Bacillus subtilis strains that could produce new truncated cyclic or linear peptides of the predicted sequence, which further provided an important insight into the regioselectivity of plipastatin TE. The TE was capable of recognizing and catalysing the lactone formation between L-Try3 with the last few residues L-Pro7 and L-Gln8 at the C-terminus. Additionally, the unmatched linkers connecting the TE region and T domain resulted in nonproduction strains, suggesting that the native T–TE linker is necessary and sufficient for the TE domain to release the products from the hybrid enzymes. This is the first report to demonstrate truncated cyclic lipopeptides production and module skipping by simply moving the TE domain forward in an NRPS system.Non-ribosomal peptide synthetases (NRPSs) are large enzymatic complexes that catalyse the synthesis of biologically active peptides in microorganisms. Genetic engineering has recently been applied to reprogram NRPSs to produce lipopeptides with a new sequence. The carboxyl-terminal thioesterase (TE) domains from NRPSs catalyse cleavage products by hydrolysis or complex macrocyclization. In this study, we modified plipastatin synthetase by moving the intrinsic TE region to the end of the internal thiolation (T) domains, thus generating  The sequence of the peptide product directly corresponds to the linear arrangement of modules and domains within the biosynthetic template. This structure enables the development of straightforward strategies for the rational design of NRPSs via the recombination of gene fragments to generate arrays of peptide derivatives and thus expand the diversity of microbial-produced lipopeptides.Plipastatin, an antifungal lipopeptide antibiotic, was originally isolated from e operon . Accordiin vitro characterization of various TE domains has been reported based on purified proteins151617in vivo studies on the TE domain of plipastatin synthetase using molecular engineering strategies are scarce. Valuable biologically active compounds synthesized by an NRPS system in hosts could be better suited for industrial production. Here, we moved the TE domain of plipastatin synthetase forward, and evaluated the effect of this change on the production of truncated active analogues in vivo. We further investigated the structure of the novel peptides generated to analyse the catalytic activity and selectivity of the TE domain in the new hybrid enzymes.In recent years, engineering of deletions, insertions, and module exchange in NRPS systems has proven to be a useful strategy to obtain lipopeptides of a targeted sequence39101113Bacillus subtilis was grown in Luria-Bertani (LB) medium at 37 °C. Landy fermentation medium buffered with 0.1 M 3-(N-morpholino) propanesulfonic acid (MOPS) was used as described previouslyEscherichia coli JM110 into Bacillus subtilis pB2-L by natural competence, as previously describedBacillus strain bearing an integrative plasmid that contained a gene replacement construct was grown in LB medium at 37 °C (a non-permissive temperature for plasmid replication). The entire plasmid was inserted into the chromosome via a single crossover between the target gene and a homologous sequence on the plasmid. In the second step, a separate clone of the integrant was cultured in LB medium at 30 °C for 48 h to induce the second exchange event and excise the plasmid. Kanamycin-sensitive (KanS) clones with either the parental or mutant sequence were obtained and verified by PCR analysis and sequencing. The kanamycin resistance gene was not amplified, but the upstream and downstream regions of the homologous sequence could be amplified, and then sequencing confirmed the mutant sequence.The bacterial strains and plasmids used in this study are listed in SalI and KpnI of pKS2.The integrative plasmids that were used to move the TE domain forward to the end of the T domain were all derivatives of pKS2Long, pKS-8GlnTELong, and pKS-9TyrTELong were constructed as follows. Gene fragments encoding the seventh T domain, eighth T domain, ninth T domain, and TELong region (harbouring the native T-TE linker) of plipastatin synthetase were amplified by PCR from chromosomal DNA of strain pB2-L using pfu DNA polymerase  with the primers 7ProT-F/R, 8GlnT-F/R, 9TyrT-F/R, and TELong-F/R, respectively. The fused fragments 7ProTTELong, 8GlnTTELong, and 9TyrTTELong were constructed by SOE-PCR using the primers 7ProT-F/P7-R and P7-F/TELong-R, 8GlnT-F/P8-R and P8-F/TELong-R, and 9TyrT-F/P9-R and P9-F/TELong-R, respectively, which were then inserted into the restriction endonuclease sites SalI and KpnI of pKS2. All inserted fragments were confirmed by sequencing .The plasmids pKS-7ProTEg for 15 min at 4 °C to remove the bacterial cells. The supernatant was adjusted to pH 2.0 with 6 N HCl to precipitate plipastatin and plipastatin-derived peptides. The pellet was then collected and extracted with methanol. The crude extract was filtered through a 0.22-μm filter and analysed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) using an LTQ Orbitrap XL hybrid mass spectrometer . Five microliters of methanolic extract was loaded onto a C18 reverse-phase column . The separation of lipopeptides was achieved using acetonitrile/H2O/0.2% formic acid as the mobile phase. The flow rate was maintained at 0.2 mL/min with a gradient of 22 min of 5% vol/vol acetonitrile for 2 min, 5–95% vol/vol acetonitrile for 13 min, 95% vol/vol acetonitrile for 3 min, and 95–5% vol/vol acetonitrile for 3 min. The elution was monitored by a total ion chromatogram at 205 nm. MS spectra were recorded in positive ion mode within a mass range of 300–1500 m/z. The molecular ions were further characterized by LTQ Orbitrap tandem mass spectrometry (MS/MS) using the following parameters: ion spray voltage at 2.5 kV, sheath gas at 20 units, capillary temperature at 300 °C, capillary voltage at 41 V, and tube lens at 110 V. Xcalibur software was used for visualization of the high-resolution spectral profile data .The recombinant strains were fermented in triplicate for 3 days at 33 °C, 180 rpm. Cultures were centrifuged at 5000× et al.In previous experiments, we found that deletion of the TE domain of plipastatin synthetase results in the synthesis of truncated enzymes that were unable to produce the corresponding peptides (data not shown). This result suggested that the TE domain is necessary for plipastatin production, which is consistent with a study on the surfactin TE domain reported by de Ferra B. subtilis pB2-L, and then integrations of the TE domain were generated using a two-step replacement procedure, as described previouslyIn this experiment, the TE domain  was respectively integrated into the end of the seventh, eighth, and ninth T domains in plipastatin NRPSs . In descLong  domain, containing a linker region between the 10IleT and TE domains in subunit PPSE, was fused to the end of the T domain that originated from 7Pro-, 8Gln-, and 9Tyr-activating modules, respectively. In the hybrid peptide synthetases, the TE domain was connected with the native T-TE linker, as shown in + peaks obtained from the different plipastatin derivatives. Comparison of these data with the molecular weight of plipastatin clearly indicated the fused position of the TELong domain, as deduced from the expected sequences of these truncated lipopeptides. Linear heptapeptides, octapeptides, and nonapeptides were respectively detected in strains LP4, LP5, and LP6 as expected, and the linear structure was further confirmed by MS/MS analysis . Similarly, To validate our speculation, we maintained the native 10IleT-TE linker in the hybrid synthetases. The TE9.66 m/z , the obs+ 1087.6, 1101.6, 1115.7, 1129.7 m/z) in the crude extracts of the recombinant strain LP4, with a mass difference of 18 Da from the characterized linear heptapeptides  and –y6 (704.36), corresponding to the macrocyclic moiety and the macrocycle along with the Orn residue, respectively. Similarly, cyclic octapeptides  were also observed in the crude extracts of the recombinant strains LP5 and LP6, which were further characterized by MS/MS -to ACP transferPlipastatin TEB. subtilis pB2-L was only 10 mg/L, which may be result in the production level of derivatives in these experiments were very low. Similar yield reduction has been reported in many engineering NRPS. For example, the production of novel antibiotics related to daptomycin generally ranged from about 1% to 50% of control, which were generated by module exchanges, NRPS subunit exchanges, inactivation of the tailoring enzyme and natural variations of the lipid tail3101123The yield of original plipastatin produced by In summary, our approach enables the construction of truncated hybrid enzymes to synthesize peptides of desired length and amino acid composition, and provides important insights into the regioselectivity of plipastatin TE, which could be used to rationally manipulate the ring size of macrocyclic product. We believe that the plipastatin TE domain has great potential in the engineering of peptide synthetases for generating many new analogues of active peptides, especially non-linear and modified peptides that are difficult and costly to produce via chemical synthesis.How to cite this article: Gao, L. et al. Translocation of the thioesterase domain for the redesign of plipastatin synthetase. Sci. Rep.6, 38467; doi: 10.1038/srep38467 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Pyruvate carboxylase (PC) catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate. The reaction occurs in two separate catalytic domains, coupled by the long-range translocation of a biotinylated carrier domain (BCCP). Here, we use a series of hybrid PC enzymes to examine multiple BCCP translocation pathways in PC. These studies reveal that the BCCP domain of PC adopts a wide range of translocation pathways during catalysis. Furthermore, the allosteric