant LdtMt2 was overexpressed and purified essentially as above.As a positive control in the m potassium nitrate, 18% (w/v) polyethylene glycol 3,350, and 10 mm EMTS). EMTS-derivatized crystals grew to approximate dimensions of 0.1 × 0.1 × 0.05 mm within a few days. Single-wavelength anomalous diffraction data were collected at 100 K from an EMTS-derivatized crystal of Csd6 using a cryoprotectant solution containing 25% (v/v) glycerol added to the reservoir solution -derivative crystals of the recombinant Csd6 were obtained at 23 °C by co-crystallization in the presence of EMTS by the sitting-drop vapor diffusion method. Sitting drops were prepared by mixing 3 μl of the protein solution and 2 μl of the reservoir solution . The microseed crystals were originally obtained with ligand-free Csd6 using the above reservoir solution. The crystals grew to approximate dimensions of 0.2 × 0.1 × 0.05 mm within a few days. X-ray diffraction data for the ligand-free Csd6 (Csd6-unbound) were collected at 100 K using a cryoprotectant solution containing 20% (v/v) glycerol added to the reservoir solution (d-Ala-bound Csd6 (Csd6-Ala) were collected at 100 K after the native crystal was preincubated for 2 min in a cryoprotectant solution containing both 20% (v/v) glycerol and 96 mmd-Ala, which were added to the reservoir solution  Tacsimate, pH 7.0, 22% (w/v) PEG 3,350, and 100 msolution . X-ray dsolution . The strsolution  using thsolution . Data coi.e. Trp-227, Lys-258, Tyr-297, Lys-313, and Glu-329) in the binding pocket. (ii) In the initial Glide docking stage, a potential docking with van der Waals radius scaling of 0.5 for the protein and ligand was performed retaining a maximum number of 20 poses per ligand. (iii) Residues within 5.0 Å of ligand poses were kept free to move in the Prime refinement step, and the side chains were further optimized. (iv) Poses within 30 kcal mol−1 of the energy cutoff in the previous step were re-docked using Glide XP. (v) The binding energy (IFDScore) for each output pose was computed as implemented in the IFD protocol. The best docked poses were also reproduced by a different docking program, Surflex-Dock of Sybyl X 2.0 . All the computations were undertaken on an Intel® XeonTM Quad-core 2.5 GHz workstation with Linux Cent OS release 5.5.Induced fit docking (IFD)  , 50 was N-acetylneuraminic acid) and Pse onto Micromonospora viridifaciens sialidase (PDB code 1EUS) (Pseudomonas aeruginosa pseudaminidase (PDB code 2W38)  in M. viridifaciens sialidase   and Pseude 2W38)  was perfialidase . The bing result . These rN-acetylmuramic acid-l-Ala1-γ-d-Glu2-mDAP3-d-Ala4) and muramyl pentapeptide (β-methyl N-acetylmuramic acid-l-Ala1-γ-d-Glu2-mDAP3-d-Ala4-d-Ala5) were prepared as described previously (A and B) (m was incubated for 2 h at 37 °C with the recombinant Csd6 protein (5 μm), which was dissolved in 20 mm sodium phosphate, pH 6.0, and 150 mm sodium chloride. A sample solution (1 μl) was mixed on the target with a fresh saturated matrix solution of 2,5-dihydroxy benzoic acid dissolved in 0.1% (v/v) trifluoroacetic acid and 50% (v/v) acetonitrile. For sample deposition, a 384-position stainless steel sample plate was used. Mass spectra were acquired on a matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometer  equipped with a nitrogen laser . Mass spectra were obtained in a positive ion mode. Helium was used for trapping and cooling ions in the ion source. The pressure in the trap was held at 4 × 10−3 torr. Each spectrum constituted an average of 200 profiles. All spectra were externally calibrated with bradykinin (757.3992 Da), angiotensin II (1046.5418 Da), angiotensin I (1296.6848 Da), Glu-fibrinopeptide B (1570.6768 Da), and N-acetyl renin substrate (1800.9432 Da) in TOFMixTM . Acquisition and data processing were controlled by the LanchpadTM software. The m/z values of multiple sodium adduct ions from the substrates and products are shown in A–E. As a positive control, the peaks were detected corresponding to the dimeric cross-linked species between the muramyl tetrapeptide and muramyl tripeptide (C), a product of the l,d-TPase reaction catalyzed by the recombinant LdtMt2 (Ala-55–Ala-408). The l,d-TPase domain  of M. tuberculosis LdtMt2 shows sequence identity and similarity of 22 and 38%, respectively, with the Csd6 l,d-CPase domain (residues Lys-67–Glu-202).As potential substrates, synthetic muramyl tetrapeptide  . The anaipeptide C, a prodl,d-CPase activity of Csd6, each of seven residues in the active site was mutated into alanine as follows: E110A, Y132A, Y133A, H155A, W158A, H160A, and C176A. The mutations were verified by DNA sequencing. The wild type and these single mutants of Csd6 were incubated with the substrate muramyl tetrapeptide for 30 min at 23 °C. Mass spectra were acquired as described above.To evaluate the importance of specific residues in the m Tris-HCl buffer, pH 7.9, containing 150 mm sodium chloride and 1.5 mm tris(2-carboxyethyl)phosphine at 20 °C. For the equilibrium sedimentation experiment, the absorbance from the Csd6 samples was measured at 235 and 280 nm using a six-sector cell at two speeds  and at three different Csd6 monomer concentrations  with a loading volume of 135 μl. For the sedimentation velocity experiment, the Csd6 samples at two different monomer concentrations (0.40 and 5.00 μm) were measured in double-sector cells at 30,000 rpm, and the collected data were analyzed by SEDFIT and SEDPHAT programs available on line. The concentration of the recombinant Csd6 protein was calculated using ϵ280 nm = 54,780 m−1 cm−1.To determine the oligomeric state of the recombinant Csd6 in solution, equilibrium sedimentation and sedimentation velocity experiments were carried out using a Beckman ProteomeLab XL-A analytical ultracentrifuge in 20 mE-S graph (A), where E is the FRET efficiency and S denotes the Cy3/Cy5 molar ratio in a dimer. The Csd6 sample was divided into 2 aliquots, each of which was labeled on the sole cysteine residue (Cys-176) with the maleimide-reactive Cy3 dye (donor) or the Cy5 dye (acceptor). Subsequently, Cy3-labeled Csd6 (Cy3-Csd6) and Cy5-labeled Csd6 (Cy5-Csd6) were mixed at 2 μm final monomer concentration in a buffer  for 3 h at 37 °C. The protein sample was diluted further to 100 pm using a single molecule buffer  glycerol, 1 mm 2-mercaptoethylamine, and 0.01% (v/v) bovine serum albumin). The single molecule data were acquired from a 10-min measurement.To analyze the dimeric form of Csd6 in solution, a single molecule FRET technique incorporating alternating laser excitation (ALEX) was employed , 55. Bri-S graph A, where N-acetylmuramic acid-l-Ala1-γ-d-Glu2-mDAP3) as a reaction product were assessed using a Reichert SR7500 surface plasmon resonance (SPR) dual channel instrument . Purified Csd6 in 20 mm sodium acetate, pH 5.5, was immobilized using the standard amino coupling at 20 μl min−1 on a carboxymethyl dextran hydrogel surface sensor chip  until saturation was achieved. The running buffer B used in all SPR experiments was 20 mm HEPES, pH 7.9, and 150 mm sodium chloride. SPR experiments were performed at 25 °C. The muramyl tetrapeptide or muramyl tripeptide at concentrations of 15.6, 31.3, 62.5, 125, 250, and 500 μm was injected over the Csd6-chip at 30 μl min−1 for 5 min for association analyses. Subsequently, the running buffer was flowed over the chip for an additional 6 min (30 μl min−1) for dissociation analyses. Regeneration of the chip was carried out using 20 mm sodium hydroxide. Binding was detected as a change in the refractive index at the surface of the chip as measured by the response unit. A reference flow cell was used to record the response by bovine serum albumin (BSA) as a positive control, and the response by BSA was subtracted from each sample. SPR data were fit using the Scrubber2 software.The kinetics and affinity of Csd6 with the synthetic muramyl tetrapeptide and muramyl tripeptide  as potential substrates using mass analyses. When the muramyl pentapeptide was incubated with the recombinant Csd6(13–330), no reaction product could be detected (C). This suggests that Csd6 does not have “endo”-, “d,d-carboxy”-, or “l,d-trans”-peptidase