Escherichia coli topoisomerase I has an essential function in preventing hypernegative supercoiling of DNA. A full length structure of E. coli topoisomerase I reported here shows how the C-terminal domains bind single-stranded DNA (ssDNA) to recognize the accumulation of negative supercoils in duplex DNA. These C-terminal domains of E. coli topoisomerase I are known to interact with RNA polymerase, and two flexible linkers within the C-terminal domains may assist in the movement of the ssDNA for the rapid removal of transcription driven negative supercoils. The structure has also unveiled for the first time how the 4-Cys zinc ribbon domain and zinc ribbon-like domain bind ssDNA with primarily π-stacking interactions. This novel structure, in combination with new biochemical data, provides important insights into the mechanism of genome regulation by type IA topoisomerases that is essential for life, as well as the structures of homologous type IA TOP3α and TOP3β from higher eukaryotes that also have multiple 4-Cys zinc ribbon domains required for their physiological functions. Escherichia coli topoisomerase I (EcTOP1) encoded by the topA gene is the prototype type IA topoisomerase required for preventing excess negative DNA supercoiling. It has an important role in removing transcription-induced negative supercoiling behind the RNA polymerase complex . This protein fold found in type IA topoisomerases is related to other transcription regulators . ExperimHere, we present for the first time the structure of completely active full-length EcTOP1 in complex with single-stranded DNA (ssDNA). This provides significant new structural information on the essential C-terminal domains of type IA topoisomerases, including homologous TOP3α and TOP3β in humans. The interactions between the type IA topoisomerase C-terminal domains and ssDNA are critical for these enzymes to recognize hypernegatively supercoiled DNA as the preferred catalytic substrate. Furthermore, the C-terminal domains’ interaction with the N-terminal domains and the flexible linkers between the C-terminal domains observed in this new structure could promote the movement of the bound DNA for passage through the DNA break in order to rapidly catalyze the relaxation of negatively supercoiled DNA. Two models of how bacterial topoisomerase I could interact with ssDNA regions in partially unwound duplex DNA for removal of negative supercoils are considered here based on the new structural information. Additional biochemical data support the model in which the enzyme interacts with both the G-strand and T-strand of underwound ssDNA.E. coli BL21 STAR (DE3) strain (Invitrogen). The pLIC–EcTOP plasmid was produced by placing the EcTOP1 coding sequence into the pLIC–HK vector that allows T7 RNA polymerase-dependent expression of EcTOP1 along with a tobacco etch virus (TEV) protease-cleavable N-terminal His6 affinity tag . The recombinant protein molecules in the soluble lysate were allowed to bind to Ni Sepharose 6 Fast Flow  before being packed into a column. After extensive washing with buffer , the topoisomerase protein was eluted with buffer , cleaved with TEV protease, and passed through the Ni Sepharose again to remove the His6 tag. Additional purification was achieved using a ssDNA cellulose column (Sigma). The topoisomerase was eluted with an increasing concentration gradient of KCl. Final purification was carried out with an S300 size exclusion chromatography column as described previously (EcTOP1 was expressed from a recombinant plasmid pLIC–EcTOP in the nity tag . Cells weviously .Oligonucleotides used in site-directed mutagenesis of EcTOP1 are listed in Supplementary Table S1. The Q5 site-directed mutagenesis kit (New England BioLabs) was used to create the F616L and I701term mutants. The Quikchange site-directed mutagenesis procedures were used for the F616E and R189A mutants.32P using γ32P-ATP and T4 polynucleotide kinase, and analyzed by electrophoresis in 15% sequencing gel against 32P-labeled O20 and O29.A partial duplex oligonucleotide substrate was produced by hybridization of a 29 base oligonucleotide (O29) 5′-GCTAAACCTGAAAGATTATGCGATTTGGG-3′ to a 20 base oligonucleotide (O20) 5′-CATAATCTTTCAGGTTTAGC-3′. The hybrid DNA O29-O20 was used for co-crystallization with EcTOP1. Since only the ssDNA corresponding to the last 11 bases of oligonucleotide O29 is visible in the crystal structure, a dozen of EcTOP1/DNA complex crystals were recovered from crystallization drops to verify the nature of bound DNA. The crystals were washed in crystallization buffer  and dissolved in TE buffer . The oligonucleotide(s) present were labeled on the 5′-end with For crystallization, EcTOP1 was concentrated to 38 mg/ml (∼0.39 mM). The protein was then mixed with oligonucleotide O29-O20 in a 1:1 molar ratio and the mixture was incubated on ice for 2 h. Hanging drop vapor diffusion setup was used for the crystallization with each drop containing 1 μl of the protein/DNA mixture and 1 μl crystallization solution. Diffraction quality crystals appeared under condition: 0.125 M ammonium sulfate, 0.1 M MES pH 6.0, 1 mM zinc sulfate, and 19% polyethylene glycol monomethyl ether 5000 at 24°C. Prior to data collection, the crystals were treated with cryoprotectant (25% glycerol) and cryocooled directly in liquid nitrogen.free = 32.4/41.1% and FOM = 66.4.X-ray diffraction data were collected at 100 K from EcTOP1/DNA crystals at the Structural Biology Center 19-ID beamline at the Advanced Photon Source at Argonne National Laboratory using the program SBCcollect . The intModel re-building of the N-terminal domains and the building of the C-terminal domains were subsequently performed manually by using the program Coot . The C-tX-ray fluorescence spectra were measured from crystals and showed the presence of zinc in the protein crystals (data not shown). Since 1 mM zinc sulfate was present in the crystallization buffer and the resolution limit of the X-ray diffraction data was 2.9 Å, one more X-ray diffraction data set was collected at zinc absorption peak (9.6688 KeV) to pinpoint anomalous scatter zinc sites inside crystal structure. The EcTOP1 structure was then refined with the low-energy data set and anomalous difference maps were subsequently calculated  were carried out with the program Phenix.refine and included TLS refinement Table  30). Re. Re30). 2 as indicated. After incubation at 37°C for 30 min, relaxation reaction products were analyzed by agarose gel electrophoresis (32P on the 5′ end (5′-GATTATGCAATGCGCTTTGGGCAACCAAGAGAGCATAAC-3′) as described previously  pLL1) )  for grow2 for 1 h at 37°C before digestion with 5 ng of DNase I or 7.5 pg of Micrococal nuclease (both from New England BioLabs) for 2 min and 1 min, respectively, at 37°C. Nuclease digestion was terminated by addition of 50 mM EDTA. The reaction products were analyzed by electrophoresis in a 15% sequencing gel, followed by Phosphorimager analysis.The production of G116S mutant of EcTOP1 was reported earlier . This muThe structure of full length EcTOP1 with bound DNA was determined at 2.90 Å resolution using MR with a TOP67 structure (PDB entry: 3PWT) as the search template. The full-length structure was completed with alternative cycles of manual model building and refinement Table . This reEcTOP1 has been previously described as a protein consisting of three domains, a 67 kDa N-terminal domain (TOP67), a zinc-binding domain and a 14 kDa C-terminal DNA-binding domain ,29,36–3832P end labeling and compared with the original O29 and O20 oligonucleotides used for co-crystallization. We confirmed that both intact strands were present in the crystals . A percentage (21%) of O29 was converted into its cleavage product (cleavage site five bases from the 3′-end of O29) by the EcTOP1 enzyme, likely after the crystals were dissolved in TE buffer for analysis.In the structure of full-length EcTOP1 in complex with DNA, the ssDNA segment is bound to TOP30C with its 3′-OH end primarily interacting with D5. The extended ssDNA also interacts with D7, D8 and D9 but not D6. Arginine 189 from TOP67 D4 contributes additional interactions with two phosphate groups of the backbone of the ssDNA chain. From the 3′-OH end a total of 11 nucleotides can be recognized and assigned a sequence (3′-GGGTTTAGCGT). The electron density for the ssDNA strand gradually becomes weak and smeared, particularly after its interaction with the last C-terminal domain D9. Two more sugar–phosphate repeats of the backbone of the strand are also visible and included in the model. The DNA present in the crystals was analyzed by gel electrophoresis with 5′-The overall arrangement of TOP67 in the full-length EcTOP1 is similar to what was observed previously in other TOP67 structures (e.g. PDB code: 1ECL)  Figure  result iE. coli topoisomerase III, a similarly charged loop, R454RDEEND, is also present  are 4-Cys zinc ribbon domains Figure . Each domerase I . The foumerase I . D5 has merase I  and 3, wmerase I .D5 makes few contacts with D4. There are only one hydrogen bond contributed by the first residue of D5 Q591) to D4 and a few van der Waals contacts between D5 and TOP67. However, it is D6 that forms extensive interactions with TOP67, mostly through its unique helix (α1 of D6)  was annealed using two oligonucleotides, O29 and O20  as well Figure . The aroAs mentioned earlier, D6 is not involved in ssDNA binding. Interestingly, this domain does not have an aromatic residue equivalent to F616 of D5, Y728 of D7 or F780 of D8 on the β3 strand  retained partial relaxation activity while a truncation further removing a C-terminal portion (corresponding to D6 and D7 based on the full-length structure) abolished relaxation activity completely  with nucleotides. To validate the importance of these stacking interactions at a distance from the active site, F616 was mutated to either a leucine (F616L) to retain hydrophobic interaction but eliminate π-stacking, or to a glutamate (F616E) to introduce a negative charge into the key ssDNA-binding site of the domain. Analysis of the relaxation activity of the F616L and F616E mutant enzymes  and the two 4-Cys zinc ribbon-like domains of the 14 kDa C-terminal fragment (D8–D9) bind the ssDNA portion of the predicted duplex DNA, while the dsDNA part of the oligonucleotide is completely disordered without any recognizable association with EcTOP1. In the single chain model Figure , the twoThe interactions between ssDNA and the C-terminal domains are dominated by multiple parallel and T-shaped π–π stackings between the bases of the ssDNA and the side chains of the aromatic residues from the C-terminal domains. Noticeably, the conserved aromatic residue from the β3-strand of each zinc ribbon domain, with a Zn(II)-binding site (D5 and D7) or not (D8), is the center of these π-stacking interactions of each domain. The importance of these π-stacking interactions in the catalytic mechanism of the enzyme is clearly illustrated by the reduction in