activator, acetyl CoA, promotes one specific intermolecular carrier domain translocation pathway. These results provide a basis for the ordered thermodynamic state and the enhanced carboxyl group transfer efficiency in the presence of acetyl CoA, and reveal that the allosteric effector regulates enzyme activity by altering carrier domain movement. Given the similarities with enzymes involved in the modular synthesis of natural products, the allosteric regulation of carrier domain movements in PC is likely to be broadly applicable to multiple important enzyme systems. Carrier domain enzymes accomplish catalysis by physically transporting intermediates long distances between remote active sites. Here the authors describe a wide range of catalytically productive translocation events during catalysis by pyruvate carboxylase and suggest a basis for its allosteric activation. The two half-reactions are coupled by a ≥60 Å translocation of the BCCP domain between the BC and CT domain active sites, facilitating the net carboxyl group transfer from bicarbonate to pyruvate.Pyruvate carboxylase  is a biotin-dependent enzyme present in a wide range of species, ranging from bacteria to mammals. In most species, PC is a multifunctional, homotetrameric enzyme with each subunit comprised of four domains: the biotin carboxylase (BC) domain, the carboxyltransferase (CT) domain, the biotin carboxyl carrier protein (BCCP) domain, and the allosteric domain  domain)  or an asymmetric tetramer (Rhizobium etli PC4), irrespective of whether acetyl CoA is present. Cryo-EM studies in SaPC more recently revealed that PC oscillates between a symmetrical and asymmetrical state during the catalytic cycle15. In SaPC, acetyl CoA was found to stabilize and promote BC domain dimerization through a subtle reorganization of the BC and allosteric (PT) domain dimers5. However, despite multiple structures, there is no evidence to support a significant remodeling of the domain organization or active site architecture in PC in response to acetyl CoA binding. Consequently, acetyl CoA is more likely to stimulate catalysis by altering dynamic processes, such as BCCP domain translocation.X-ray crystallographic studies of PC cloned from multiple source organisms have provided structural snapshots of the enzyme both in the presence and absence of acetyl CoA. PC structures reveal either a symmetric (Aspergillus nidulans PC (AnPC) and R. etli PC (RePC), two enzymes that respond very differently to acetyl CoA activation. We report that carrier domain translocation is regulated by acetyl CoA in RePC, an acetyl CoA sensitive PC enzyme. However, BCCP domain translocation is unaffected by the presence of the allosteric inhibitor, l-aspartate. These findings alter the classical description of catalysis in PC and suggest that carrier domain motions in swinging domain enzymes may be far more complex than previously imagined.Here, we use a series of mutated, hybrid PC enzymes to perform a detailed analysis of multiple translocation pathways for the BCCP domain during catalytic turnover in PC. We demonstrate that, surprisingly, the BCCP domain is capable of a much broader range of catalytically productive movements than previously recognized. We also describe the effect of allosteric effectors on BCCP domain translocation during catalysis by both 16. The translocation of the BCCP domain has been assumed to occur exclusively in trans, traveling from the BC domain on its own subunit to the CT domain on a neighboring subunit , is dimeric in solution and that it catalyzes the biotin-dependent, oxamate-induced decarboxylation of oxaloacetate nearly three times faster than wild-type RePC17. We attempted to generate pure heterodimers of ΔBC T882A and ΔBCΔBCCP RePC by co-expressing these with separate N-terminal affinity tags -binding peptide) and sequentially purifying them using Ni2+- and CaM-affinity resins. Unfortunately, the purified heterodimers quickly redistributed back into a mixed population of homo- and heterodimers (data not shown). To circumvent this problem, a population of hybrid dimers was generated by mixing together ΔBC T882A RePC with ΔBCΔBCCP RePC at a 1:1 ratio. Affinity co-purification was used to demonstrate that these dimers recombined into a fully heterogeneous population , while a second construct was C-terminally truncated to eliminate the BCCP domain entirely (ΔBCΔBCCP RePC). Both of these mutations have previously been shown to result in a complete loss of activity in the oxamate-induced oxaloacetate decarboxylation reactioncis, accessing the CT domain through an intramolecular interaction, we created a series of mixed heterodimer populations by combining a fully inactive ΔBCΔBCCP T882A RePC with a fully active ΔBC RePC. As above, the redistribution of purified heterodimers into a mixed population precluded an analysis of purified heterodimers. Instead, inactive ΔBCΔBCCP T882A RePC was mixed at increasing ratios with active ΔBC RePC as outlined in Fig. cis, or inactive if the BCCP domain is unable to act in cis. The predicted curves for a system that acts 100 and 0% in cis are plotted in Fig. cis, with the BCCP domain accessing the CT domain through an intramolecular interaction. Together, these experiments demonstrate that, in the context of the ΔBC RePC dimer, the BCCP domain exhibits a much wider range of catalytically productive conformations than previously envisioned.To determine whether the BCCP domain on the ΔBC RePC dimer can act in R. etli PC; RePC) that is sensitive to acetyl CoA activation and a fungal PC enzyme  that is insensitive to acetyl CoA activation.The kinetic studies of heterodimeric ΔBC RePC confirmed that the BCCP domain can adopt a wide range of catalytically productive motions. The subunits within the PC tetramer, however, exist in a more sterically constrained environment, necessitating a detailed evaluation of BCCP translocation in the context of the tetrameric enzyme. Furthermore, to evaluate the general applicability of these findings and to determine whether allosteric activation influences BCCP translocation, this phenomenon was studied in two different PC enzymes: a bacterial PC enzyme  were co-expressed using a pETDuet-1 vector in Escherichia coli BL21(DE3). In this system, wild-type RePC can only be co-purified if it recombines with (His)9-tagged RePC ×BC×CT×BCCP to form hybrid tetramers. The degree of recombination into hybrid tetramers was assessed using an avidin gel shift assay. Successful recombination was expected to result in a purified population containing both untagged wild-type RePC (biotinylated) and (His)9-tagged ×BC×CT×BCCP (unbiotinylated). A mobility shift in the presence of avidin confirmed the co-purification of wild type, untagged, biotinylated RePC, and demonstrated that hybrid tetramers were successfully generated by co-expression ion Fig. . Using tion Fig. .Fig. 5Hy9-tagged AnPC ×BC×CT×BCCP. For reasons that are not clear, co-expression of mutated AnPC genes in the pETDuet-1 vector, in some cases, resulted in very poor expression levels for one of the two genes. This issue was resolved by converting to a co-expression method that involved co-transforming with two compatible plasmids, with each plasmid carrying an individually mutated PC gene. This approach successfully generated well-combined hybrid tetramers of AnPC nPC Fig. .The steady-state catalytic activity of hybrid tetramers was measured with saturating concentration of substrates, in order to determine which translocation pathways are functional during catalysis. The system is designed such that, while all translocation pathways are available to any particular hybrid enzyme, only one pathway is catalytically productive. For example, the carrier domain of hybrid A may proceed through all four translocation pathways represented in Fig. For both RePC and AnPC, the activities recovered in the hybrid tetramers were much higher than the activities of the individual PC mutants Tables  and 3. T1 and M2 18. The present study confirms and validates the catalytic relevance of this structural snapshot and extends the implications to a broad cross-section of PC enzymes.This study sought to unambiguously assess the productivity of three intermolecular BCCP translocation pathways in catalytically distinct PC enzymes from two different species. Contrary to the accepted dogma, which states that the BCCP domain transits exclusively between a single combination of BC and CT domains, the present study reveals a much more fluid and flexible carrier domain that travels in catalytically productive pathways between multiple combinations of active site pairs. Considering that RePC and AnPC are evolutionarily and functionally divergent, and that the kinetic and thermodynamic features of the vast majority of PC enzymes are highly conserved, we believe that these findings are broadly applicable to all PC enzymes. Notably, a recently reported structure of PC from The three hybrid tetramers assessed in this study were designed to examine three possible intermolecular translocation pathways Fig. , indicat13. Several studies have focused on specific mechanistic features in both the BC and CT domains that contribute to the enzyme’s high-coupling efficiency19. These studies point to specific gatekeeping features in the individual active sites that reduce the potential for abortive ATP cleavage and that ensure a high probability of carboxyl-group transfer from carboxybiotin to pyruvate in the CT domain. Interestingly, in the absence of acetyl CoA, the efficiency with which MgATP cleavage at the BC domain is coupled with oxaloacetate formation at the CT domain is severely reduced, even at saturating concentrations of all substrates12. It is known that acetyl CoA accelerates MgATP cleavage in the BC domain10, but the enhanced-coupling efficiency in the presence of acetyl CoA indicates that there are