activities with the muramyl pentapeptide. The l,d-CPase domain of Csd6 shows limited but significant sequence identity (22%) with the l,d-TPase domain of M. tuberculosis LdtMt2. M. tuberculosis LdtMt2 is a functional l,d-TPase, catalyzing the formation of 3→3 cross-links between the muramyl tetrapeptide and the muramyl tripeptide  but also the dimeric cross-linked species  were detected as the reaction products. In comparison, when the muramyl tetrapeptide was incubated with the recombinant Csd6, the muramyl tripeptide was detected as the sole reaction product of trimming without any dimeric cross-linked species . This result implies that Csd6 would not be active for cross-linking, if the muramyl tripeptide were used as a substrate. The present data, together with the previous report that Csd6 converts monomeric tetrapeptides of the peptidoglycan sacculus into tripeptides  and in the d-Ala-bound state (“Csd6-Ala”)  A and in d6-Ala”) . Refinedl,d-CPase domain (Lys-67–Glu-202), and the C-terminal NTF2-like domain (Thr-209–Lys-330) (A). The NTD consists of three α-helices (αA–αC), and the helix αC is positioned at one end of the antiparallel coiled-coil formed by helices αA and αB. The l,d-CPase domain consists of two curved β-sheets (β3↓-β2↑-β1↓-β8↓-β4↑ and β4↑-β5↓-β6↑-β7↑), which share the β4-strand, as well as two α-helices (αD and αE). The NTF2-like domain forms an antiparallel four-stranded β-sheet (β9↓-β10↑-β11↓-β12↑), whose concave side is packed by three α-helices (αF–αH). DALI structural similarity searches , the middle Lys-330) A. The NTsearches  revealeddatabase .A). Virtually no interaction exists between the NTD and the l,d-CPase domain; the buried surface area at the interface is only 36 Å2, as calculated by the PISA server . The NTD (helix αC) and the NTF2-like domain (helix αF) interact with each other, burying a surface area of 410 Å2 at the interface.In the Csd6 monomer structure, three domains are arranged in a tripartite leaf-like fashion with a single chain connecting two adjacent domains A. VirtuaA server . Much mom). The data fit well to a dimer model, and a representative result measured at 16,000 rpm using the 3.65 μm monomer concentration is presented in Fig. 2B. Further sedimentation velocity experiments at 0.40 and 5.00 μm Csd6 monomer concentrations also indicate the oligomeric state of Csd6 as a dimer, without any discernable dissociation of a dimer into monomers, down to 0.40 μm . In the crystal of Csd6, two possible models of the dimer could be identified. The dimer model I has a buried surface area of 1,080 Å2 per monomer (6.7% of the monomer surface area) at the interface, as analyzed by the PISA server  (2 (4.5% of the monomer surface area) . In the dimer model II, the two monomers are related by an exact crystallographic 2-fold symmetry. The ΔiG values (solvation free energy gain upon formation of the interface) are −15.8 and −2.1 kcal mol−1 for models I and II, with the complexation significance scores of 1.000 and 0.000, respectively. These complexation significance score values imply that the interface in model I, but not in model II, plays an essential role in complex formation. Therefore, we conclude that the dimer model I in the crystal represents the biologically relevant Csd6 dimers in solution. This conclusion is supported experimentally by the distance between two Cys-176 residues in the dimer as estimated using the single molecule ALEX-FRET technique (A). The single molecule ALEX-FRET experiment was performed at 100 pm monomer concentration, which is much lower than that used for analytical ultracentrifugation, to avoid ensemble averaging (i.e. to avoid the case of having two noninteracting species) (Ecorr ≈0.03), using oR (Förster distance) of 60 Å for the Cy3 and Cy5 pair. In the crystal structure of Csd6-unbound, the distance between sulfur atoms of two Cys-176 residues is 96 and 66 Å in dimer models I and II, respectively. This result unambiguously favors model I, supporting the assignment of dimer model I in the crystal as the dimer in solution .Equilibrium sedimentation