relaxation activity from the F616 mutations introduced. In addition, some basic residues, arginines or lysines, contribute a couple of cation–π interactions to the bases of ssDNA. They also form salt bridges to some backbone phosphate groups.It should be noted that all these dominant π-stackings and cation–π interactions, together with van der Waals interactions mentioned earlier, between EcTOP1 TOP30C and ssDNA are independent of the ssDNA sequence. Only a few hydrogen bonds were observed in the ssDNA sequence and most of them appear nonspecific. For example, the residue N635, which forms two hydrogen bonds to the guanine G28, could also form at least one hydrogen bond with an adenine at the same position, and potentially with thymine or cytosine as well. The DNA sequence-independent interactions between TOP30C and ssDNA suggest that these domains can bind ssDNA of a wide range of sequences, as expected from the function of EcTOP1 in global gene expression. However, we cannot exclude any potentially preferred nucleotide sequences at given binding sites for maximal interactions between the C-terminal domains and ssDNA.The lack of sequence specificity for ssDNA binding to EcTOP1 C-terminal domains might cause problems in the trials of the co-crystallization of EcTOP1 and ssDNA. The success of EcTOP1/O29-O20 co-crystallization was likely due to (i) the right length of ssDNA in the substrate, which covered the C-terminal domains from D5 to D9; (ii) the presence of a dsDNA segment in the substrate, which does not bind the C-terminal domains and therefore prevents random binding registration of ssDNA along the C-terminal domains. This limited the binding mode of the substrate to the C-terminal domains and helped the co-crystallization with EcTOP1.The presence of Zn(II) has been proven to be critical to the structure and function of EcTOP1. In the In the proposed model for the DNA relaxation mechanism, the movement of the passing strand of DNA is guided by the C-terminal domains. We believe the interdomain flexibility of D7 to its preceding and succeeding domains and the coordination of the C-terminal domain movement and the opening-closing of the N-terminal toroid hole would greatly facilitate cycles of the passing strand of DNA entering and exiting the toroid hole for multiple processive cycles of removal of negative supercoils with only short pauses, as observed in single-molecule studies . The chaT. maritima topo I, have only one zinc ribbon domain that may or may not bind Zn(II). EcTOP1 and its most closely related bacterial topoisomerase I enzymes have additional zinc ribbon-like domains without the hallmark of four zinc-binding cysteines. In the EcTOP1/ssDNA structure, it is clear that both the 4-Cys zinc ribbon domains and zinc ribbon-like domains bind ssDNA in similar fashions involving primarily multiple π-stacking interactions without direct involvement of the Zn(II)-binding sites. Zn(II) binding appears to be important for the structural stability of a zinc ribbon domain, but is not always necessary for ssDNA binding. It is possible that during evolution when a Zn(II)-binding domain acquires other structural element(s) that may help its stable folding, it may lose its Zn(II)-binding site. D8 and D9 of EcTOP1 may be examples of such an evolution of a ssDNA binding domain, in which a unique helical hairpin linker eventually clamps the two domains when they lose Zn(II)-binding cysteines.The N-terminal domains (D1–D4) of type IA topoisomerase enzymes, found among protein family (Pfam)  members Actinobacteria, including Mycobacterium tuberculosis, Mycobacterium smegmatis -dependent ssDNA binding activity and may be involved in DNA repair  with accession code 4RUL.The atomic coordinates and structure factors of the full-length


The activity of beta-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is elevated during aging and in sporadic Alzheimer’s disease (AD), but the underlying mechanisms of this change are not well understood. p25/Cyclin-dependent kinase 5 (Cdk5) has been implicated in the pathogenesis of several neurodegenerative diseases, including AD. Here, we describe a potential mechanism by which BACE activity is increased in AD brains. First, we show that BACE1 is phosphorylated by the p25/Cdk5 complex at Thr252 and that this phosphorylation increases BACE1 activity. Then, we demonstrate that the level of phospho-BACE1 is increased in the brains of AD patients and in mammalian cells and transgenic mice that overexpress p25. Furthermore, the fraction of p25 prepared from iodixanol gradient centrifugation was unexpectedly protected by protease digestion, suggesting that p25/Cdk5-mediated BACE1 phosphorylation may occur in the lumen. These results reveal a link between p25 and BACE1 in AD brains and suggest that upregulated Cdk5 activation by p25 accelerates AD pathogenesis by enhancing BACE1 activity via phosphorylation. APP or presenilin genes produce more Aβ. However, the mechanisms underlying sporadic AD, which occurs in the majority of AD cases, are not well understood.Alzheimer’s disease (AD) is an irreversible, progressive brain disorder that is characterized by dementia. The brains of AD patients have pathological hallmarks, including amyloid plaques and neurofibrillary tangles, insoluble deposits made of proteins called Aβ and hyperphosphorylated tau, respectively. Beta-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1), a β-secretase, cleaves APP during an initial step in Aβ generation  and affinity-purified first with a cognate nonphosphopeptide affinity column and then with a phosphopeptide column (Peptron).BACE1 and the p25/Cdk5 complex were from Invitrogen  and Upstate Biotechnology  respectively. p25 and Cdk5 antibodies were obtained from Santa Cruz Biotechnology ; APP and phospho-APP antibodies, from Cell Signaling Technology ; anti-actin antibodies, from Sigma ; anti-BACE1 antibodies to the C-terminal peptide, from Chemicon International (Temecula USA), Calbiochem , and Santa Cruz;, and the peptide 296–310 (RLPKKVFEAAVKSIK) was custom-made . A phosphospecific BACE1 (P-BACE1) antibody was raised against a synthetic phosphopeptide ATP. The reaction mixtures were separated on SDS-polyacrylamide gels. For analysis by immunoblotting with a P-BACE1 antibody, BACE1 protein (0.32 μg) was incubated with the recombinant p25/Cdk5 complex (50 ng) in kinase buffer containing 200 μM ATP at 30°C for 2 h.For autoradiography analysis, purified BACE1 protein (1.5 μg) was incubated for 1 h at 30°C with the p25/Cdk5 complex (40 ng) in kinase buffer . Fluorescence was measured at an excitation wavelength of 320 nm and an emission wavelength of 405 nm using a SPECTRAmax Gemini XS fluorometer with Softmax PRO 4.3 LS software . The amount of Aβ peptide was measured using a sandwich enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s recommendations . For the measurement of Aβ in the brain, SDS (2%)-extracted hippocampal lysates were used . The amo2 in Dulbecco’s modified Eagle medium (DMEM) or DMEM/F-12 medium , respectively, supplemented with 10% fetal bovine serum (FBS). PC12(Tet-Off) cells were cultured at 37°C and 5% CO2 in RPMI1640 medium supplemented with 10% FBS, 5% horse serum (HS), and 100 μg/mL G418. Cells were transiently or stably transfected using Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocol. PC12(Tet-Off) cells stably expressing p25 were generated as described previously ATP. A band corresponding to the molecular size of BACE1 was detected by autoradiography only when both BACE1 and p25/Cdk5 were present in the reaction mixture (To determine whether the p25/Cdk5 complex can phosphorylate BACE1  mixture . This in mixture .252(PO4)PIRR (P-BACE1 antibody) . The BACE1 dimer was resistant to nonionic detergents and reducing and denaturing conditions, such as boiling in SDS, which suggests the existence of strong intermolecular interactions of BACE1 proteins. These results were consistent with previous reports on SDS- and boiling-resistant BACE1 dimers [An examination of the amino acid sequence of BACE1 revealed the presence of a consensus Cdk5 phosphorylation site at Thr252, which was followed by proline and basic amino acids that are conserved in humans, rats, and mice . To detentibody) . To detecovitine , indicatcovitine . In mouscovitine . This wacovitine  and 4. I1 dimers –33 and sTo explore the interaction between BACE1 and p25/Cdk5 proteins, co-immunoprecipitation assays were performed with the lysates of HEK293T cells transfected with plasmids encoding p25 and Cdk5. BACE1 was coimmunoprecipitated with Cdk5  and 13% ± 2% , respectively , whereas transient expression of the T252A mutant had little effect on the amount of Aβ C human neuroblastoma cells were transfected with either a control plasmid or with plasmids encoding full-length wild-type BACE1 (WT) and a phosphorylation-defective BACE1 mutant (T252A). The expression of WT BACE1 and BACE1T252A enhanced the BACE1 activity by 50% ± 10%  and 15% ± 2%  respectively  in the presence or absence of detergent, followed by immunoblotting analysis . Surprisin vitro experiments using purified BACE1 and p25/Cdk5 provide direct evidence that phospho-BACE1 increases BACE1 activity; (2) the BACE1T252A mutant inhibits BACE1 activity and basal secretion in cells compared to that of WT BACE1, suggesting that Thr252 phosphorylation is important for BACE1 activity/function; and (3) BACE1 phosphorylation, BACE activity, and Aβ levels are enhanced in p25-overexpressing cells, p25 TG mice, and human AD brains, supporting a link between phospho-BACE1 and BACE1 activity. BACE1 activity, but not the amount of BACE1, increases in humans, monkeys, and mice during aging, the major risk factor for AD [In this study, we demonstrate for the first time that BACE1 is phosphorylated by Cdk5. We conclude that BACE1 phosphorylation by p25/Cdk5 contributes to the observed increase in BACE activity based on the following data: (1) ion; and  BACE1 phr for AD , 14. Perr for AD . The phoin vivo proof for the link between BACE1 activity and p25 protein. Further investigations measuring BACE1 activity in BACE1 Thr252A knock-in p25 TG mice may further substantiate the functional importance of this phosphorylation event.The inconsistencies in reports of p25 levels in AD brain samples , 17, 19 Previous studies have shown that miscompartmentalization occurs in patients with AD, including mislocalization of extracellular heparan sulfate to the cytoplasmic compartment , 43; howOne possibility is that under cellular stress caused by p25 overexpression, autophagosomes containing various cytosolic proteins together with p25 may fuse with BACE1-containing endosomes, and p25/Cdk5-mediated BACE1 phosphorylation may then occur in the lumen of amphisomes. The autophagosomes can fuse with endocytic structures, such as multivesicular bodies (MVBs), generating the amphisome, which fuses with the lysosome to degrade the cytosolic materials . In AD b1–42 in p25 TG mice may disrupt endosomal/lysosomal membrane impermeability and cause membrane leakage [The second possibility is that cytosolic p25 and Cdk5 pass through leaky endosomal membranes and phosphorylate BACE1 in the lumen of endosomes because increased Aβ leakage , 52.1–42 in p25 TG mice may cause leakage of p25/Cdk5-containing small vesicles, and p25/Cdk5 can then phosphorylate BACE1 in the lumen of multivesicular endosomes. Previous studies have shown that Aβ1–42 is localized predominantly to MVBs in the AD brain [Another possibility is that p25/Cdk5 may be localized inside small vesicles of multivesicular endosomes by some unknown mechanism, perhaps in a similar way as the sequestration of glycogen synthase kinase 3 (GSK3) from the cytosol into MVBs upon Wnt stimulation . ElevateAD brain .An alternative possibility is that p25/Cdk5-mediated BACE1 phosphorylation may occur in the cytosol during or after translation of the BACE1 protein on free ribosomes, as reported in a previous study in which Dyrk2 was shown to be phosphorylated before leaving the ribosome . The N-tFrom the results herein, we propose the following mechanism for the regulation of BACE1 activity in AD brains. Aberrant Cdk5 activation by p25 upregulates BACE activity through enhanced phosphorylation of BACE1, and this process contributes to the pathogenesis of sporadic AD. Furthermore, the elevated p25/Cdk5 levels could contribute to the phosphorylation of STAT3, which in turn transcriptionally upregulate both BACE1  and γ-se