additional factors, beyond substrate-mediated control of the individual active sites, which contribute to efficient intermediate channeling in PC. Since intermediate transfer via carrier domain translocation is essential to link the catalytic activities of the individual half-reactions, we reasoned that acetyl CoA might enhance the coupling efficiency by mediating BCCP translocation in PC. The intramolecular translocation of BCCP in the dimeric enzyme form of RePC (ΔBC RePC) was not sensitive to acetyl CoA in the oxamate-induced oxaloacetate decarboxylation reaction .Intermediate transfer via carrier domain translocation is essential to ensure a high-coupling efficiency between the individual half-reactions in PCion Fig. . However20 and S. aureus PC13, indicating that acetyl CoA facilitates a more ordered thermodynamic state during catalytic turnover. This reduction in activation entropy was originally attributed to more compact quaternary structures that were observed in low-resolution cryo-EM images in the presence of acetyl CoA21. However, several more recent high-resolution X-ray crystal and cryo-EM structures in the presence and absence of acetyl CoA have not provided any corroborating evidence to support a more compact PC quaternary structure in the presence of acetyl CoA5. Furthermore, the acetyl CoA insensitive AnPC was also shown in cryo-EM studies to adopt a more compact structure in the presence of acetyl CoA22. This leads us to believe that the compact structure observed in the presence of acetyl CoA is more likely to be a general structural feature of all PC enzymes and that it is not related to the observed reduction in the activation entropy. Rather, the decreased activation entropy in the presence of acetyl CoA is more easily rationalized by the constrained freedom of the BCCP domain during catalytic turnover. Taken together, we propose that, for acetyl CoA sensitive PC enzymes like RePC, the BCCP domain is flexible in the absence of acetyl CoA and is able to adopt at least four separate catalytically productive translocation pathways. In the presence of acetyl CoA, a single translocation pathway 24, non-ribosomal peptide synthetases (NRPS)25, pyruvate dehydrogenase26, and the broader family of biotin-dependent enzymes27. In all cases, the transfer of reaction intermediates between spatially distinct active sites requires long-range movements of the carrier domain during catalytic turnover. In NRPS, for example, the peptidyl carrier protein domain rotates ~75° and translocates ~60 Å as it transports intermediates between active sites25. Such long-range motions between multiple active sites require the carrier domain to combine remarkable flexibility with exquisite selectivity in order to appropriately present the proper intermediates to the correct active sites. For type I modular PKS and NRPS, the carrier domain transfers reaction intermediates among multiple modules and mediates specificity through protein-protein interactions between the carrier domain and the individual catalytic domains29. Iterative enzyme complexes, such as yeast type I FAS, use separate reaction chambers to constrain and control intermediate transfer and avoid off-pathway reactions with improperly positioned intermediates30. Consistent with the model established for FAS, it has long been envisioned that PC confines the carrier domain to a single reaction chamber between one set of active site pairs. Unlike most assembly line enzymes, however, PC is not at risk for off-pathway reactions when the carrier domain travels between alternate combinations of active site pairs. Instead, PC presents a new example for catalytic control in a swinging domain enzyme, through regulated movement of the carrier domain in the presence of the allosteric activator, acetyl CoA. Instead of constraining carrier movements as a means to control specificity, altered carrier domain movements in PC contribute to enhanced catalytic efficiency. These studies reveal a new mechanism for carrier domain movement and allosteric regulation in swinging domain enzymes.Carrier proteins and carrier domains are commonly used to shuttle covalently bound intermediates between remote active sites in large, multienzyme complexes, such as fatty acid synthases (FAS)19 were re-cloned into compatible expression vectors. To ensure vector compatibility, the pET-28a vector encoding ΔBCΔBCCP RePC was modified to replace the pBR322 origin of replication with a pSC101 origin of replication. The gene encoding ΔBCΔBCCP RePC was re-cloned into vector pKLD66nCBP31. The T882A mutation was introduced into both ΔBC RePC and ΔBCΔBCCP RePC by whole plasmid mutagenesis using the Quikchange method. The N-terminally poly(His)-tagged ΔBCΔBCCP RePC was expressed in E. coli BL21Star(DE3) cells from a modified pET-28a-(His)8-TEV vector4. ΔBC RePC with an N-terminal CaM binding domain was expressed from vector pKLD66nCBP and co-expressed with BirA encoded by the pCY216 vector (4). Proteins were produced using a 20 L batch culture in M9 minimal medium . After reaching an optical density (600 nm) of 1.0, the culture was induced for 24 h at 16 °C with 1 mM isopropyl 1-thio-β-D-galacto-pyranoside (IPTG) and 20 mM l-arabinose4. The cell paste from the equivalent of 6 L of M9 minimal media was resuspended in 150 mL of lysis buffer  butane (E-64); 1 μM pepstatin A; 1 mM phenylmethylsulfonyl fluoride; and 200 μg/mL lysozyme). The cell suspension was lysed by sonication and pelleted by centrifugation. The supernatant was loaded on a 10 mL column of CaM resin at a flow rate of 1.5 mL per minute. The column was subsequently washed with 300 mL of lysis buffer at a flow rate of 1.5 mL per minute. Following the wash, the protein was eluted with 50 mL of elution buffer -N,N,N′,N′-tetraacetic acid (EGTA); 200 mM NaCl; 6 mM β-mercaptoethanol) at a flow rate of 1.5 mL per minute. Fractions containing the protein were pooled and dialyzed  for 13 h. The protein was concentrated to 7.3 mg/mL (ΔBC RePC) and 1.5 mg/mL (ΔBC T882A RePC) as determined by the protein’s predicted extinction coefficient and absorbance at 280 nm. The concentrated protein was drop frozen in liquid nitrogen and stored at −80 °C.The original ΔBC RePC and ΔBCΔBCCP RePC constructs in pET-28a9 vector were site-specifically mutated by whole plasmid mutagenesis using the Quikchange method. The specific mutations used to inactivate individual domains of both RePC and AnPC are provided in Supplementary Table The RePC and AnPC wild-type genes in a pET-17b-(His)9-tagged RePC domain inactive mutants were inserted into the pETDuet-1 vector in tandem by PCR-based amplification from pET-17b followed by ligation-based cloning into the pETDuet-1 vector. In all cases, one of the domain-inactivated RePC genes was inserted into the first multiple cloning site (MCS) using the XbaI/NotI restriction endonuclease sites; the second gene was inserted into the second MCS using the NdeI/PacI restriction endonuclease sites 9-tag at the protein N-terminus, while those genes encoded on pRSFDuet-1 did not include an affinity tag.The co-expression of pairs of AnPC mutants for the generation of hybrid tetramers was performed by the co-transformation of two compatible plasmids, pSFDuet-1 and pET-17b, with one member of the pair encoded on pSFDuet-1, while the other member of the pair was encoded on pET-17b (Supplementary Table E. coli biotin protein ligase A (BirA) on vector pCY216 (ref. 32). Transformed E. coli BL21(DE3) cells were cultured in M9 minimal media at 37 °C to an optical density (600 nm) of 0.8 −0.9, whereupon cells were induced by adding IPTG and l-arabinose to a final concentration of 1 and 25 mM, respectively. In addition, the culture was supplemented with d-(+)-biotin and MnCl2 to a final concentration of 3 mg/L and 0.1 mM, respectively. Induced cells were incubated with shaking at 16 °C for 20 h before harvesting by centrifugation.All full-length PC genes were expressed and purified in the same manner. In order to ensure complete biotinylation, all PC clones were co-transformed and co-expressed with 2+-affinity and anion exchange chromatography. The cell paste from 12 L of cell culture was resuspended in 200 mL Ni-lysis buffer . Cells were disrupted by sonication at a temperature not exceeding 10 °C and pelleted by centrifugation. The supernatant was loaded on a 10 mL Ni2+-nitrilotriacetic acid profinity resin column . The column was washed with 20× column volume of wash buffer buffer  and the protein was eluted with a gradient from 20 to 250 mM imidazole using wash buffer and elution buffer . Purified protein was pooled and dialyzed against a buffer compatible for anion exchange chromatography ) at 4 °C overnight. The dialyzed protein was loaded on a 10 mL Q-Sepharose Fast Flow resin column  and was eluted from the column in buffer  with a gradient from 50 mM to 1 M NaCl. The elution peak of PC full-length protein was typically between 400 and 800 mM NaCl. The purified protein was pooled and dialyzed against storage buffer  glycerol, and 2 mM DTT) for >4 h with at least two successive changes. The protein was concentrated to a final concentration of 6–10 mg/mL and was drop frozen in liquid nitrogen prior to storage at −80 °C freezer. All protein concentrations were determined by the predicted extinction coefficient and absorbance at 280 nm33.All PC full-length enzymes were purified using sequential NiAvidin gel shift assays were performed to measure the degree of PC biotinylation. Avidin is a homotetramer of molecular weight ~60 kDa with four very high-affinity biotin binding sites per avidin tetramer. Chicken egg white avidin  and denatured PC protein (1 mg/mL) were prepared in buffer  and mixed in equal volumes to a final molar ratio of 10:1, avidin:PC. The mixture was incubated at room temperature (25 °C) for 40 min. Biotinylated PC binds with avidin irreversibly, retarding its migration