measurements indicate that the Csd6 protein exists as a homogeneous dimer in solution (at the tested Csd6 monomer concentration range of 2.74–4.56 μr panel) . In the echnique A. The sispecies) . After di.e. dimer model I) is elongated with approximate dimensions of 100 × 80 × 50 Å (B). In this dimer, two l,d-CPase domains are well separated from each other, and their active sites are open toward the bulk solvent (B). The bulk of the buried surface area at the dimerization interface is contributed by the NTD (82.7%), with the linker between the NTD and the l,d-CPase domain contributing 5.7% and the NTF2-like domain contributing 11.6%. The N-terminal helix αA of the NTD takes slightly different conformations in the two monomers, with a maximum Cα deviation of 8.1 Å at Leu-15. The two NTDs pack against each other through their antiparallel coiled-coils (helices αA and αB), at an angle of about 60°, to form a four-helix bundle (B); numerous hydrophobic side chains are present at the interface. The αC-helices from two NTDs cover the sides of the four-helix bundle. Seven hydrogen bonds  and a salt bridge (involving Arg-26 and Glu-53) exist at the interface (C). Interestingly, the Csd6 dimer has a deep crevice of an approximate size of 20 × 20 Å, with a widened bottom . The NTDs provide the bottom of the crevice, and the C-terminal NTF2-like domains form the two sides of the crevice. The sides of the crevice are lined with positively charged residues  but no negatively charged residues .The Csd6 dimer . The Csd6 NTD is perhaps functionally more related to the L27N domain of Mus musculus PALS1-associated tight junction protein  (e.g. PALS1-L27N domain and PATJ-L27) heterodimerize by building a compact four-helix bundle structure through the first two helices from each L27 domain  alone showed very remote structural similarities to other known protein structures. The highest  of 12%) . L27 dom of 12%) , 63. Lik7 domain .H. pylori Csd6 to have an overall fold of the l,d-TPase catalytic domain over residues 67–200. The structure reported here validates this prediction (A and 4A). However, this catalytic domain is referred to as the l,d-CPase domain, because Csd6 is shown to function only as an l,d-CPase and not as an l,d-TPase against the muramyl tetrapeptide. According to the DALI search, the overall fold of the Csd6 l,d-CPase domain (Lys-67–Glu-202) (A) resembles those of well characterized l,d-TPases such as B. subtilis LdtBs (B)  (E. faecium Ldtfm (C)  (M. tuberculosis LdtMt2 (D)  X14–19C (E), where X stands for any amino acid and the strictly conserved residues are in boldface. This motif is conserved among proteins containing an l,d-TPase catalytic domain and provides the catalytic triad residues, which form a hydrogen bond network in a wide range of Cys/Ser-based proteolytic enzymes , corresponding to His-336, Ser-337, and Cys-354 of LdtMt2  from P. aeruginosa  from Streptococcus pneumoniae, Bacillus anthracis, and Bacillus subtilis  family of Zn2+-dependent peptidases  A resemblis LdtBs B (PDB cositions) , E. faecum Ldtfm C (PDB cositions) , and M. s LdtMt2 D (PDB cositions) . Unlike  enzymes , 30, 64.)X14–19C E, where  enzymes . The catruginosa  and Novosubtilis . LdcA isptidases . This ral,d-CPase domain with l,d-TPase domains reveals that the shape of the active site in the Csd6 l,d-CPase domain seems to be tailored for the l,d-carboxypeptidation, i.e. the active site is a deep pocket and the catalytic triad positioned at the bottom of this pocket is accessible via a single narrow path (A), as in typical carboxypeptidases  ; loop II ; loop III ; and loop IV .A structural comparison of Csd6 row path A, as in ptidases . In contwo paths , B–D  and deacylation by the acceptor substrate for cross-linking (“transpeptidation”) , thereby allowing an approach of the acceptor substrate upon acylation of the donor substrate by the catalytic Cys residue for transpeptidation. For a better understanding of the substrate-binding site (A–D), meropenem (mimicking the substrate) was modeled into Csd6 by superimposing its l,d-CPase