The allostery of P-selectin has been studied extensively with a focus on the Lec and EGF domains, whereas the contribution of the CR domain remains unclear. Here, molecular dynamics simulations (MDS) combined with homology modeling were preformed to investigate the impact of the CR domain on P-selectin allostery. The results indicated that the CR domain plays a role in the allosteric dynamics of P-selectin in two ways. First, the CR1 domain tends to stabilize the low affinity of P-selectin during the equilibration processes with the transition inhibition from the S1 to S1’ state by restraining the extension of the bent EGF orientation, or with the relaxation acceleration of the S2 state by promoting the bending of the extended EGF orientation. Second, the existence of CR domain increases intramolecular extension prior to complex separation, increasing the time available for the allosteric shift during forced dissociation with a prolonged bond duration. These findings further our understanding of the structure-function relationship of P-selectin with the enriched micro-structural bases of the CR domain. First, the different states of the Lec domain R3 loop resulted in distinct P-selectin-PSGL-1 interactions, where the unfavorable conformation of the S1’ state showed the weaker non-covalent interaction energy (middle) compared with those for the S1 (left) and S2 (right) states  and S1’ (3 runs) states than for the S2 (6 runs) state, and there was therefore no significant difference in interaction energy between S1 and S1’, even though the former showed slightly stronger interactions and a lower standard deviation compared with the S2 state (S1 (1G1Q/P-LECC-SGP-3), S1’ (1G1Q/P-LEC-SGP-3) and S2 (1G1S/P-LECC-SGP-3), six independent runs of forced dissociation under a constant force of 300 pN for each system were carried out with D158-Cα fixation of the EGF domain C-terminus. The bond dissociation time of S1’ decreased significantly compared with those of S1 and S2 states  states B. It is1 to S1’ , leadingS2 state B. Secon2 states C. The c1 and S2 .S1 state to S1’ state after gradual evolution over 2.5–8.0 ns   and the adopted conformation of the Lec domain R3 loop (cyan). The forward and backward order of the time-dependent evolution for the transition of the CR1 domain orientation, EGF domain orientation and Lec domain conformation could be observed intuitively during the dynamic process of 20-ns equilibration  B. It wac domain A-B. Howc domain C. The fentation G, cyan  R3 loop H, cyan.ibration .R3 loop from the S2 state (ns (purple) and the R3 loop conformation of the Lec domain (purple). Again, the forward and backward order of the time-dependent evolution for the transitions of the CR1 domain orientation, EGF domain orientation and Lec domain conformation could be observed intuitively during the dynamic process of 20-ns equilibration  for the CR1 end-fixed simulations (black) of the CR2 end-fixed system showed a peak force of ~ 600 pN (black) to ~ 50 Å for CR1 end fixation (red) and ~ 63 Å for CR2 end fixation (blue), for most dissociations of the 1G1Q/P-LECC-SGP-3 system (inserted left three arrows). The two exceptions to the force profiles (inserted right arrow). The observed intramolecular extensions were mainly due to the hinge opening of P-selectin during the normal dissociation process with intact domain structures , CR1 (red) and CR2 (blue) end-fixed dissociations of the 1G1S/P-LECC-SGP-3 system (inserted arrow) with intact domain structures.The force profiles of the 1G1Q/P-LECC-SGP-3 system under y 200 pN A. Both y 200 pN B and CRy 200 pN C simulaulations B, and t~ 600 pN C. Accorprofiles B-C exhiructures A, D, buructures B, C. Inructures E, CR1 eructures F and CRructures G simulawhite) and CR2-end fixed (gray) simulations compared with the EGF end-fixed (black) simulations of the 1G1Q/P-LECC-SGP-3 system (left three bars). The results for the 1G1S/P-LECC-SGP-3 system showed comparable dissociation times among the EGF end-fixed (black), CR1 end-fixed (white) and CR2 end-fixed (gray) simulations (right three bars). The transition time statistics for intermolecular separation (inserted arrows) revealed the same trends as those for dissociation time  corresponding to the transition of complex unbinding (red and blue) and the extension of the EGF domain (red) from the initial bent orientation occurred earlier than the allosteric shift (black) of the Lec domain, the allosteric shift took place differently. Because the external force acted on the EGF domain directly in the EGF end-fixed dissociation, the EGF orientation was disturbed immediately, which resulted in fast allostery of Lec domain R3 loop without intra-structural destroy of EGF domain , the 1G1Q/P-LEC-SGP3 system with an initial conformation of the S1’ state (middle two bars), and the 1G1S/P-LECC-SGP-3 system with an initial conformation of the S2 state (right three bars). Combined with the finding that the peak forces under cv-0.01 Å/ps forced dissociation processes were mostly below 300 pN for the 1G1Q/P-LECC-SGP-3 system but fluctuated approximately 300 pN for the 1G1S/P-LECC-SGP-3 system . This means that the first CR domain showed less independence than the other CR domains because Ala is the second smallest amino acid with a short side chain. The first CR domain uniquely exhibits two charged amino acids (Arg and Glu) immediately after the Val residue, which would increase the hinge interaction between the first CR domain and the EGF domain. The reduction of independence and the increase in the hinge interaction would enhance the ability of the first CR domain to regulate the EGF domain orientation and, moreover, the Lec domain conformation through its own movement. The observed regulation of allostery by the first CR domain was consistent with the experimental observation whereby increasing the flexibility of the Lec-EGF domain hinge induces an earlier transition from a catch bond to a slip bond, as the changed flexibility of the EGF domain induced by the CR1 domain was equivalent to the changed hinge flexibility of the EGF-Lec domain induced by hinge mutant [The allostery of the P-selectin Lec domain and the regulation of allostery through the orientation of the EGF domain have been proposed upon experiments  and alsoe mutant . In facte mutant .The occurrence of intramolecular extension prior to intermolecular dissociation during forced dissociation processes was predicted through MD simulations using P-LE systems for both hinge opening between the Lec and EGF domains and disruption of the secondary structure of the EGF domain ,34. WhilS1’, between S1 and S2. Our findings also revealed the regulatory ability of the EGF domain in relation to allostery through changes in its orientation and its long distance correlation with the Lectin domain, and predicted the function of the CR domain in the P-selectin allosteric shift during both equilibration and dissociation processes. Our results further the understanding of the structure-function relationship of P-selectin in the viewpoint of CR presence.Conclusively, this study further confirmed the allostery of P-selectin and the existence of a transition state, S1 FigA) The EGF orientation was defined by the relative angle of the target EGF domain to the reference EGF domain based on the alignment of the rigid parts of the Lec domain, which was quantified using the two vectors connecting the geometric center of the heavy atoms of the Lec-EGF hinge (residues A120 and S121) of reference structure to that of main EGF domain (residues C122 to T141) of reference structure (blue) and the target structure (silver). (B) The CR1 orientation was defined by the relative angle of the target CR1 domain to the reference CR1 domain based on the alignment of the EGF domain, which was indicated by the angle between the two vectors connecting the geometric center of the heavy atoms of the EGF-CR1 hinge (residues C159 and G160) of the reference CR1 domain to that of the CR1 domain (residues E161 to L217) of the reference (blue) and the target (silver) structures.((TIF)Click here for additional data file.S2 FigA) Conformational consistency among the homology-modeled structures of the first CR domain of human P-selectin upon different templates, which was quantified using the backbone Cα distances of every residue between the reference based on the template of 2RLQ and each of other five structures. (B) A typical conformation of the P-selectin first CR domain modeled based on the template of 2RLQ. The first residue of the N terminus and the last residue of the C terminus were presented in CPK format with a distance of 34.6 Å between their Cα atoms, and the β-sheet, loop and turn are illustrated in yellow, cyan and blue, respectively. (C) Conformational comparison between modeled P-selectin CR1 domain upon 2RLQ template (cyan) and crystallized E-selectin CR1 domain (purple), three disulfide bonds were highlighted in CPK and licorice, respectively. (D) Conformational difference between crystallized E-selectin CR1 (purple) and CR2 (silver) domains. (E) Orientation consistency of CR1 domain relative to EGF domain between modeled P-selectin (cyan) and crystallized E-selectin (purple) upon alignment of EGF domains. All structures were presented as in the NewRibbons format. The conformational comparisons  were all upon the backbone atom alignments of target regions. The E-selectin structure was adopted from PDB code of 4CSY.