on SDS-PAGE. In contrast, unbiotinylated PC does not bind with the avidin and migrates on SDS-PAGE at a molecular weight of a single subunit, ~125 kDa.2, and 0.1 M KCl. All substrates and coupling reagents were mixed in a 10× stock solution that containing components at the following concentrations: 250 mM NaHCO3, 25 mM MgATP, 120 mM pyruvate, 2.4 mM NADH, and 100 units/mL malate dehydrogenase. For titration of acetyl CoA and l-aspartate, the final concentration of acetyl CoA ranged from 0 to 0.5 mM and l-aspartate ranged from 0 to 300 mM. All reactions were performed in 96-well plate format at 25 °C in a total working volume of 200 μL. The final PC concentration in the assay ranged between 5 and 50 μg/mL (0.04–0.4 μM) per reaction. Reactions were initiated by addition of 20 μL of the 10× substrate stock solution.Pyruvate carboxylation activity was monitored spectrophotometrically at 340 nm by following the conversion of oxaloacetate to malate using the coupled enzyme malate dehydrogenase (MDH). Reactions were performed in 0.1 M Tris-HCl (pH 7.8), 7 mM MgCl16. Unless otherwise noted, reactions were performed in 0.1 M Tris-HCl (pH 7.8), 0.24 mM NADH, lactate dehydrogenase (10 U), 0.2 mM oxaloacetate, 0.5 mM oxamate, and 0.25 mM acetyl CoA. All reactions were initiated by the addition of oxaloacetate and performed in 1 mL quartz cuvettes. The final PC concentration ranged from 50 to 200 μg/mL (0.4–1.6 μM). All kcat values were calculated by dividing the experimentally determined Vmax values by the total protein concentration, defined as the subunit molar concentration . Protein concentrations were determined spectrophotometrically by measuring the absorbance at 280 nm for wild-type and hybrid enzymes. The extinction coefficients for the fully reduced enzyme were calculated from the primary sequence.The PC-catalyzed rate of oxaloacetate decarboxylation was determined by measuring the reduction of pyruvate to lactate using lactate dehydrogenaseTo assess the activity of the ΔBC T882A/ΔBCΔBCCP RePC hybrid dimer, both ΔBC T882A and ΔBCΔBCCP RePC were diluted to 1 mg/mL in 20 mM Tris-HCl, pH 7.8; 200 mM NaCl and 6 mM β-mercaptoethanol. The two enzymes were mixed together at a 1:1 ratio. The ΔBC T882A, ΔBCΔBCCP, and ΔBC T882A/ΔBCΔBCCP RePC mixture, all at a final concentration of 1 mg/mL, were incubated for 2 h at room temperature before assessing their oxaloacetate decarboxylation activity, as described above, in both the presence and absence of 0.5 mM oxamate. All reactions were performed in 1 mL reaction volumes at 25 °C in 100 mM Tris-HCl, pH 7.8. The reaction was initiated by the addition of 150 μg of ΔBC T882A, ΔBCΔBCCP or the ΔBC T882A/ΔBCΔBCCP mixture.To generate mixed combinations of the wild-type ΔBC RePC with the inactive CT dimer ΔBCΔBCCP T882A RePC, the procedure for remixing was as follows: ΔBC RePC and ΔBCΔBCCP T882A were diluted to 3 mg/mL in 100 mM Tris (pH 7.8) before being combined together at a given ratio to a final protein concentration of 1.0 mg/mL. The mixtures were incubated for 2 h at room temperature before assessing their oxaloacetate decarboxylation activity, as described above, in the presence of 0.5 mM oxamate. All reactions were performed in 1 mL reaction volumes at 25 °C in 100 mM Tris-HCl, pH 7.8. The reaction was initiated by the addition of 150 μg of ΔBC, ΔBCΔBCCP T882A or the ΔBC/ΔBCΔBCCP T882A mixture.cis translocation in the BCCP domain is shown in Eq. (cis translocation in the BCCP domain is shown in Eq. (n represents the fraction to which the data by a 100% cis translocation.The relative recovered activity measured from the mixture comparing to RePC ΔBC dimer were plotted against the ratio 1:x) for ΔBC:ΔBCΔBCCP T882A. The equation describing the theoretical curve for a 100% :x for ΔBn in Eq. , where nl-aspartate/acetyl CoA concentrations, in order to determine the Ki for l-aspartate and the Ka for acetyl CoA. The Ka value for acetyl CoA was determined from a plot of the initial velocity  against acetyl CoA concentration and by fitting the data to Eq.  for acetyl CoA; and h is the Hill coefficient with respect to acetyl CoA.The activity of pyruvate carboxylation was measured over a range of a to Eq.  using leKi for l-aspartate was determined from a plot of the initial velocity  against l-aspartate concentration and fitting the data to Eq. (l-aspartate concentration, kcat0 is the kcat value in the absence of l-aspartate; kcatasp is the kcat value at saturating concentrations of l-aspartate; Ki represents the inhibition constant for l-aspartate; and h is the Hill coefficient with respect to l-aspartate.Similarly, the a to Eq.  using leThe data that support the findings of this study are available from the corresponding author upon reasonable request.Supplementary InformationPeer Review File


Leptospira infection. The ligands for TLR2 harbor several conserved patterns such as lipidation molecules, leucine-rich repeat (LRR) domains, TLR2 binding motifs, and TLR2 binding structure. In Leptospira, LipL32 interacts with TLR2 on human kidney cells concomitantly stimulating inflammatory responses. However, the binding mechanism of LipL32 to TLR2 is unknown. The computational prediction suggests that β1β2, loop-α3-loop, and α4 domains of LipL32 play vital roles in LipL32-TLR2 complex formation. To test these predictions, protein truncation experiments revealed that LipL32ΔNβ1β2 significantly decreased the affinity to TLR2 while LipL32ΔCα4 slightly reduced it. Interestingly, LipL32ΔCenα3 retained affinity to TLR2 in the absence of Ca2+ ions, indicating that Cenα3 play a role preventing the interaction between LipL32 and TLR2. Furthermore, the critical residues of LipL32 involved in interacting with TLR2 suggested that V35S, L36S and L263S variants significantly decreased the affinity to TLR2. The results further confirm that LipL32 interacts with TLR2 through Nβ1β2 and Cα4 domains of LipL32 as well as LipL32-TLR2 complex formation results from hydrophobic interactions. This study provides a detailed mechanism of the interaction between LipL32 and TLR2 and the residues involved in complex formation.Proteins belonging to the toll-like receptor (TLR) family, particularly TLR2, are the major components of innate immunity against  Humans become infected through contact with Leptospira containing water, food, soil or urine from infected animals through exposed mucosal surfaces or broken skin6. Clinical manifestations of Leptospirosis range from mild to severe or even potentially lethal as characterized by high fever, intense jaundice, bleeding, renal and pulmonary dysfunction, neurologic alterations, and cardiovascular collapse7. Kidneys are one of the main target organs of Leptospira, especially the proximal tubule epithelial cells (PTECs), resulting in tubulointerstitial nephritis and acute renal failure8.Leptospirosis is an overlooked risk in acute and chronic kidney diseases, which contributes to high mortality and morbidity worldwide, particularly in warm and humid areasLeptospira have evolved to complete the infectious cycle, which includes entering the host, evading the immune responses, adhering and colonizing to tissues, and finally exiting the host to initiate a new infection, while maintaining the capacity to survive in the environment9. At the adhesion stage, outer membrane components mediate the major interactions between the pathogen and the host cell. The important outer membrane components from Leptospira potentially involved in virulence have been recognized including major outer membrane lipoprotein 32 (LipL32)10, lipopolysaccharides (LPS)11, Leptospira immunoglobulin-like proteins (Lig)12, Leptospira endostatin-like proteins (Len)14, and Leptospira OmpA-like lipoprotein (Loa22)15. Notably, the most abundant outer membrane component found in pathogenic Leptospira is LipL32, a lipoprotein with a lipid modification at its Cys20 residue and a signal peptide tag at its N-terminus18. The crystal structure of LipL32 reveals a jellyroll-fold structure and a calcium ion was shown to be important for structural and thermal stability21. In addition, LipL32 has been validated by the affinity to different components of extracellular matrix (ECM), including collagen I, collagen IV, collagen V, laminin, and plasma fibronectin while the C terminal and intermediate domain of LipL32 are responsible for the interaction to ECMs22. In addition, purified LipL32 protein increased the permeability and decrease the expression of zonula occludens-1 (ZO-1) and F-actin in human umbilical vein endothelial cells (HUVEC)23. Recently, post-translational modification (PTM) of LipL32 was observed to include either acetylation or tri-methylation of lysine residues within multiple peptides, and PTM was further demonstrated to be related to immune-reactivity24. These evidences demonstrate that LipL32 stimulates inflammatory responses, however, the exact mechanism is still unclear. Our previous studies demonstrated that LipL32 induces inflammatory signals through the innate immune component, Toll-like receptor 2 (TLR2)26. Among these inflammatory signals, chemotactic chemokines play vital roles in the recruitment of macrophage or neutrophil to the site of inflammation and the level of these chemotactic factors are up-regulated in a variety of glomerular and interstitial renal injuries27. IL-8 is a chemokine contains ELR (glutamic acid-leucine-arginine) motif with ability to induce neutrophil activation and MCP-1 is monocyte chemoattractant protein-1 that is secreted by several cell types including renal cells28. Tumor necrosis factors alpha (TNF-α) and matrix metalloproteinase-7 (MMP-7) have been demonstrated to up-regulate when treatment of LipL32 to renal cells25. In additions, other macrophage chemotatic factors including CXCL1/GRO-α and Osteopontin (SPP1) are also selected to measure the renal interstitial inflammation30.Pathogens have