domain and the l,d-TPase domain of the meropenem-complexed LdtMt2 . In LdtMt2 and Ldtfm, the extended loop II (green colored in C and D) forms a lid to cover the active-site cleft, while exposing two discrete paths for the access of two substrates. However, the loop II of LdtBs is much shorter, and the active-site cleft is not covered with a lid . Like LdtBs, the loop II is much shorter in Csd6, and the active-site cleft is not covered with a lid. Moreover, in the Csd6 structure, the corresponding paths to the active-site cleft for access of two substrates in three l,d-TPases are blocked due to the distinct length and conformations of the loops I–IV, and the active-site cleft opens vertically toward the solvent , allowing access of only one muramyl peptide for carboxypeptidation. This difference in the shape of the active-site cleft is the likely reason for the enzyme serving as a carboxypeptidase, to the exclusion of the transpeptidase activity.The reaction catalyzed by dation”) . As a coing site , A–D, med LdtMt2 . The posops I–IV , B–D. InH. pylori Csd6 than in the l,d-TPases (E). Loop III contains the helix αD, which protrudes from the active site. Together with loop I, it provides one side of the active-site cleft to recognize a substrate. The C. jejuni Pgp2, a Csd6 homolog, is predicted to contain an l,d-TPase domain on the basis of its amino acid sequence (E), but it was shown to function as an l,d-CPase (E). Therefore, it is anticipated that the observed unique structural features of these four loops around the active-site cleft are conserved among Csd6 homologs in other ϵ-proteobacteria.Among the four loops I–IV, the loops I and III show the most distinct variations in sequence length and conformation. They are considerably longer  in d-TPases E. Loop Isequence E, but it,d-CPase . We noti,d-CPase E. TherefMt2, His-160 of Csd6 serves as a proton acceptor to assist a nucleophilic attack by Cys-176. In the present Csd6 structure, the side chains of His-160 and Cys-176 are suitably predisposed for a cooperative action with the main-chain carbonyl of Gly-161 (A). His-160Nϵ2 interacts with Cys-176Sγ with a distance of 3.96 Å and His-160Nδ1 interacts with the main-chain oxygen atom of Gly-161 with a distance of 2.75 Å (A). In addition, the l,d-CPase domain of Csd6 shows a well defined oxyanion hole to facilitate catalysis. The backbone amide nitrogen atoms of Lys-174, Gly-175, and Cys-176  could stabilize the transient oxyanion species of the polarized carbonyl bond, as in LdtMt2 , whereas only one d-Ala molecule is present in monomer B at the corresponding position as d-Ala′ in monomer A. The different binding states of monomers A and B in the crystal seem to be correlated with the oxidation state of Cys-176. Its side chain is oxidized to sulfenic acid only in monomer A, as indicated by the extra electron density . An oxygen atom of sulfenic acid possibly stabilizes the weak binding of the product d-Ala″N in monomer A. d-Ala′ is located between loops I and III, whereas d-Ala″ is trapped by loops II–IV, right in front of the nucleophile Cys-176. d-Ala′ is too far from the predicted site of the scissile bond for Cys-176 to act, but it interacts with Lys-107Nζ, Glu-110Oϵ2, His-155N,O,Nδ1, and Gly-111N, as well as Lys-174N, and the main-chain oxygen atom of Leu-113 via a water molecule. Interestingly, the side-chain portion of mDAP, part of the Csd6 substrate muramyl-l-Ala1-γ-d-Glu2-mDAP3-d-Ala4, is structurally identical to a d-Ala molecule. Therefore, we suggest that d-Ala′ and d-Ala″ represent the portion of the substrate and the end product, d-Ala, respectively, for the link between mDAP3 and d-Ala4 (the scissile peptide bond) of the muramyl tetrapeptide. mDAP was modeled into the Csd6 l,d-CPase domain on the basis of the positions of d-Ala′ and Wat1 bound in the oxyanion hole. The modeled mDAP fits well into the active-site cleft . It makes proper interactions with the oxyanion hole and is suitably positioned for a nucleophilic attack by Cys-176, thus providing the substrate-binding mode. Dissociation constants (DK) of