((TIF)Click here for additional data file.S3 FigA) Crystallized unligated (silver) (PDB code: 1G1Q) and SGP-3-ligated (blue) (PDB code: 1G1S) P-LE domains are superposed and are presented in newcartoon format. The loop from P81 to D89 is labeled as the R3 loop, and the calcium ion is illustrated in VDW. (B) The conformational differences in the R3 loop between 1G1Q and 1G1S are highlighted in topview for the Lec domain and are labeled as S1 and S2, respectively.  The conformational dynamics of the R3 loop during free equilibrations of P-LE domains from 1G1Q (C) and 1G1S (D) are quantified based on RMSD evolution via alignment to both 1G1Q (black) and 1G1S (blue). The conformational features are illustrated by superposing the typical 30 ns snapshots  or final states (G) with the original conformations of 1G1Q (silver) and 1G1S (blue). The conformational superposition shown in  and the RMSD calculations for (C-D) are all based on alignment of the alpha carbon atoms of the rigid regions of the Lec domain. The Lec domain, except for the R3 loop and calcium ion, is presented in transparent format for clarity in .((TIF)Click here for additional data file.S4 FigR3 loop with respect to the reference S1 (black), S1’ (red) and S2 (blue) states (left column), and the EGF orientation with respect to the references of crystallized 1G1Q (black) and 1G1S (red) (right column), were quantified for each of six repeated runs of the 1G1Q/P-LE systems.The RMSD of the Lec domain (TIF)Click here for additional data file.S5 FigR3 loop with respect to the reference S1 (black), S1’ (red) and S2 (blue) states (left column), the EGF orientation with respect to the references of crystallized 1G1Q (black) and 1G1S (red) (middle column), and the CR1 orientation in relation to the reference of the respective initial conformation (right column) were quantified for each of six repeated runs of the 1G1Q/P-LEC systems.The RMSD of the Lec domain (TIF)Click here for additional data file.S6 FigR3 loop with respect to the references of S1 (black), S1’ (red) and S2 (blue) states (left column), and the EGF orientation to the references of crystallized 1G1Q (black) and 1G1S (red) (right column) were quantified for each of six repeated runs of 1G1S/P-LE systems.The RMSD of the Lec domain (TIF)Click here for additional data file.S7 FigR3 loop with respect to the references of S1 (black), S1’ (red) and S2 (blue) states (left column), the EGF orientation to the references of crystallized 1G1Q (black) and 1G1S (red) (middle column), and the CR1 orientation to the reference of respective initial conformation (right column) were quantified for each of six repeated runs 1G1S/P-LEC systems.The RMSD of the Lec domain (TIF)Click here for additional data file.S8 FigA, B) First (A) and second (B) runs of the CR1 end-fixed dissociation process.  First (C) and second (D) runs of the CR2 end-fixed dissociation process.((TIF)Click here for additional data file.S9 FigThe interaction energy of each trajectory was averaged first, and the results were then averaged over the different equilibration simulation runs. Consequently the interaction energy between the Lec and EGF domains was the average of the first two repeated runs for all 0CR, 1CR and 2CR sets. The interaction energy of the EGF-CR1 hinge was the average of the first two repeated runs of all 1CR and 2CR sets, and that of the CR1_CR2 hinge was the average of all 2CR set. The data were presented as the mean ± SD.(TIF)Click here for additional data file.S1 Movie(AVI)Click here for additional data file.S2 Movie(AVI)Click here for additional data file.S3 Movieleft), SGP-3 ligand unbinding and allostery of the Lec domain (middle), and evolution of the EGF domain orientation (right) were highlighted, respectively.Whole molecular extension ((AVI)Click here for additional data file.S1 Table¶: ≥10%.Blast score represents the sequence similarity between the first CR domain of P-selectin first CR and respective template, and the other data represent the percentage of non-optimal stereochemistry structures. *: ≥ 5.0%; (DOCX)Click here for additional data file.S2 Tableblue boxes, and partially conserved sites are indicated with red boxes. Three disulfide bonds are formed in each CR domain between 1–5 cysteines, 2–3 and 4–6 cysteines. (B) Disulfide bond distributions for various homology modeling templates. The templates with two disulfide bond pairs which close to the human P-selectin CR domain are highlighted with gray boxes.Fully conserved sites throughout the nine domains are indicated with (DOCX)Click here for additional data file.


Previous studies suggested that expression of cyclin-dependent kinase 5 (CDK5) may promote the migration and invasion of human glioma cells. In this study, we aimed to evaluate the clinical value of CDK5 in different grades of glioma in relation to Ki-67 labeling index (LI).We firstly assessed by immunohistochemistry the expression of CDK5 in 152 glioma tissues and 16 normal brain tissues and further explored the relationship between CDK5 expression and other clinical features.P = 0.001). Difference of CDK5 expression among four World Health Organization (WHO) grades was statistically significant (P = 0.001). The significant differences of CDK5 expression were also observed between WHO I glioma (34.8 %) and WHO III glioma (62.5 %), as well as WHO IV glioma . Furthermore, Spearman’s rank correlation confirmed that CDK5 was positively correlated with the pathological grade of glioma . The CDK5 expression was also positively correlated with Ki-67 LI .The positive ratio of CDK5 in gliomas (57.2 %) was higher than that in normal brain tissues (12.5 %, The current result suggests that CDK5 may play an essential role in the tumorigenesis and aggressiveness of gliomas. Gliomas, the most frequent intracranial malignant tumor, are categorized as grade I to grade IV based on histological and clinicopathological criteria, as provided by the World Health Organization (WHO) . Gliomas2+-dependent protease calpain to p25, which aberrantly activates CDK5, thereby promoting the phosphorylation of substrates implicated in neurodegeneration [Cyclin-dependent kinase 5 (CDK5), which is a proline-directed serine/threonine kinase, expresses predominately in mature neurons and is involved in neurite extension, neuronal migration, and neuronal differentiation . Cellulaneration , 9. Actuneration –13. Prevneration . Additioneration . More reneration . These aThis retrospective study included a total number of 152 glioma samples that were surgically resected from the First Affiliated Hospital of the Guangxi Medical University  during the period from January 2008 until September 2013. The patients in the current study included 106 males (69.7 %) and 46 females (30.3 %) with a mean age of 40.50 ± 16.6 . According to the classification criteria of nervous system tumors , 23 cases were WHO grade I as pilocytic astrocytomas, 44 cases were grade II , 56 cases were WHO III (including 42 cases of anaplastic astrocytoma and 14 cases of anaplastic oligodendroglioma), and 29 cases were WHO IV . Another 16 cases of normal frontotemporal brain tissues under intracranial decompression were collected from the First Affiliated Hospital of the Guangxi Medical University as a control group. Thirteen male and three female individuals were enrolled in the study as healthy controls, with ages between 23 and 59 years old (with the median age of 48 and mean age of 43.75 ± 15.34). The clinical data was obtained from the patients’ medical records and included age, gender, and WHO grade.Polyclonal antibody anti-CDK5  and monoclonal antibodies Ki-67  were used to perform the immunohistochemical detection as previously described . To scorH test was performed to detect the differences between CDK5 expression and pathological grades of glioma. The significance of difference of LI between two groups was analyzed by unpaired Student’s t test. The statistical significance of the difference of LI between more than two groups was assessed by using ANOVA test. The predictive values of CDK5 and Ki-67 LI in diagnosis and tumor differentiation were evaluated by the receiver operating characteristic (ROC) curves. Spearman’s correlation was used to study the correlation between CDK5 expression, Ki-67 LI, and tumor grade. P values were two-sided, and significance level of P < 0.05 was set up in all tests.For statistical analysis, SPSS 20 software was used. The difference of CDK5 expression between two patient subgroups was determined by using chi-square test. Kruskal-Wallis Immunohistochemical staining results showed that CDK5 protein was expressed in glioma cell cytoplasm Fig. .Fig. 1ExZ = −3.400, P = 0.001). The CDK5-positive expression in WHO III glioma (62.5 %) and WHO IV glioma (82.8 %) was observed to be higher when compared to WHO I glioma .CDK5-positive expression (57.2 %) was higher in glioma tissues than in normal brain tissues . Ki-67 LI also showed significant differences among the four WHO grades . The Ki-67 LI also showed a significant difference between CDK5-positive group and CDK5-negative group .The result of Ki-67 LI was shown in Table P = 0.003). CDK5 expression level significantly contributed to diagnosing high-grade glioma . The ROC curve indicated that Ki-67 LI ≥ 0.7 could significantly diagnose gliomas with 93.42 % sensitivity and 75 % specificity and with AUC as 0.903  0.610, 0.837; Glioma encompasses different histological subtypes with high variability in prognosis and accounts for almost 80 % of primary malignant brain tumors . At presn = 152) tested. Consistent with our study, CDK5 expression was observed to be overexpressed in NSCLC and breast cancer, which implies that CDK5 serves as an important role in the tumorigenesis of different malignancies [CDK5, one of the major kinases activated by its regulators p35 or p39, directly phosphorylates various residues and simultaneously regulates various substrates . Evidencgnancies , 25. Evignancies –28. Moregnancies , 29. CDKgnancies . With fugnancies . Later, gnancies . Researcgnancies . In lighFurther, we continued to investigate the relationship between CDK5 expression and clinicopathological factors of gliomas, mainly the tumor grades. In the current study, we observed that the increasing expression of CDK5 protein was significantly related to the pathological grade in our set of 152 glioma cases. In accordance with our study, Catania et al. conducted the study that CDK5 expression in the astrocytomas of grades II–IV was consistently stronger than that in a single grade I pilocytic astrocytoma, which was the first study to examine CDK5/p35 expression in human astrocytic tumors of varying grades. Nevertheless, the study only enrolled a small size of 12 human astrocytic tumor specimens . Liu et Our results suggest that CDK5 may represent a valuable predictive marker of tumorigenesis and progression in glioma. Larger studies are still desirable in the future to provide stronger evidence for CDK5 as a candidate diagnostic and prognostic biomarker of glioma.