developed a variety of mechanisms to survive in the host environment and pathogenic species of 31. Association of TLR2 with TLR1 or TLR6 is essential for sensing bacterial lipoproteins and lipopeptides33. TLRs containing leucine-rich repeats (LRRs) are responsible for pattern recognition of the extracellular portion and a Toll/IL-1 receptor (TIR) domain is responsible for signal transduction through the cytoplasm34. The crystal structure of TLR2/1 complex with its ligand, Pam3CSK4, revealed that two fatty esters of the glycerol moiety are embedded into a hydrophobic pocket of TLR2, while the amide-bound lipid chain fits into the hydrophobic channel of TLR135. The lipid molecule of lipoprotein presumably supports the binding domain of TLR2. Recently, the complex structure of TLR2 in association with staphylococcal superantigen-like protein 3 (SSL3) revealed that the interaction is predominantly mediated by hydrophobic contacts between the two proteins36. In addition, the PorB protein from Neisseria meningitidis was suggested to act as a TLR2 ligand with the binding mechanism hypothesized to involved electrostatic interactions that contribute to ligand/receptor interaction37. Moreover, the BspA protein from Tannerella forsythia which contains a LRR domain, was also suggested to be a TLR2 ligand38. Furthermore, the pentameric B subunit of type IIb E. coli enterotoxin (LT-IIb-B5) uses its hydrophobic upper-pore region to directly bind TLR2 and the ring with four residues  are considered to be TLR2-binding sites for induction of inflammatory responses39. These TLR2 ligands use various binding mechanisms to interact with innate immune components that induce inflammatory responses. In Leptospira, it has been proven that the LipL32 stimulated inflammatory responses through TLR2 and the calcium binding cluster  of LipL32 can regulate the affinity between LipL32 and TLR2 during infection with Leptospira26. However, the TLR2 binding domains and mechanisms for binding with LipL32 are unclear. Accordingly, identification of the binding mechanisms for LipL32-TLR2 interaction will provide new insights into innate immunity.TLR family proteins play a pivotal role in innate immunity by recognizing conserved patterns in diverse microbial moleculesIn this study, we predicted the LipL32-TLR2 complex from the Cluspro website and the binding domains were confirmed by truncation experiments. We demonstrated that the active components of LipL32, including N terminal β1β2 (Nβ1β2), central loop-α3-loop (Cenα3), and C terminal α4 helix (Cα4) domains, play critical roles involving in TLR2 interaction. In combination with site-directed mutagenesis and protein-protein interaction studies, we find the essential residues within active components of LipL32 participating in TLR2 binding. Our study suggested a novel working mechanism of a potential TLR2 ligand.3CSK4 complex suggests a mechanism for recognition of lapidated ligands by TLRs35; however, the protein ligands of TLR2 are still unknown. To identify the binding mechanism, we modeled the interaction between LipL32 (2WFK) and TLR2 (2Z7X). The top ten scoring models of TLR2/LipL32 were retrieved from ClusPro40 protein-protein docking website and were divided into three groups on the basis of LipL32 orientation in interact with TLR2  protein was purified in a series of purification procedures including anti-V5 antibody activated NHS resin affinity purification, mono Q ion exchange column, and Superdex 200 gel filtration column , respectively25 , transmembrane domain (TMD), and Toll/interleukin-1 receptor domain (TIR)umn Fig. . The molumn Fig. . The funy25 Fig. . The intE. coli ClearColiTM BL21(DE3) and BL21(DE3)pLys to obtained the recombinant proteins  assay was used to measure the endotoxin contamination of these recombinant proteins. The rLipL32 proteins from E. coli BL21(DE3)pLys contained high level of endotoxin that >100000EU/mg, while the protein from E. coli ClearColiTM BL21(DE3) significant decreased the endotoxin contamination to 688.5 ± 23.4 EU/mg  contain relatively low endotoxin and suitable for mammalian cell applications. In order to improve the purify and removed endotoxin contamination of the recombinant proteins, Mono Q ion exchange chromatograph was further used to purify the LipL32 protein and its variants as well as the proteins were assayed by Superdex 200 gel filtration column  and the different fragments including rLipL32ΔNβ1β2 (51–272), rLipL32ΔCenα3 (deletion the residues of 195–200), and rLipL32ΔCα4 (21–247), respectively, were constructed according to the modeling results and these variants were used to evaluate the affinity to TLR2 Fig. . In addiins Fig. . The rec/mg Fig. . The recumn Fig. 41. Mono low Fig. . To redulow Fig. . The mollow Fig. . The strlow Fig. . Furtherved Fig. . The Ca2Leptospira, outer membrane proteins of Leptospira are the first line to contact the host cell and interact with the cell membrane components. LipL32 is the most abundant outer membrane lipoprotein and occupied about 75% of the total outer membrane protein profile43. Previous studies have illustrated that LipL32 binds to different components of the ECM, including fibronectin, collagen, and laminin, respectively22. Besides ECM molecules, LipL32 also interacts with innate immune components, such as TLR2, and induces inflammatory responses26. However, the domains of LipL32 responsible for interacting with TLR2 are unclear. Therefore, confocal microscopy was used to observe the co-localization of truncated rLipL32 variants with the TLR2 receptor on the HEK293 cell surface , hCCL2/MCP-1 (MCP-1), hTNF-α (TNF-α), hMMP7 (MMP-7), hCXCL1/GRO-α (GRO-α), and osteopontin/SPP1 (SPP1)27 were measured to investigate TLR2 activation by its ligands  and proteinase K (20 µg/ml at 63 °C for 18 h). The results showed that rLipL32 protein treated with heat and proteinase K had a significantly lower level of inflammatory induction compared to that of WT  and mitogen-activated protein kinases (MAPKs), and induces the expression of chemokines and cytokinesnds Fig. . Two houPP1 Fig. . The two WT Fig. . These rTo confirm the N terminal β sheet as the binding domain, the synthesized peptide including N20 peptide (1–20 residues of LipL32), N50 peptide (1–50 residues of LipL32), C25 peptide (248–272 residues of LipL32), and a scrambled peptide with the sequence of VSLEGLTKVLAGSKFSESPILIKTVCDMILGASFANPALKISTASTLFTG, were used to induce inflammatory responses. With the exception of the N50 peptide, all these synthesized peptides stimulated less than 2-fold inflammatory responses compared to that stimulated by the mock control Fig. . The N5025. Using ELISA, we first analyzed the binding of rTLR2 protein to its ligands, including Lipoteichoic acid (LTA), Pam3CSK4, and peptidoglycan (PGN), and these results indicating that the rTLR2 protein from our system was suitable for ligand binding assays  was calculated as 0.13 ± 0.02 µM from Eq. 2+ regulates the interaction of rTLR2 and rLipL32 as well as poly-D motif variants of rLipL32, which reduce regulation ability25. The role of Ca2+ ions was further investigated in these deletion variants of rLipL32. In the presence of ethylene glycol tetraacetic acid (EGTA), the affinity of rLipL32WT to rTLR2 was decreased to 0.45 ± 0.12 µM as compared to that in the presence of Ca2+ ions, demonstrating that sequestration of Ca2+ ion reduced the binding affinity of rLipL32 to rTLR2 proteins , and the affinity of rLipL32 to rTLR2 was measured in the absence and presence of Ca2+ , L36S (p < 0.01), and L263S (p < 0.05) showed significantly reduced interaction force compared to WT, whereas F34S showed no significant difference compared to WT measurements Fig. . Additio50) of the rLipL32-rTLR2 interaction. The IC50 of N20, N50, C25, and scrambled peptides were calculated as 0.09 ± 0.05 μM, 0.07 ± 0.04 μM, 0.14 ± 0.01 μM, and 13.9 ± 1.2 μM, respectively 35, leucine-rich repeat family proteins (BspA)38, proteins with TLR2 binding sequence (PorB)45, and proteins with the specific structures for TLR2 binding (enterotoxins)39. Crystal structure of the TLR2/1 complex associated with the ligand, Pam3CSK4, revealed that the lipid molecule of the lipoprotein supported the binding to TLR235. Previous studies showed that the LipL32 protein is modified with lipid molecules at the N-terminus18. Interesting, other study showed that non-lipidated polypeptides lack stimulation activity via TLR2 pathway46. However, an increasing studies revealed that polypeptides ligands could interact to TLR2 and stimulate or inhibit the inflammatory responses through TLR2 pathway45.The TLR family comprises pattern recognition receptors and their ligands originate from microbes and contain multiple conserved patterns that can be recognized by the TLR LRR domain28. The mature form of rLipL32 protein is a lipoprotein in which the first 20 residues are removed. Therefore, the rLipL32ΔN20 construct is considered as wild-type rLipL32 protein for comparison with other truncated variants. Crystal structure of rLipL32, a jellyroll conformation without the LRR domain in its structure21, appears unlikely to use the LRR domain to interact with rTLR238. In addition, structure alignment showed that the structure of rLipL32 is substantially different from that of enterotoxin B39. Moreover, sequence alignment of rLipL32 with and other TLR2 binding proteins such as BspA, PorB, and LcrV suggested that the homology of these proteins are low (<10%), and TLR2 binding motifs from BspA, PorB, and LcrV were not found in LipL32. These results indicate that rLipL32 is a unique protein with specific sequence and structure that different from the known TLR2 ligands. As a novel TLR2 ligand, rLipL32 is suggested to utilize its distinctive structure and mechanism to interact with TLR2.In our study, deletion of the signal peptide of rLipL32 did not prevent binding to rTLR2, and it was