Csd6 for its complex with d-Ala (or mDAP) have also been measured to be 224 (or 94) μm using SPR measurements. The measured association and dissociation rate constants (ak and dk) are 1.05 m−1 s−1 and 2.30 × 10−4 s−1 for d-Ala and 1.49 m−1 s−1 and 1.40 × 10−4 s−1 for mDAP, respectively.To understand the structural determinants for ligand binding, the structure of Csd6 bound with the reaction product l,d-carboxypeptidation activities of the wild-type Csd6 and the mutants against the muramyl tetrapeptide for a limited time (30 min) were compared, mutation of Glu-110, Tyr-133, His-155, and Trp-158, as well as His-160 and Cys-176 that belong to the catalytic triad, resulted in a complete loss of the l,d-carboxypeptidation activity (B). Glu-110, Tyr-133, and Trp-158 are strictly conserved in C. jejuni Pgp2 as well (E). Lack of l,d-carboxypeptidation activity in E110A and H155A mutants supports that Glu-110 and His-155 recognize the side chain of mDAP, as shown in d-Ala′ bound to Csd6 and mDAP modeled into the position of d-Ala′ (A). Trp-158 holds the aliphatic side chain of d-Ala″, whereas Tyr-133 likely interacts with the main chain of mDAP, possibly via a water molecule such as Wat2 in Fig. 5A. Meanwhile, the retained activity of the Y132A mutant indicates that Tyr-132 is not important for the l,d-carboxypeptidation activity of Csd6 . The l,d-carboxypeptidation activity of Csd6 is independent of metal ions, as there is no change upon EDTA treatment .To further confirm the role of potential key residues in the active-site cleft, seven single amino acid mutant proteins  were prepared. When the activity B. Glu-11 as well E. Lack of d-Ala′ A. Trp-15ak) and the dissociation rate constant (dk) for the muramyl tripeptide are roughly 3 and 50× larger than those for the muramyl tetrapeptide, respectively (C). As a consequence, the DK values for the muramyl tetrapeptide and the muramyl tripeptide are 27.0 and 510 μm, respectively, indicating that the muramyl tripeptide dissociates readily from Csd6 as the product of Csd6-catalyzed l,d-CPase reaction. This result is also consistent with the muramyl tripeptide not being used as the substrate for the possible l,d-TPase cross-linking reaction in the subsequent step.To estimate the kinetics and affinity of Csd6 with the muramyl tetrapeptide (as a substrate) and muramyl tripeptide (as a product), SPR experiments were conducted. The association rate constant , following its l,d-CPase domain. The combination of these two domains is conserved in the so-called ErfK/YbiS/YcfS/YnhG family proteins from both Helicobacter and Campylobacter species  , Pseudomonas putida Δ5-3-ketosteroid isomerase , and Rhodococcus sp. naphthalene dioxygenase  (A). None of these enzymes bears any significant sequence or functional homology to each other, except that they share common hydrophobic substrates. This suggests that the fold of NTF2-like proteins may be a suitable scaffold for building a pocket to bind hydrophobic ligands. Although the Csd6 NTF2-like domain differs considerably from these enzymes in the sequence and the potential function, it shares a deep, largely hydrophobic pocket that is open to the solvent (A and C). This potential ligand-binding pocket is lined with numerous hydrophobic residues . In addition, four positively charged residues , and a negatively charged residue (Glu-329) are located within the pocket (D). The bold-face residues are strictly conserved in Csd6 homologs in ϵ-proteobacteria (5-3-ketosteroid isomerase (A and C). The Csd6 NTF2-like domain is highly basic with a calculated pI of 9.4; the electrostatic potential surface shows a highly positively charged surface around its deep pocket (A), compared with the above three NTF2-like superfamily enzymes.The present structure shows that the overall fold of the Csd6 NTF2-like domain differs from other nonviral sialidase or pseudaminidase of the six-bladed β-propeller fold , 71. Insydratase  (PDB codsomerase  (PDB codxygenase  (PDB codsitions) A. None o solvent , A and Ce pocket D. The bobacteria . The pocsomerase , A and Cp pocket