Polycomb repressive complex 2 (PRC2) silences gene expression through trimethylation of K27 of histone H3 (H3K27me3) via its catalytic SET domain. A missense mutation in the substrate of PRC2, histone H3K27M, is associated with certain pediatric brain cancers and is linked to a global decrease of H3K27me3 in the affected cells thought to be mediated by inhibition of PRC2 activity. We present here the crystal structure of human PRC2 in complex with the inhibitory H3K27M peptide bound to the active site of the SET domain, with the methionine residue located in the pocket that normally accommodates the target lysine residue. The structure and binding studies suggest a mechanism for the oncogenic inhibition of H3K27M. The structure also reveals how binding of repressive marks, like H3K27me3, to the EED subunit of the complex leads to enhancement of the catalytic efficiency of the SET domain and thus the propagation of this repressive histone modification. Polycomb repressive complex 2 (PRC2) silences gene expression through trimethylation of K27 of histone H3 (H3K27Me). Here, the authors report the structure of the human PRC2 complex bound to the oncogenic H3K27M mutant, and suggest a mechanism for its potency in childhood brain cancers. Drosophila development. A second WD40 repeat protein RbAp46/48 binds to this catalytic core to target nucleosomesEpigenetic mechanisms play an important role in setting and maintaining gene expression profiles that define the fate of a cell2In addition to crystallographic data on peptide binding by EED, important insights into the PRC2 mechanism have come from a number of structures of other components of the complex. Structures of RbAp48 subunit, and homologues, in complex with a SUZ12 peptide and a histone H3 peptide helped explain PRC2 recognition of its nucleosomal substrate11The gain of function mutations in the SET domain of EZH2 were found to be associated with a subtype of B-cell lymphomas, follicular lymphoma and melanoma1517Chaetomium thermophilum, has been reported in both an activated and unactivated conformation with a peptide representing H3K27M bound to the SET domain. Here we present the crystal structure of the human PRC2 with bound H3K27M peptide that, in contrast to the C. thermophilum structure, shows the substituted methionine residue occupying the lysine access channel of the catalytic SET domain supporting a model of inhibition consistent with the known biochemical data. The structure also suggests a mechanism for the allosteric activation of PRC2 by neighbouring repressive marks.A recent landmark achievement was the report of a crystal structure of an active fragment of PRC2 ref. . The strC. thermophilum, which were subsequently used to guide final building. For clarity, in our description of the human structure, we have adopted, where appropriate, the domain nomenclature used in the earlier paper. A detailed comparison between the human and C. thermophilum PRC2 structures is presented in We have solved the crystal structure of a human PRC2 complex that contains the essential elements of the EZH2 and EED polypeptides with the Vefs fragment of the SUZ12 subunit . In addiThe complex forms a compact arrangement of three lobes with overall dimensions of approximately 120 × 75 × 50 Å . The firN lobe. The first subdomain of EZH2, termed SBD, forms a long helix that packs against the SANT1 domain, the final subdomain of the EZH2 N-terminal fragment, completing a four-helix bundle. Thus, the first and last of the domains from the EZH2 (N-domain) interact to close the ‘lasso' that EZH2 makes around the EED subunit. The EBD and BAM domains, helical and β-structures respectively, pack against the bottom and side of EED and are currently of unknown function. The function of the next two elements of EZH2, referred to as SAL and SRM, is clearer. Together they link one side of the SET domain to the EED subunit and communicate the binding of repressive trimethyl signals to EED to the catalytic centre of the SET domain, stimulating activity. The detailed mechanism of this process is discussed below. The SAL chain runs between the SET, EED and Vefs domains before switching back, passing behind the SET-I helix and forming an extended chain and helix (SRM) that sits across the top ‘open end' of the EED barrel making extensive interactions. The SRM helical segment runs adjacent to the Jarid2 peptide at the top of EED with the main chains about 8 Å apart and making extensive side-chain interactions between the two segments. Similarly, most of the contacts made by the SRM helix with the SET-I helix are mediated by side chains.Middle lobe. The middle lobe probably has regulatory and targeting functions. For example, a helical segment (residues 330–343) at the C-terminal of the SANT2 domain is known to be involved in RNA bindingN construct and the SANT2 from the beginning of the EZH2C construct into an extended helical domain. There are two zinc-binding motifs in the middle lobe; one (Zn1Cys3His1) is formed solely by MCSS and the second (Zn1Cys4) by two cysteines contributed by SANT2 and two from the MCSS domain.Catalytic lobe. The C terminus of EZH2 contains the pre-SET and SET domains; the former is characterized by two zinc-binding motifs, both containing three Zn2+ ions but with distinct coordination—the first Zn3Cys8His1 and the second Zn3Cys9. This cysteine-rich pre-SET domain makes extensive interactions with the Vefs helical domain and the MCSS domain on one side and with the SET domain on the other. The position and structure of the SET domain in the complex has features that explain the basis of allosteric activation by repressive mark binding to the EED, and suggests a mechanism for oncogenic inhibition.Given that even low level of expression of the histone variant H3K27M is oncogenic and prevents methylation of wild-type histone by PRC2, we determined the structure of human PRC2 with a histone peptide containing the H3K27M substitution. There is unambiguous electron density, in unbiased Fourier maps, for residues 22 to 30 of the peptide . Signifi22Kd=3.3 μM) compared with wild-type  when the Arg26 is substituted with an Ala , with the tryptophan side chain oriented away from the SET domain into a hydrophobic pocket composed of residues Val-107 to Met-110 from the EZH2 SAL, Phe-566 to Ser-568 from SUZ12 Vefs, and Leu-616 to Pro-618 from SET-N and Phe-686 from SET-I . In the The EZH2 SET-I region in the human PRC2 complex adopts an anti-clockwise rigid-body rotation relative to its position in isolated structures as viewed in Finally, the structures of the EZH2 post-SET regions, from the isolated and complex forms, differ substantially after residue Tyr-726 . In the C. thermophilum and human PRC2, reveals that the SET-I region in the former case is significantly displaced outwards, away from the active site  residue is oriented away from the SRM ((EED) and the carbonyl of the trimethyl lysine residue hydrogen bonds with Arg-414(EED). In addition, the side chains of alanine (−3) and phenylalanine (+1) sit in shallow hydrophobic pockets on the surface of EED. The alanine at (−3) also packs against Ile-131 of the extended strand of SRM (The side chain of Arg(−1) , makes a salt bridge to acidic residues located in the turn between the SRM extended chain and helix, and two hydrogen bonds with glutamine side chains from the peptide at the (−4) and (+3) positions . The imp the SRM , reflect the SRM . The pepd of SRM  providind of SRM , have an(SET), the aliphatic moiety of Lys-660(SET) and the phenolic ring of Tyr-661(SET). Additionally, Lys-660(SET) makes a salt bridge with Asp-142(SRM) and Lys-656(SET) makes a hydrogen bond with Asn-152(SRM).The stabilization of the SRM by peptide binding to EED leads to activation of EZH2 through the SET-I . The intC. thermophilum PRC2, determined in the presence and absence of EED bound trimethyl lysine peptide, reveals that the extended chain and helix of the SRM are disordered in the absence of peptide. There is very little change in the position of the SET-I helix but the analysis of normalized thermal factors . Thus, the observed levels of stimulation of PRC2 activity is consistent with the stabilization of the SET-I structure, and its contribution to the active site of the enzyme, and does not necessitate a substantial conformational change in the SET domain or its binding partners. As in the other SET domains, the PRC2 SET-I region contributes many of the residues that interact with the substrate peptide , removing the linker between the end of MCSS and the beginning of SANT2, thus 28 residues are missing from the middle lobe as indicated in The human PRC2 core was co-expressed in Methyltransferase assays−1 with 1:10 molar ratio H3K27M peptide, 1:5 molar ratio Jarid2 peptide and 1:10 molar ratio SAM. The crystals were grown at 18 °C using the vapour diffusion technique in MRC sitting drop plates (Hampton Research). The drops were prepared by mixing equal volumes of PRC2 complex with a reservoir solution containing 400 mM ammonium citrate pH 6.5 and 25% PEG3350. The crystals were transferred into reservoir solution containing 20% ethylene glycol before flash cooling in liquid nitrogen. The diffraction data were collected at Diamond Light Source on beamline IO2 at wavelengths 0.980 and 1.283 Å (Selenium and Zinc edges). The data were integrated using XDSPurified PRC2 complex was prepared with H3K27M peptide (H3 residues 22–42), trimethylated K116 Jarid2 peptide (residues 111–121) and S-adenosylmethionine (SAM). The complex was prepared at a protein concentration of 4.8 mg mlrOBS) is given by:Equilibrium dissociation constants for the interaction of PRC2 (P) with FAM-H3K27M peptide (L) to form the complex PRC2:FAM–K27M (PL) were determined using anisotropy titrations. For any mixture of PRC2 and FAM-H3K27M, the observed anisotropy  is the fluorescence intensity of the complex divided by that of the free peptide. The data were analysed using nonlinear least-squares fits to rPL, rL and the dissociation constant  as variables, and PL and L calculated in the usual way:where where the subscript 0 indicates the total concentration of the species.Equilibrium dissociation constants for the interaction of PRC2 (P) with the unlabelled peptides (K27M and R26AK27M) were determined using a displacement assay in which aliquots of the unlabelled peptide (N) were added to a preformed PRC2:FAM–K27M complex. In the case of unlabelled K27 methylation could be a factor. We therefore prepared separate solutions of the pre-formed complex for each point of the titration. We monitored anisotropy over time and noted that the reading remained stable for at least 60 s following addition of the K27. The anisotropy reported was taken within 15 s and we are therefore confident that methylation is not affecting the data. The data were analysed using nonlinear least-squares fits to The free protein concentration ([P]) is the root of the cubic equation:whereC3=1C2=−[P0]+Kd,L+Kd,N+[L0]+[N0]C1=−[P0]Kd,L−[P0]Kd,N+Kd,LKd,N+[N0]Kd,L0=−[P0]Kd,LKd,NCKd,N is the equilibrium dissociation constant for binding of N to PRC2 and [N0] is the total concentration of N. PL and L are then calculated using:where All anisotropy titrations were performed at 20 °C using a Jasco FP-8500 fluorimeter. The buffer (25 mM Hepes (pH 7.25), 25 mM NaCl, 1 mM TCEP, 0.01% Brij-35) contained SAM, SAH or SAM plus trimethylated K27 peptide as required.Accession Code: The structure is deposited with the PDB under accession code 5HYN.How to cite this article: Justin, N. et al. Structural basis of oncogenic histone H3K27M inhibition of human polycomb repressive complex 2. Nat. Commun. 7:11316 doi: 10.1038/ncomms11316 (2016).Supplementary Figures 1-6 and Supplementary Table 1.