still able to stimulate inflammatory responses, suggesting that other domains of rLipL32 are responsible for interacting with rTLR2TM BL21(DE3) was used to produced endotoxin free recombinant proteins47. The yield of protein expression are similar to that expressed in BL21(DE3) competent cell. In addition, the anion-exchange chromatography (Mono-Q) and polymyxin B resin were further used to remove contaminating endotoxin from the purified rLipL32 variants41. In the LAL assay, it was observed that the endotoxin was significantly removed. In order to prevent the interference of the resident endotoxin, the HEK293-TLR2 cell naturally not expressing TLR4 was used to measure the inflammatory responses. Therefore, the responses from the stimulation of HEK293-TLR2 were directly from the rLipL32 and its variants.The endotoxin contamination from the recombinant protein interfere the intrinsic inflammatory properties of the innate immunity system. To overcome this problem, the endotoxin locked expression system, ClearColiThe synthesized peptides derived from LipL32 were used to interact with rTLR2. However, the affinity of synthesized peptides to rTLR2 was low as determined in ELISA and AFM analysis. Previous study in BspA protein revealed that the synthesis truncated peptides (LRR domains) could induce responses to interact with rTLR2. We speculated that the synthesis peptides derived from LipL32 lacking suitable conformation to interact with rTLR2. Interestingly, the synthesized peptides, N20 and N50 peptides were used to compete the interaction between rLipL32 and rTLR2 Fig. . However35, Leu36, and Leu263, are involved in rLipL32-rTLR2 complex interface. Similarly, protein complex of SSL3-TLR2 was solved and the residues involved in complex formation were proposed36. The interface of the SSL3-TLR2 complex is a hydrophobic region which contains Phe156, Phe158, Leu160, and Pro194 of SSL3 as well as Phe349, Leu350, Gln375, Tyr376, and Asn379 of TLR2. In addition, the pentameric B subunit of type IIb E. coli enterotoxin (LT-IIb-B5) also suggests its hydrophobic upper-pore region directly binds to TLR2 and four residues, including Met69, Ala70, Leu73, and Ser74 are responsible for TLR2-binding. Therefore, protein ligands for TLR2 with the hydrophobic patch may play vital roles in interacting to TLR2.Furthermore, the residues at N- and C-termini of rLipL32 are investigated to find out the residues responsible for interacting to rTLR2. In the predicted complex, several resides of rLipL32 with nearest distance to rTLR2 were replace to Serine residues and the mutation variants were purified to measure the affinity to rTLR2. Interestingly, our prediction rLipL32-rTLR2 complex and experimental data also suggest that three hydrophobic residues, including Val35, Leu36, and Leu263 at N and C terminus play pivotal roles in interaction to TLR2, respectively. The corresponding binding residues of rTLR2 were predicted using nearest distance prediction and the results show the hydrophobic interaction of Phe349 and Leu371 of rTLR2 to Leu36 of rLipL32; Leu350, Gln357 of rTLR2 to Leu263 of rLipL32, respectively , Dulbecco’s modified Eagle’s medium (DMEM), and F-12 nutrient mixture (HAM) were obtained from Invitrogen Corp. . The anti-TLR2 IgG clone T2.5 neutralizing antibody (MAb-mTLR2) and isotype mouse IgG1 were provided by Invivogen . The anti-His tag antibody (Ab18184) was purchased from Abcam . All other chemicals were supplied by Sigma .lipl32 gene was cloned from pathogenic L. santarosai serovar Shermani str. LT821  genomic DNA with pfu-Turbo DNA polymerase  according to prior investigation26. Wild-type and truncated lipl32 variants were amplified by PCR and inserted into the expression vector pRSET-c . The point mutation variants were performed by Q5® Site-Directed Mutagenesis kit . The sense and antisense primers used in this research were listed in Table E. coli and the DNA sequences were verified by DNA sequencing.The tlr2 gene was amplified by PCR using the primers listed in Table E. coli were transformed with pLenti6.3/V5-TOPO-TLR2. The real cloned DNA with the right size and right orientation was verified by restriction digests and confirmed by DNA sequencing. The correct clone of p-TLR2-Lenti plasmids was used for stable gene expression in culture HEK293 cells.The human 2. All stimulation experiments are performed under serum free conditions to avoid the influence of serum on cell function and investigated events.Human embryonic kidney cells  were maintained in Thermo Scientific HyClone Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% Thermo Scientific HyClone Defined Fetal Bovine Serum. Cells were grown in an incubator at 37 °C and the humidified atmosphere of 5% CO2.The vector p-TLR2-Lenti was transfected into HEK293FT cells with ViraPower packaging mix  to generate the lentivirus according to manufacturer’s protocol. HEK293FT cells were transfected with the lentivirus and stable cell lines were generated by selecting with blasticidin. Cells were collected and named HEK293-TLR2. Cells were grown in DMEM with 10% FBS and maintained in a 37 °C humidified atmosphere containing 5% COlipl32 and its variants were transformed into the expression host cell E. coli ClearColiTMTMBL21 (DE3) pLys . The transformants of His6-LipL32 and its variants were grown in Luria broth medium with 50 μg/ml ampicillin at 37 °C to an OD600 of ~0.6. Isopropyl 1-thio-β-galactopyranoside (500 μM) was subsequently added to induce the expression of LipL32 protein for additional 24 h at 16 °C. E. coli cells were harvested by centrifugation at 4,000 x g for 15 min and then sonicated in phosphate buffered saline (PBS buffer). The cell debris was discarded after centrifugation at 14,000 xg for 30 min and the supernatant absorbed to Ni2+-nitrilotriacetic acid (Ni2+-NTA) agarose resin  for affinity chromatography purification48. rLipL32 protein and its variants were eluted in PBS buffer containing 250 mM imidazole and the elution fractions were dialysis against the PBS buffer to remove the imidazole and applied to mono Q column ion exchanged column  for further removal the contaminated protein and endotoxins. Furthermore, to detach His6-tag, the rLipL32 protein and its variants were incubated with 0.2 mg/ml enterokinase  at 37 °C for 16 h49. The tag peptide was further removed by size exclusion chromatography using Superdex 200 pg 16/600 column . The purified rLipL32 and its variants (100–150 μg) were injected into the column and the flow rate was set at 1 ml/min at 4 °C. To validate the inflammatory effects of rLipL32, the protein were subjected to polymyxin B (Invitrogen), heat, and proteinase K (Invitrogen) treatments, respectively, as described previously25. The Limulus Amebocyte Lysate (LAL) test was further used to detect the recombinant proteins and measured the endotoxin contamination according to the manufacturer’s guidelines .The DNA plasmid of 2 for human rTLR2 protein expression. The cell was suspended in PBS buffer containing 0.5% CHAPS for rTLR2 protein solubilization. The cell debris was removed after centrifugation at 14,000 × g for 30 min and the supernatant absorbed to anti-V5 antibody activated NHS resin  for affinity chromatography purification. rTLR2 protein was eluted in 25 mM Glycine-HCl buffer (pH 2.5) and following by neutralization in the PBS buffer containing 0.5% CHAPS.HEK293-TLR2 was grown in the medium of DMEM with 10% FBS and maintained in a 37 °C humidified atmosphere containing 5% COThe secondary structures of rLipL32WT and deletion variants were analyzed at 25 °C on an CD Aviv 202 spectrometer  using a 1-mm path length cuvette. The resulting spectra were corrected to remove buffer signal and smoothened using SigmaPlot 10.0. The obtained CD spectra were reported as averages of no less than three scans for each sample.2+ binding protein. rLipL32WT and its variants (15 μM) were mixed with stains-all dye solution (500 μM) in 2mM MOPS (pH 7.2) containing 30% (v/v) ethylene glycol and incubated for 10 min. The CD spectra was used to scan from 700 to 400 nm and the characteristic peak at 660 nm (J band) was recorded. The obtained CD spectra were reported as averages of no less than three scans for each sample.Stains-all is a carbocyanine probe and could be used to detect the Ca6 . The secondary antibodies for confocal laser scanning microscopy were Alexa594 conjugated anti-mouse and Alexa488 conjugated anti-rabbit secondary antibodies . Cells were imaged by confocal laser scanning microscopy .HEK293 and HEK293-TLR2 cells were fixed, permeabilized and incubated with appropriate primary and secondary antibodies: mouse monoclonal anti-TLR2 antibody , rabbit monoclonal anti-His28. Real-time PCR was performed following manufacturer’s instructions using an ABI Prism 7700 with SYBR green I as a double-stranded DNA-specific dye . Primers of IL-8, TNF-α, MCP-1, and hMMP7 were listed in Table GRO-α  and SPP1  mRNA expression. The results of the mRNA levels for the different genes are displayed as transcript levels of the analyzed genes relative to GAPDH. The statistical analyses were performed with Student’s t test. Statistically significant were considered as p-values of ≤0.05.Total RNA was extracted according to the previous protocol with minor modifications2+-bound (PDB:2WFK20) and Ca2+-free (PDB:3FRL19) LipL32 and TLR2 LRR domain  were submitted to automated docking using the ClusPro 2.0 server (available on-line52). Each protein was analyzed both as receptor and ligand. The 10 highest scoring models were analyzed using PyMOL (available on-line). The 10 possible binding models were divided in to 3 groups according to the binding domains of LipL32 interacted with TLR2.The PDB files corresponding to Ca25. The ELISA plates (Nunc-Immuno Plate MaxiSorp surface) were primarily coated with 1 μg rTLR2 protein in 100 μl PBS buffer and incubated for 2 h at 37 °C. The wells were