A, comparH. pylori Csd6 is involved in the deglycosylation of flagellin (in silico docking study has been performed. It shows that Pse can fit well into the pocket of the NTF-like domain (C). However, it remains to be determined whether Csd6 has pseudaminidase activity.In light of the previous report that lagellin  and the e domain C. HoweveH. pylori. Each of its three domains is associated with a distinct function. The NTD plays a dominant role in homodimerization. The C-terminal NTF2-like domain has a putative binding pocket for Pse. The central l,d-CPase domain has the l,d-TPase fold but the shape of its active-site pocket explains why Csd6 possesses only the l,d-carboxypeptidation activity, despite the presence of the conserved catalytic residues for the potential transpeptidase reaction.We have determined the first crystal structure of Csd6, one of the cell shape-determining proteins in H. pylori Csd6, as well as its close homolog Pgp2 from C. jejuni, contributes to the helical morphology of the cells. Interestingly, H. pylori Csd6 and C. jejuni Pgp2 are unrelated in their sequence and structure to previously characterized l,d-CPases such as LdcA and LdcB from other microorganisms. They are thus presumed to form a third family of l,d-CPases. However, it is currently impossible to assign and distinguish genuine l,d-TPases from the functional l,d-CPases via bioinformatics analyses of amino acid sequences alone. Our finding that Csd6-specific active-site loops I and III are much longer than the corresponding loops in known l,d-TPases (E) enables us to distinguish l,d-CPases possessing the l,d-TPase fold from bona fidel,d-TPases. Indeed, when amino acid sequences of Csd6 homologs in ϵ-proteobacteria are aligned, Csd6-specific loops I and III display high levels of sequence conservation (H. pylori Csd6, as well as its homologs in ϵ-proteobacteria, as belonging to the protein family containing an l,d-TPase catalytic domain needs to be revised. We propose that they should be classified as a third distinct family of l,d-CPase, as initially noted by others (d-TPases E enableservation . In lighy others .H. pylori leads to an increased immune response of the host (H. pylori Csd6 is known to be involved in regulating motility through trimming of the peptidoglycan-derived peptides as an l,d-CPase or through affecting the Pse glycosylation level of flagellin. This study shows that the active-site of the Csd6 l,d-TPase domain is tailored to function as the l,d-CPase, which converts muramyl tetrapeptides into muramyl tripeptides. As Csd6 is nonfunctional as l,d-TPase, muramyl tripeptides in the peptidoglycan cannot be 3→3 cross-linked. This is consistent with the previous reports that the Helicobacter peptidoglycan layer lacks 3→3 cross-links (in silico docking shows that the Csd6 NTF-like domain presents a potential binding site for Pse, supporting its role in the control of the glycosylation level of flagellin. Relaxation of peptidoglycan 4→3 cross-linking was reported to promote H. pylori's helical shape and stomach colonization (H. pylori, because it can limit the supply of substrates for peptidoglycan 4→3 cross-linking. In conclusion, our study on the multidomain, multifunctional Csd6 provides further insights into the strategy of H. pylori for regulating its helical cell shape and motility, which are crucial for its virulence.Enhanced cell attachment of the host . Too mucss-links , 20, 33.H. S. K., H. N. I., S. M., S. C., N. K. L., S. J. K., J. Y. K., B. W. H., B. I. L., and S. W. S. designed the study and wrote the paper. H. N. I., D. R. A., J. Y. Y., J. Y. J., and H. J. Y. purified and crystallized Csd6 protein and determined its x-ray structure. S. M., D. H., and M. L. synthesized the muramyl peptides. J. Y., M. C., and S. C. conducted docking calculations. C. K. and N. K. L. performed the ALEX-FRET experiment. S. J. K. performed the equilibrium sedimentation experiment. J. Y. K. and G. B. performed mass spectrometric assays of enzyme activities. All authors analyzed the results and approved the final version of the manuscript.