Yersinia pestis) but existing methods are not yet accurate enough to inform public health planning. The present study aimed to identify characteristics of individual mammalian host (Rhombomys opimus) burrows related to and potentially predictive of the presence of R.opimus and the dominant flea vectors (Xenopsylla spp.).The wildlife plague system in the Pre-Balkhash desert of Kazakhstan has been a subject of study for many years. Much progress has been made in generating a method of predicting outbreaks of the disease (infection by the gram negative bacterium Over four seasons, burrow characteristics, their current occupancy status, and flea and tick burden of the occupants were recorded in the field. A second data set was generated of long term occupancy trends by recording the occupancy status of specific burrows over multiple occasions. Generalised linear mixed models were constructed to identify potential burrow properties predictive of either occupancy or flea burden.Rhombomys, and remaining occupied, were both related to the characteristics of the sediment in which the burrow was constructed. The flea burden of Rhombomys in a burrow was found to be related to the tick burden. Further larger scale properties were also identified as being related to both Rhombomys and flea presence, including latitudinal position and the season.At the burrow level, it was identified that a burrow being occupied by Therefore, in advancing our current predictions of plague in Kazakhstan, we must consider the landscape at this local level to increase our accuracy in predicting the dynamics of gerbil and flea populations. Furthermore this demonstrates that in other zoonotic systems, it may be useful to consider the distribution and location of suitable habitat for both host and vector species at this fine scale to accurately predict future epizootics. Yersinia pestis infection) in modern human populations, historical outbreaks such as the Black Death across medieval Europe clearly demonstrate the disease’s devastating potential [Despite the relative rarity of plague  by a variety of fleas, although Xenopsylla spp. generally dominate. R.opimus is the dominant rodent in the landscapes of the Pre-Balkash area, inhabiting complex burrow systems as extended family groups. Previous work has identified that plague spreads through gerbil populations in a manner well described by percolation theory [The plague system in the Pre-Balkhash region of Kazakhstan has been subject to extensive study over the past 15 years. The desert and semi-desert belt across the region consists of a vast area of mixed vegetation and shrubs that extends from the Caspian Sea in the west to the Chinese border in the south east of the country, in the Almaty province. The northern desert subzone of the Almaty province is the largest of 20 endemic plague foci in the country. The hosts from this focus have been recently suggested to have had involvement in initiating the European outbreaks of plague in the 1300’s , 7. Acron theory . Importan theory  in predin theory . HoweverR.opimus burrows are visible on satellite images due to depletion of vegetation in the active  centre of the burrow, and the surrounding area where activities are most intense [ intense , 11, 12. intense  and prov intense . The gre intense  and as s intense , 9.Y.pestis. Further, for plague to spread across a landscape, burrows occupied by the host must be distributed with sufficient connectivity to permit percolation through the population. Preference for burrows with specific attributes may cause clustering of burrows supporting gerbils and fleas within the landscape, with extensive gaps in between, potentially preventing long distance spread of plague [The ultimate aim of the present study is to improve our ability to predict plague incidence in the Pre-Balkhash region, through increased ability to predict the presence of the rodent host and the abundance of the flea vector at the level of the individual burrow. Given their importance for the presence of plague, we can consider that a burrow that is highly preferred by both the host and vector may be a perfect burrow for f plague .R.opimus burrow properties and ecological preferences have been reported previously [Descriptive accounts of eviously . More reeviously ). HoweveHaloxylon and Calligonum species.The study sites are located in the Pre-Balkhash endemic plague focus in south eastern Kazakhstan . The caIn four seasons  2011, summer (July) 2012, autumn (September) 2012 and spring (April-May) 2013), all burrow systems located within randomly chosen 500m x 500m (25ha) squares spread over the 6 sectors were mapped. In total, 2113 burrows were mapped. Of these, occupancy status was recorded twice (in two seasons) for 894 burrows and just once for 1219 burrows. The GPS location of the burrow system was recorded along with its altitude and its occupancy status.Individual burrow systems were identified by locating the central burrow structures and the outer limits of each discrete burrow unit . For each burrow a description of its occupancy status and the landscape of the area surrounding the burrow was created. If direct evidence of gerbils was found at the burrow  then it was classified as occupied. Signs of gerbil activity limited to feet/tail markings in the sand at the entrances of burrows would classify a burrow as visited. If no gerbil activity was identified then the burrow was classified by its condition; if in usable condition but otherwise not inhabited the burrow was classified as unoccupied, varying states of disrepair would identify the burrow as old or disappeared.A landscape description of the burrow was then generated. This was based on visual inspection of landscape properties of the local area in which the burrow had been constructed, in particular guided by granulometry to classify sediments. Classification of landscape properties was thus conducted by methods detailed in Pogrebinsky (1963) , 21 and R.opimus) were caught using local traps placed at burrow entrances; trapping procedures and laboratory protocols were performed according to Russian standard methods [Y.pestis. Further details of the laboratory techniques can be found in the supplementary material  was collected to examine occupancy over multiple seasons. Methods differed little from those above except that the squares monitored were 200m x 200m, and rodents were not trapped. 513 unique burrows were revisited in each of six seasons  and their occupancy recorded each time. Some of these burrows were trapped as per the protocol above in Spring 2013 and these records added to the main data set.All animal trapping and handling procedures were standard practice for Kazakh fieldworkers, requiring no specific permission and none involved endangered or protected species. These procedures were additionally approved by the University of Liverpool Animal Welfare Committee.A unique burrow landscape description was generated for each of the 2113 mapped burrows. Some of the landscape characteristics are mutually exclusive, but many can occur together and so as many characteristics as were needed to fully describe the burrow were noted in each case. The resulting data set was comprehensive and highly detailed, but the large variety (171 unique descriptions) of individual burrow landscape descriptions required condensing in order to identify key aspects of the landscape linked with the outcome variables .a priori indication of the coarseness of landscape description relevant to the outcome variable. Therefore an initial exploratory quantitative analysis, designed specifically for this data set, was conducted to identify general trends in the outcome variables across different landscape descriptions and groups of characteristics. For the two general outcome variables  the level of an outcome response associated with each of the burrow landscape descriptions across the data set was estimated. For fleas this was measured as the mean flea index associated with burrows possessing each landscape description, and for occupancy as the proportion of burrows possessing this description that were occupied. In each case this was normalised by the number of burrows possessing each description to avoid over-representation of the effect of burrow properties that were simply common throughout the landscape.Landscape descriptions could be considered at their finest scale  or at a coarser level (the burrow is on a dune). There was little All of the unique individual burrow descriptions in the data set were ranked by their level of outcome response . The ranked descriptions were then split into a high, medium and low group by their associated response level. These groups each contained roughly one third of the descriptions, with some allowance for groups of descriptions with the same level of response to be grouped together. For each of these groups, the proportion of descriptions in the group containing each of the 15 characteristics was calculated. Where the proportion of descriptions containing a particular characteristic differed by approximately 5–20% between the high and medium or low and medium groups, the characteristic was considered either as a predictive landscape factors to be used in statistical analysis or for inclusion within a predictive factor, (for more details see the supplementary document ). In theGeneralised linear mixed models (GLMMs) were used to explore the relationship of the model variables described in In particular for models of occupancy, apart from individual landscape factors, the interaction (and respective main effect terms) between burrows being on clay sediment and the presence of dunes was included to test the hypothesis that their potential advantages  would make their combined presence of greater benefit. Tree presence was included to test the hypothesis that trees are indicators of moisture concentration in the environment, and therefore in turn indicate fertile areas with ready food supply. For models of flea burden, the tick index was tested as a predictor to test whether certain burrows were more likely to be generally infested with invertebrates. In addition the percentage of burrows occupied in the surrounding sector (in the same season as the flea record was taken) was tested as a predictor of flea index to identify whether the flea burden was related to gerbil abundance.The field recording of occupancy status was condensed to a binary record of occupied or unoccupied . GLMMs using a binomial error distribution were constructed to predict occupancy for the full data set of 3011 observations (i.e. trapping events) of 2113 unique burrows, including repeat visits. Additionally, as multiple visits to sectors were not randomised, both burrow identity and sector were included as random effects (with burrow identity nested by their relevant sector).A second GLMM was constructed to identify factors predictive of burrows remaining occupied using the long term occupancy data set, using, as the dependent variable, the proportion of the six monitoring periods where each of the revisited burrows was occupied. As above, sector was included as a random effect. The same selection of landscape factors was used in both cases.Xenopsylla spp. dominated as