washed with PBST (PBS containing 0.05% (v/v) Tween 20) and then blocked with 1% (w/v) bovine serum albumin (BSA) for 1 h at 37 °C. The plates were incubated overnight at 4 °C. Protein samples  in PBS buffer were added to the attached rTLR2 protein for 90 min at 37 °C and then washed with PBST. The bound rLipL32 protein was detected by adding 100 μl anti-His6 antibody   incubated for 1 h and then washed with PBST. Subsequently, 100 μl horseradish peroxidase conjugated donkey anti-rabbit immunoglobulin G  was added and incubated for 1 h at 37 °C. After washing with PBST, the 3,3′,5,5′-tetramethylbenzidine (TMB) was added for color development. The reaction was allowed to proceed for 15 min and then terminated by adding 50 μl 2 N H2SO4. The absorbance was measured at 450 nm in an ELISA reader . These experiments were accomplished in three independent experiments and the mean absorbance ± S.D. of three independent data sets was calculated. The absorbance at each data point was corrected by subtracting a negative control from BSA coated well. The dissociation constant (Kd) according to the prior protocol53, based on the following equation:A is the absorbance at a set protein concentration, Amax is the maximum absorbance for the ELISA plate reader (equilibrium), [protein] is the protein concentration, and Kd is the dissociation equilibrium constant for a given absorbance at a set protein concentration (ELISA data point). The binding data were assayed via SigmaPlot 10.0 program by fitting the data to the optimal equation54 assuming that ligands bound to one independent site. The statistical analyses were performed with Student’s t test. Statistically significant were considered as p-values of ≤0.05.The interaction between rLipL32 and rTLR2 was investigated by ELISA according to the prior method with minor modifications55. For anchoring proteins, the tips were rinsed with PBS to remove the glutaraldehyde remainders and subsequently incubated with proteins, including BSA, Pam3CSK4, rLipL32 and its variants for 1 h at room temperature. The functionalized AFM tips were then rinsed three times with PBS to remove the unbound proteins56. The mica surface was modified for better deposition of proteins according to previous reports58. The unbound proteins were removed and the proteins were fixed on the mica for later AFM measurements.Silicon nitride AFM cantilever, PNP-TR , was cleaned by sonication in a series of solvents and functionalization in 3-aminopropyl-trimethoxysilane and glutaraldehyde  according to the method reported previously with minor modifications26. For antibody neutralization analysis, the protein modified mica was treated with anti-TLR2 antibodies   for 1 h followed by five washes with PBS to remove unbound antibodies. For the binding events, force curves from at least 10 areas on each surface were independently selected for analysis. All the measurements described above were performed with modified tips and showed repeatedly similar results. The statistical analyses were performed with Student’s t test. Statistically significant were considered as p-values of ≤0.05.The functionalized cantilever tips used in this work had a spring constant in the range of 0.02–0.08 N/m as determined from the amplitude of their thermal vibrations. A commercial atomic force microscope  with a E type scanner was employed throughout this study. The force volume software takes a force curve at each of the 999 points during a two dimensional scan over a sample surface. The X-Y scan size was 150 μm, and the Z scan distance was 5 μm at a rate of 1 Hz. The force applied to the protein modified mica surface was kept below 400 pN. The distance-force curves and force parameters were obtained according to the methods described previouslyAll experiments were performed at least three independent processes and the variables are expressed as mean ± SEM and compared by using Student’s t-test or one-way ANOVA. P-values < 0.05 are considered statistically significant. All analyses were performed using the Graphpad Prism 5.1 .Supplemental information


A well-designed macrocycle affords nanomolar binding to a cancer biomarker lysophosphatidic acid, showing potential application in the diagnosis of gynecologic cancers. We designed a water-soluble guanidinium-modified calix[5]arene to target lysophosphatidic acid (LPA), an ideal biomarker for early diagnosis of ovarian and other gynecologic cancers, achieving binding on the nanomolar level. An indicator displacement assay, coupled with differential sensing, enabled ultrasensitive and specific detection of LPA. Moreover, we show that using a calibration line, the LPA concentration in untreated serum can be quantified in the biologically relevant low μM range with a detection limit of 1.7 μM. The reported approach is feasible for diagnosing ovarian and other gynecologic cancers, particularly at their early stages. With respect to the efficient host–guest interactions between macrocycles and biological substrates in aqueous media, their molecular recognition has gained considerable attention and demonstrated various applications in, but not limited to, the fields of disease diagnosis and therapy, such as sensing of biomarkers,via an indicator displacement assay (IDA) in aqueous media  family, we screened C5A on account of its size fit. The alkyl chain effectively threads C5A, but not the smaller C4A, while C6A and C8A are relatively larger and have complex conformations.via the synergistic effect among several interactions . The GC5A was prepared primarily according to the syntheses of reported C4A analogues  unlimited possibilities” benefiting from their facial modification.nalogues .15 In bret al. as a standard strategy for molecular sensing, complementary to the approach of direct sensing.The GC5A host and the LPA guest are both amphiphilic with critical aggregation concentrations (CACs) of 0.4 and 0.35 mM Fig. S9, respectKa), extracted from the fluorescence titration, was fitted as (5.0 ± 1.0) × 106 M–1, which was further validated by UV-vis titration  was screened as the optimal reporter dye, owing to its high brightness, strong binding with GC5A and drastic complexation-induced quenching of fluorescence . The bin Fig. S11. In partKa value of (1.6 ± 0.1) × 108 M–1. The 1 : 1 binding stoichiometry was clearly verified by the inflection point at 1 : 1 molar ratio between GC5A and LPA in the competitive titration. To validate the synergistic effect of several interactions on nanomolar binding between GC5A and LPA, we measured the binding of phosphate and 6 : 0 LPA  with GC5A, giving Ka values of (4.6 ± 0.6) × 104 M–1 and (4.8 ± 1.0) × 105 M–1  to Fl than GC5A  and anionic (LPA) surfactants that leads to weak complexation.3, the control C5A host with conformational flexibility, quenches the fluorescence of Fl to a much lower extent and affords the corresponding weaker affinity ((4.5 ± 0.3) × 104 M–1) than GC5A .vide infra), leading to a relatively weak ring current effect. The shifts of H3–H5 signals are larger than the rest, indicating their location in the center of the cavity. However, H1 and H2 are presumably located at the upper rim, H6 and H7 are close to the lower-rim oxygen mean plane and thread out of the cavity, which are away from the region of maximum shielding provided by the aromatic rings. Moreover, NMR measurements were performed above CAC, so it is also possible that GC5A and LPA form a co-assembly, but not an endo-complex. If GC5A forms a co-assembly with LPA, it is possible that GC4A also forms a co-assembly. We further executed the NMR measurements of GC4A with LPA as a control, observing no appreciable complexation-induced shifts /6-31G(d)/SMD(water) method.R2 = 0.997). The limit of detection (LOD) for LPA is calculated to be 5.6 nM by utilizing a 3σ/slope method,The IDA principle based on the GC5A·Fl reporter pair allows for a fluorescence “switch-on” sensing of LPA. As shown in Fig. S21,Ka = (4.7 ± 1.4) × 108 M–1, Fig. S24iii) phthalocyanine chloride tetrasulfonic acid  in the case of GC5A is thus much larger than that in the case of GC4A.We further tested changes in the fluorescence intensity of GC5A·Fl caused by other biologically important species  in plasma to evaluate the sensing selectivity for LPA . In most Fig. S22. We ther pattern  and S25† pattern .23 LPA w4 reporter pair was still observed upon the gradual increase in LPA concentrations (0–80 μM) (R2 = 0.998) allows us to construct a calibration line for the fluorescence intensity to accurately determine unknown concentrations of LPA down to the low μM range, which is of practical diagnostic relevance. Furthermore, we applied the GC5A·AlPcS4 reporter pair in analyzing cancerous and non-cancerous blood samples. The blood samples were obtained from healthy mice and mice with ovarian tumour, which was created by inoculating ID8 cells subcutaneously. A significant difference was observed, whereby the cancerous group had a greater fluorescence response than the non-cancerous group  . The LOD Fig. S26. This reIn conclusion, we designed an artificial receptor GC5A for LPA, a cancer biomarker, with nanomolar affinity in aqueous media. Through IDA coupled with differential sensing, we achieved ultrasensitive and specific detection of LPA. For accurately determining unknown concentrations of LPA down to the low μM range, which is of practical diagnostic relevance, a calibration line was successfully set up in serum. To the best of our knowledge, although calibration lines of LPA have been obtained among several known assay approaches,All animal studies were performed in compliance with the guidelines set by the Tianjin Committee of Use and Care of Laboratory Animals and the overall project protocols were approved by the Animal Ethics Committee of Nankai University.There are no conflicts to declare.Supplementary informationClick here for additional data file.