anticipated (making up 98.9% of the observations). A GLMM was constructed using records of flea burden, using a poisson error distribution, and using a quasi-poisson model as a correction of standard errors due to over dispersion in the data [The data set contained 999 observations of flea burden, of which the data , while iIn each case, a model was first constructed using all of the burrow level landscape traits hypothesised to be related to the outcome. This model was minimised as detailed below. A second model was then built using the significant landscape factors with the addition of further environmental factors, as detailed in However, while the AIC scores provide a method of comparing the different models, in each case they are estimated for the non-mixed version of the model and so we consider them to be approximate measures of the fits of the models with random effects included. They were therefore considered alongside the complexity of the model and the significance of the parameters. Specifically, simpler models, and those where all terms were significant, notwithstanding AIC score, were prioritised as detailed below.In the final parsimonious static occupancy and flea models, checks of the nested random effects structure were performed by iteratively running the model, using a subset of the data consisting of one record from every instance where a burrow was sampled more than once, alongside all records of burrows only recorded on one occasion. The range and average of the coefficients and P values in these models were compared to those produced by the models using the nested random effects . These All statistical analysis was conducted using the R statistical computing environment , using tOf the terms included in the landscape factor model, the interaction term between clay sediments and dunes was found to be significant. However the ΔAIC between the models with and without the interaction term did not differ significantly . Hence, the simpler model without the interaction term was chosen as a minimal landscape model. Within this only the clay sediment term was significant and this was carried forward to the second model. Of the additional terms then tested, latitudinal position, the presence of trees and season were found to be significant. Backwards selection did not suggest removal of any of these would create significant improvement in the AIC scores, and as all terms were significant this was maintained as the minimal model. All of the model terms were validated by the iterative sampling method . DescripThe same landscape factors were tested as in the occupancy model. The interaction term between clay sediment and dunes, and its constituent main effects were found to be significant alongside sand. Here the model with the interaction had a significantly lower AIC  compared to the simpler model with just the main effects, and so as the terms were significant this was taken as the minimal landscape model. Of the additional environmental factors tested alongside these landscape factors, only latitude was found to be significant. The model including the non-significant environmental terms  was significantly better in terms of AIC , but the reduced model was selected due to its increased simplicity and exclusion of insignificant terms. Again, backwards selection did not indicate that removal of any of these terms would significantly improve the model. Descriptions of these relationships, and their associated coefficients are detailed in Of the four landscape factors tested, only clay was found to be a significant predictor. Of the additional environmental factors alongside clay, only the tick burden and season were found to be significant. The model including all of the additional environmental factors  had a significantly lower AIC , but the simpler model with only significant parameters was taken as the final parsimonious model as detailed in Here we have identified that both static gerbil occupancy, and the maintenance of burrow occupancy in the long term are related to the burrow’s latitudinal position. Further, static occupancy is found to be related to the presence of trees, clay soils and the season, and the maintenance of occupancy is related to the presence of sand, and an interaction between the presence of clay sediments and dunes.G. busarius prefer sandier loam sediments, T.bottae and P.castanops prefer clay-loam sediments [Dipodomys genus (kangaroo rats) construct longer, more complex burrows where there are higher percentages of silt, and Microtus montanus (Montane vole) construct deeper, narrower burrows in firmer sediments with more sand [Previous research examining burrow location preferences and burrow construction in a range of fossorial mammals has identified connections between sediment properties and both the resulting burrow structure and the likelihood that a burrow is occupied. In particular, at the genus level members of the Geomyidae family harbour preferences for burrows with particular environmental properties; for example, while ediments –33. Addiore sand . Other sore sand .Our results identified that when observed in a single season, burrows in clay sediments are less likely to be occupied. Digging new burrows incurs a cost in any fossorial species, but over the lifespan of the animal this is generally low in comparison to the resultant benefits created by the burrow . The optIn contrast, although neither clay nor dunes on their own had a significant relationship with long term occupancy, the significant interaction term between them indicated that burrows in clay sediments are less likely to remain occupied for longer periods when there are also dunes present, and conversely that burrows in areas with dunes are less likely to remain occupied where clay is also present. The potential benefits one might imagine of sturdier clay soils, and those observed in previous studies  may becoMeriones crassus burrows constructed in sediments with artificially controlled proportions of sediment types, those constructed in the presence of high proportions of sand result in a higher average temperature of the burrow microclimate compared to other substrate compositions, suggesting sand plays a role in the insulation of the burrow [We also observed a significant positive association between a burrow being located in an area of sand and long term occupancy. In e burrow . Our resThe significant relationship with latitude may reflect the effect of an unmeasured environmental variable that changes along a latitudinal gradient (such as climate), or indeed unmeasured isotropic spatial variation. Gerbil occupancy across the region is known to fluctuate , but theMicrotus pinetorum, identified preference for moderate soil moisture, likely to be a balance between digging ease and risk of flooding [The positive relationship between the presence of trees and static occupancy is perhaps suggestive of a necessary level of moisture (supporting both tree growth and occupancy) and/or protection from excess heat through shading, the latter fitting with previous observational records . In addiflooding . Albeit flooding . Bioturbflooding . Thus, tflooding .Lower occupancies in spring in particular are likely simply to reflect the natural fluctuations of gerbil populations, where numbers are generally highest in autumn , followXenopsylla spp. are the most important [Coptopsylla lamellifer playing a role in winter transmission) and are the dominant species in our data set. The different species themselves differ in their seasonal dynamics and details of their habitat requirements. Here, latitude, season and tick burden associated with a burrow were identified to be significantly related to the burden of fleas in the burrow.Although several flea species in this system are potential plague vectors, mportant ,  and Argasidaee ticks  in Central Asian desert foci [That tick burden is a significant predictor of flea burden suggests that there may be burrow conditions not monitored here that create conditions permissive of a wide variety of ectoparasites and that are conducive to the spread of plague. Alternatively, since the flea and tick measurements relate to numbers found on individual gerbils, the association may reflect characteristics of these individuals rather than the burrows in which they were trapped. Certainly, flea infestation is known to affect the metabolism of the mammalian host . Furtherert foci –49, and ert foci .Finally, migrating fleas may provide insight into the specific role of fleas in determining the timing and size of epizootics. A theoretical study based on the American prarie dog plague system identified a potential role for migrating fleas in enzootic persistence between epizootic events . HoweverY.pestis epizootic events to spread and be sustained, both the mammalian and insect host must be present across the landscape. This would then suggest that a burrow in the northern region, with surrounding vegetation, and perhaps located in a sandy areas, would be at the highest risk for plague presence given the significant positive relationships between these properties and host and vector presence identified here. Plague prevalence during the study period was not high enough to test this directly. There may also be other factors that need to be present or absent for a burrow to be permissive of Y.pestis itself. The question also remains of where the bacterium survives in interepizootic periods , which may itself be controlled by abiotic environmental conditions. Overall, this study gives finer resolution to our ability to predict distributions of both of the key host and vector species in this system, so improving our ability to predict plague epizootic occurrence and behaviour. As a starting point to investigate this, a closer analysis could be conducted of environmental factors linked to specific burrow transition events from season to season.At the most basic level, for S1 FileFurther detail on laboratory and statistical procedures. Table I, Landscape descriptor codes used to record landscape description in the field. Figure A, Normalised log proportion of occupied burrows for each of the landscape descriptions, in rank order. Figure B, Frequency of single letter landscape descriptor codes across 3 groups of proportion of occupied burrows. Figure C, Median flea index associated with burrows possessing each of the unique landscape descriptors where a flea index had been recorded. Figure D, Frequency of each of the landscape code across three strata of flea index scores. Table II, Landscape descriptor codes included in predictive factors. Figure E, Predicted proportion of seasons occupied for burrows with and without dunes or clay, and combinations thereof. Figure F, Plots showing coefficient estimates and P values for the model using the sector and burrow random effects and for the iterative model using sector random effects but random samples of burrows. Figure G, Plots showing coefficient estimates and P values for the model using the sector and burrow random effects and for the iterative model using sector random effects but random samples of burrows. Figure H, Flea index and tick index scores, with predictions from the minimal model.(DOCX)Click here for additional data file.


Pseudomonas aeruginosa led to synthesis of modified pyoverdines in a minority of cases, but that more often the recombinant enzymes were non-functional. One possible explanation was that the majority of introduced C domains were unable to effectively communicate with the thiolation (T) domain immediately upstream, in the first module of PvdD.Non-ribosomal peptide synthetase (NRPS) enzymes govern the assembly of amino acids and related monomers into peptide-like natural products. A key goal of the field is to develop methods to effective recombine NRPS domains or modules, and thereby generate modified or entirely novel products. We previously showed that substitution of the condensation (C) and adenylation (A) domains in module 2 of the pyoverdine synthetase PvdD from N5-formyl-N5-hydroxyornithine residue in place of the terminal threonine of wild type pyoverdine, in neither case did the T-C-A domain substitution strategy lead to substantially higher product yield. To more comprehensively examine the abilities of non-native T domains to communicate effectively with the C domain of PvdD module 2 we then substituted the module 1 T domain with 18 different T domains sourced from other pyoverdine NRPS enzymes. In 15/18 cases the recombinant NRPS was functional, including 6/6 cases where the introduced T domain was located upstream of a C domain in its native context.To test this we first compared the effectiveness of C-A domain substitution relative to T-C-A domain substitution using three different paired sets of domains. Having previously demonstrated that the PvdD A/T domain interfaces are tolerant of domain substitution, we hypothesised that T-C-A domain substitution would lead to more functional recombinant enzymes, by maintaining native T/C domain interactions. Although we successfully generated two recombinant pyoverdines, having a serine or a Our data indicate that T domains are generally able to interact effectively with non-native C domains, contrasting with previous findings that they are not generally portable upstream of epimerisation (E) or thioesterase (TE) domains. This offers promise for NRPS recombination efforts, but also raises the possibility that some C domains are unable to efficiently accept non-native peptides at their donor site due to steric constraints or other limitations.The online version of this article (doi:10.1186/s12866-015-0496-3) contains supplementary material, which is available to authorized users. Non-ribosomal peptide synthetases (NRPSs) produce a wide range of small peptide products of biotechnological interest. The peptide products can contain a highly diverse array of amino acids or related monomers, with over 600 different monomers having been detected in non-ribosomal peptides . The divin vitro e.g., [in vivo [Pseudomonas aeruginosa PAO1 that incorporates two L-threonine residues at the C-terminus of the peptide siderophore pyoverdine [in vivo, whereas nine out of nine non-synonymous A domain substitutions were inactive [The modular nature of the NRPS assembly line offers promise for the creation of rationally modified peptides, or even combinatorial libraries of novel products, by recombining NRPS domains or modules at a genetic level. The primary substrate-specifying role of the A domain has made this domain a key target for NRPS recombination experiments. However, efforts to generate novel peptide products by modifying A domain substrate specificity, or by substituting in a non-synonymous A domain  have generally been unsuccessful , 8. Numero e.g., , 10 and [in vivo , 12 stud[in vivo . Nonetheoverdine ) yieldedinactive , 12. Thein vitro via T-C-A domain substitution [To overcome C domain proof-reading constraints, we next attempted to substitute cognate C-A domain partners into the second module of PvdD. This strategy successfully yielded two modified pyoverdine products, with L-serine or L-lysine in place of the terminal L-threonine present in the native pyoverdine. However, six of the eight C-A domain substituted constructs we generated were inactive, including two bearing threonine-specifying A domains that had previously been active when the A domain was substituted alone . We proptitution , a stratin vivo due to the characteristic absorbance and fluorescence of its chromophore [P. aeruginosa host in an iron-restricted environment, and that our previous work has identified recombination sites between the T-C, C-A, and A-T domain boundaries that permit effective substitution of alternative domains into PvdD [To evaluate the general portability of T domains, we again turned to the pyoverdine synthetase PvdD as a model NRPS enzyme for domain substitution experiments. This system offers the key advantages that pyoverdine is easily detectable omophore , 21 . As a preliminary test for pyoverdine production, each strain was spotted onto iron-limiting King’s B agar plates in either the absence  . Of the C-A domain substitution strains, those bearing constructs CA-Ser2 and CA-fhOrn were fluorescent under UV and able to grow in the presence of EDDHA, whereas the strain containing construct CA-Ser1 was neither fluorescent nor able to out-compete EDDHA for iron. The T-C-A domain substitution strains showed similar results to the corresponding C-A domain substitution strains; that is, the strains bearing domain substitution constructs TCA-Ser2 and TCA-fhOrn were both fluorescent and able to grow in the presence of EDDHA, whereas the strain containing construct TCA-Ser1 was not.Each C-A and T-C-A domain substitution construct was transformed into a nce Fig.  or presence Fig.  of 200 μTo more accurately quantify the levels of pyoverdine production, the absorbance Fig.  and fluoThe identity of the pyoverdine produced by each strain was then confirmed by MALDI-TOF mass spectrometry (MS) Fig. . The strThe lack of evidence for T-C-A domain substitution being a fundamentally superior strategy to C-A domain substitution for generating functional recombinant PvdD enzymes, together with a relative paucity of suitable contiguous T-C-A domain combinations available for substitution, and technical difficulties in amplifying and cloning such large DNA inserts, dissuaded us from building additional T-C-A domain substitution constructs. Instead, to focus more specifically on whether transplanted T domains might have difficulty in communicating effectively with non-native C domains, a series of T domain substitutions were made by replacing the first T domain of PvdD according to the substitution scheme represented in Fig. When considered in the context of altered domain interactions, T domain substitutions are fundamentally similar to C-A domain substitutions; that is, both establish a scenario in which there is perturbation of (i) native A/T domain interactions; (ii) upstream C and downstream T domain interactions; and (iii) upstream T and downstream C domain interactions Fig. , 2c. As P. aeruginosa PAO1, P. syringae 1448a, P. putida KT2440 and P. fluorescens SBW25 pyoverdine NRPS gene clusters  and the restriction site positive control (T1), were spotted onto KB agar plates both with and without EDDHA  were fluorescent at levels indistinguishable from control strain T1 and were all highly viable in the presence of EDDHA , while others  were severely impaired in their level of pyoverdine production. However, the remaining TCt, TE and TTE substitution strains produced near wild type levels of pyoverdine.When grown in liquid media, the levels of absorbance Fig.  and fluoMALDI-TOF MS was used to analyse the supernatants from cultures that exhibited limited or no apparent pyoverdine production. For the apparent non-producing strains E4 and TE1, no pyoverdine-like products could be detected. However, the other apparent non-producing strain Ct3 was detected to synthesise the same truncated 991.5 m/z product Fig.  that hadc domain of PvdD had been substituted by another Tc domain. These data suggest that T domains transplanted upstream of a non-native C domain generally retain functionality, contrasting with earlier studies in which TC domains placed upstream of non-native E [TE domain from the single module indigoidine synthetase BpsA from Streptomyces lavendulae was unable to substitute for the TTE domain of the closely related single module indigoidine synthetase IndC from Photorhabdus luminescens until optimal recombination sites were identified [Thus, only 3 out of the 18 T domain substitution strains were completely unable to synthesise pyoverdine, none of these being strains in which the native Tnative E  or TE [1native E –19 domaientified .in vitro [In previous work we found that a majority of C-A domain substitutions in the second module of PvdD yielded inactive recombinant enzymes . We hypoin vitro , the autin vitro , we did Ct, TE and TTE domains that shared the highest sequence identity with the PvdD T domain were actually the least active when substituted into PvdD , and P. fluorescens SBW25 by Prof. Paul Rainey . All strains were grown in LB media with shaking at 200 rpm, at 28 °C for P. syringae pv. phaseolicola 1448A and 37 °C for other strains. For maintenance of plasmids, tetracycline was added to a final concentration of 15 μg/ml for E. coli and 100 μg/ml for P. aeruginosa PAO1. Where necessary, to prevent passive uptake of iron, EDDHA was added to a final concentration of 200 μg.ml−1.P. syringae 1448a), Moon et al. [P. fluorescens SBW25), and Ravel and Cornelis [P. putida KT2440) as well as the NRPS analysis tools available online . PCR reE. coli DH5α. C-A domain constructs were created as previously described [pvdD were PCR amplified using primers CATfwd and CA-Wt_Rev and ligated into pSW196 using the NotI and SacI restriction sites. The reverse primer introduced a NotI site in exactly the same location that was used for A domain substitutions previously [pvdD were introduced into the construct as previously described for the C-A domain substitution plasmid [NotI restriction digest and ligation. Correct orientation of the introduced T-C-A domain amplicons was confirmed by sequencing. The resulting plasmids were then transformed into P. aeruginosa PAO1 for single-copy integration at the attB locus [C-A and T-C-A domain substitution plasmids were constructed from pSW196  in E. coescribed , with theviously . Then, t plasmid . Target tB locus .pvdD was amplified using the primers CATTE_Fwd and TTeRev. Next the PCR product and pTCA vector were each digested with NotI and SacI, and the PCR product was ligated into pTCA. This replaced the T-Te domains of pTCA with the PCR amplified second module of pvdD. The primer CATTE_Fwd had added NotI and SpeI restriction sites immediately upstream of the C domain of the second module of pvdD, which allowed the subsequent insertion of T domains into these restriction sites. T domains for substitution into pST1 were amplified from the genomes of different fluorescent pseudomonads as indicated in Fig. P. aeruginosa PAO1.The domain substitution plasmid pST1 was derived from the plasmid pTCA. The second module of For measuring pyoverdine levels in liquid media, strains were grown in 200 μL of LB in a 96 well plate for 24 h at 37 °C. This starter culture was used to inoculate M9 media containing 0.2 % (w/v) L-arabinose and 4 g/l succinate (pH 7.0) at a 20x dilution and total volume of 200 μL. After 24 h of growth, cultures were centrifuged to pellet bacteria, and then 100 μL of supernatant transferred to a fresh 96 well plate. Supernatant was diluted 2x in fresh M9 media to give a total volume of 200 μL. Fluorescence (ex. 400 nm/ em. 440 nm) and absorbance (400 nm) were measured using an EnSpire 2300 Multilabel Reader .Cultures were grown according to the methods for measurement of pyoverdine production, above. Cells were pelleted by centrifugation and a sample of supernatant was mixed with matrix solution  in a volumetric sample to matrix ratio ranging from 1:5 to 1:20. Aliquots of 1 μl from each sample were spotted in duplicate onto an Opti-TOF® 384 well MALDI plate  and allowed to dry at room temperature. Spots were analyzed using a MALDI TOF/TOF 5800 mass spectrometer (Applied Biosystems) in positive ion mode. Each spot was externally calibrated using cal2 calibration mixture (Applied Biosystems). Peaks in spectra were labelled in Data Explorer (Applied Biosystems).The data sets supporting the results of this article are included within the article and its additional files (Additional file