Acanthamoeba polyphaga mimivirus (mvUNG). In the crystal structure of mvUNG, residues 95–130 in the N-domain bind to a hydrophobic groove in the catalytic domain, and motif-I forms a short β-sheet with a positively charged surface near the active site. Circular dichroism spectra showed that residues 1–94 are in a random coil conformation. Deletion of the three additional fragments reduced the activity and thermal stability, compared to full-length mvUNG. The results suggested that the mvUNG N-domain and motif-I are required for its structural and functional integrity.Cytosine deamination induced by stresses or enzymatic catalysis converts deoxycytidine into deoxyuridine, thereby introducing a G to A mutation after DNA replication. Base-excision repair to correct uracil to cytosine is initiated by uracil-DNA glycosylase (UDG), which recognizes and eliminates uracil from DNA. Mimivirus, one of the largest known viruses, also encodes a distinctive UDG gene containing a long N-terminal domain  and a motif-I (residues 327–343), in addition to the canonical catalytic domain of family I UDGs . To understand the structural and functional features of the additional segments, we have determined the crystal structure of UNG from  Although uracil is a base component of RNA, it is occasionally introduced into DNA by hydrolytic deamination of cytosine . Uracil UDG genes are found ubiquitously in cellular organisms from prokaryotes to humans, and have also been identified in some large DNA viruses, such as poxviruses and herpesviruses , 4, 5. UAlthough UNG removes uracil included in single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), it shows a preference for ssDNA than dsDNA as a substrate , 11. TheAcanthamoeba polyphaga mimivirus (APMV) was identified recently by its genome sequencing [Mimiviridae family of nucleocytoplasmic large DNA viruses (NCLDV), is one of the largest known viruses, comparable to bacteria in size [The UNG gene in quencing . APMV, w in size . It has  in size , 19, 20.6x-thioredoxin. The plasmid was then transformed into Escherichia coli strain BL21-star(DE3) , and the cells were grown in Luria-Bertani medium. Protein expression was induced using 0.4 mM isopropyl-β-D-thiogalactoside at 15°C. After overnight induction, cells were harvested by centrifugation, and clarified cell lysates were prepared in buffer A (20 mM hydroxyethyl piperazineethanesulfonic acid (HEPES) pH 7.5, 0.5 M NaCl, 5% glycerol, and 0.2 mM tris(2-carboxyethyl)phosphine). The mvUNG protein was purified using immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC). Proteins purified by IMAC were dialyzed twice in buffer A and then treated with thrombin to cleave the Hisx6-thioredoxin tag from mvUNG. After complete cleavage of the tag, the protein solution was passed through IMAC resin to remove His6x-thioredoxin. mvUNG was further purified using a Superdex-75 size exclusion column equilibrated with buffer A, followed by a Superdex-200 column equilibrated with buffer C phosphine). Purified mvUNG was concentrated to 10 mg/mL and estimated to be >95% pure by SDS-PAGE. Mutant proteins  were expressed and purified by the same procedure as that used for native mvUNG.The gene encoding mvUNG  was synthesized artificially after codon optimization  and inserted into pET-22b  with DNA encoding His2) under a layer of Al’s oil . Needle-shaped crystals of mvUNG were fully grown in 2 months. Crystals were picked up using a cryo-loop (Hampton Research) and then flash-frozen in a cold nitrogen stream without the addition of a cryoprotectant. Diffraction data were collected at PLS-BL7A  and were indexed, integrated, and scaled using HKL2000 software [Crystallization of mvUNG was performed using the micro-batch method at 20°C. The crystallization drop was prepared by mixing 1 μL of protein solution (10 mg/mL) and 1 μL of crystallization solution  Polyethylene glycol 3350, 4% (v/v) Isopropanol, and 0.1 M CaClsoftware .Leishmania naiffi UDG (PDB ID: 3CXM) was used as a template for MR. Two mvUNG proteins were found in an asymmetric unit. Cycles of refinement and model rebuilding were performed at 2.3 Å resolution using phenix.refine [free values of 17.3 / 21.6% without residues in the disallowed region of the Ramachandran plot. Data collection and refinement statistics are summarized in The crystal structure of mvUNG was determined by the molecular replacement (MR) method using PHASER . The strx.refine  and COOTx.refine . ResidueUAT TCG AGC TCG GTA CCC GGG G-3’5’-GTA AAA CGA CGG CCA GTG ) as a substrate (Cosmo Genetech) [The activity of mvUNG was measured using ssDNA and dsDNA containing a single uracil (enetech) . The reaenetech) . The quaTo measure its molecular size, purified mvUNG was injected into a Superdex-200 analytical column connected to Purifier FPLC  and the elution products were applied to inline miniDAWN TREOS MALLS and Optilab digital signal processing refractive index detectors . Data were collected at 1.0 s intervals at a flow rate of 0.4 mL/min and were analyzed using the ASTRA V software package (Wyatt Technology Corporation).CD spectra were obtained using the JASCO J-810 spectropolarimeter . For far-UV CD spectra, 10 μM protein in a 1 mm cuvette was exposed to a wavelength range of 200–260 nm, and the data were recorded at a 1 nm intervals with a scanning speed of 5 nm/min. Three spectra from a protein sample were collected and averaged. The spectra were smoothed after subtraction of the buffer spectrum. For the thermal melting spectra, 10 μM protein in a 1 mm cuvette was exposed to a wavelength of 220 nm. The thermal melting curves were recorded at 0.5°C intervals in the temperature range of 20–90°C. Temperature was increased at a rate of 2°C/min.5X55).The final coordinate and structure factor for mvUNG were deposited into the Protein Data Bank  contains a catalytic domain conserved among UNG family proteins. Additionally, it has an N-terminal domain  and an insertional motif  and the solvent content under crystal packaging of full-length mvUNG were calculated as 1.8 Å3/Da and 31.8%, respectively, which are out of the range commonly observed for protein crystals, while those of mvUNG95-370 were 2.4 Å3/Da and 48.8%, which falls within the range. A solvent space that could accommodate residues 1–94 was insufficient in the crystal. Moreover, mvUNG95-370 facilitated the growth of the needle-shaped crystal under same crystallization conditions as full-length mvUNG. Thus, the N-terminal residues seem to be truncated before crystallization.Needle-shaped crystals were grown from full-length mvUNG, and the diffraction data were collected up to 2.3 Å resolution. The phase was obtained by molecular replacement, using the structure of  density , and the.3/21.6% . MatthewTwo mvUNGs in the asymmetric unit were superimposed with a root-mean-square deviation (RMSD) of 0.4 Å for 276 Cα atoms, indicating that the two mvUNGs are in the same conformation. The oligomeric state in solution was determined by SEC. In SEC, mvUNG (molecular weight of a monomer: 42.2 kDa) was eluted as a monodisperse protein between ovalbumin (44 kDa) and conalbumin (75 kDa), which means that it was slightly larger size than a monomer of mvUNG . To measE. coli [10-helix. Additionally, two short β-strands . Residue R96 in the N-domain forms an ionic interaction with E169 in the catalytic domain, and the segment is fitted into a hydrophobic groove of the catalytic domain. Residues 95–130 of the N-domain seem to stabilize the catalytic core domain by binding to its hydrophobic surface  and an insertional motif  located between S6 and H12 . The N-d surface . Motif-I surface  and prov surface . Taken tE. coli, and herpesviruses contain six conserved active site residues that are involved in uracil-binding specificity and catalysis (Bacillus subtilis UNG and by positively charged Arg in vaccinia virus UNG (vacvUNG). mvUNG also does not contain the Leu residue (B. subtilis UNG) in the minor groove intercalation loop is a critical residue for the inhibition of UNGs by Ugi and p56, as well as for catalytic activity. The Leu or Phe residue is recognized by the hydrophobic pockets of Ugi [UNG proteins from human,  residue . The cat residue  and 3c.  residue . The Leus of Ugi , 35–37 as of Ugi . The vacs of Ugi . This imIn addition to active site residues, UNGs share five conserved motifs required for the formation of a uracil-binding pocket and DNA-binding groove Figs  and 3. CΔ327–343) and two N-terminal deletion mutants (mvUNG95-370 and mvUNG122-370) were prepared using the same procedure as the full-length mvUNG. The activity assays showed that UNG activity of the three deletion mutants was significantly lower than that of the full-length mvUNG  on UNG activity, we performed activity assays with the mutant mvUNG proteins. A motif-I deletion mutant  of mvUNG is located around the Gly-Ser and minor-groove intercalation loops, without disrupting the active site. Its surface distributes a weakly positive charge . In the 95-370. In the far-UV CD spectra, the deletion of residues 1–94 did not change the pattern and scale of the CD spectrum in the range between 210–250 nm, indicating that residues 1–94 have a random coil conformation (m). The Tm value of full-length mvUNG (55.9°C) was 4.4°C higher than that of mvUNG95-370 (51.5°C)  , indicat95-370). Thus, the structured part of the N-domain seems to stabilize the catalytic domain by binding directly to its hydrophobic surface. Motif-I, inserted into the catalytic domain, forms a short β-sheet near the active site  and motif-I (residues 327–343) are required for the structural and functional integrity of mvUNG.UNG is a ubiquitous enzyme for base excision repair. All UNGs, from human to large viruses, contain a conserved catalytic domain that removes uracil from DNA. Recently, a gene was identified from a mimivirus that encodes an UNG containing a long N-domain and motif-I in addition to the catalytic core domain . To undeive site . The delive site  and therive site . The UNGIn contrast to the structured part of the N-domain and the motif-I, residues 1–94 in N-domain are not included in the crystal structure of mvUNG. They seem to be truncated before crystallization. Indeed, all reported crystal structures of UNG proteins display only a catalytic domain. CD spectra showed that residues 1–94 in mvUNG are close to a random coil . AlthougS1 FigThe UDG activity of mvUNG was measured using ssDNA and dsDNA as a substrate. Uracil was removed from ssDNA but not dsDNA.(TIF)Click here for additional data file.S2 Fig(a) Ribbon model of two monomers in an asymmetric unit. (b) Cα trace model of two monomers superimposed.(TIF)Click here for additional data file.S3 FigmvUNG was eluted as a monodisperse protein between ovalbumin  and conalbumin  in both the Superdex-200 (a) and Superdex-75 (b) analytical columns.(TIF)Click here for additional data file.S4 Fig(a) The visible N-domain and catalytic domain in mvUNG are drawn as stick and surface models, respectively. (b) The mvUNG monomer (residues 95–370) was drawn as a surface model with the charge distribution.  The panels show that the ordered fragment (residues 95–130) in the N-domain binds to the hydrophobic surface of the catalytic domain.(TIF)Click here for additional data file.S5 FigΔ327–343 on the structure of hUNG/DNA containing an abasic site (PDB ID: 2SSP). mvUNGs and DNA were drawn as surface and backbone models, respectively. Motif-I (residues 327–343) does not disturb DNA-binding and provides a positively charged surface near the active site.The models were generated by superimposing mvUNG and mvUNG(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.