Phosphopeptide-binding domains mediate many vital cellular processes such as signal transduction and protein recognition. We studied three well-known domains important for signal transduction: BRCT repeats, WW domain and forkhead-associated (FHA) domain. The first two recognize both phosphothreonine (pThr) and phosphoserine (pSer) residues, but FHA has high specificity for pThr residues. Here we used molecular dynamics (MD) simulations to reveal how FHA exclusively chooses pThr and how BRCT and WW recognize both pThr/pSer. The work also investigated the energies and thermodynamic information of intermolecular interactions.Simulations carried out included wide-type and mutated systems. Through analysis of MD simulations, we found that the conserved His residue defines dual loops feature of the FHA domain, which creates a small cavity reserved for only the methyl group of pThr. These well-organized loop interactions directly response to the pThr binding selectivity, while single loop (the 2nd phosphobinding site of FHA) or in combination with α-helix (BRCT repeats) or β-sheet (WW domain) fail to differentiate pThr/pSer.Understanding the domain pre-organizations constructed by conserved residues and the driving force of domain-phosphopeptide recognition provides structural insight into pThr specific binding, which also helps in engineering proteins and designing peptide inhibitors. Protein phosphorylation is widely exploited in DNA damage repair, signal transduction, cell growth and cell cycle regulation; the cascades of downstream signals can be triggered by grabbing a certain phosphoprotein -6. EluciThe FHA domain is associated with proteins of diverse functions in different organisms. For example, the Rad53-FHA1 domain interacts with phosphorylated Rad9 in response to DNA damage, and the Dun1-FHA domain interacts with SCD1 of Rad53, which leads to activation of Dun1in response to DNA damage response . InsteadThe structure of the FHA domain is mostly organized into a twisted β sandwich of 11 well-defined β sheets, five in the front and six at the back Figure  12,14,1,19-23. TBRCT repeats in BRCA1 are considered to be related to breast cancer -27. The The WW domain in Pin1 is essential for mitotic progression . The domBecause FHA domains are pThr specific modular domains, this study focused on how FHA domains display selective for pThr/pSer residues and comparison with BRCT and WW domains. We study the dynamic and conformational changes of the free domain and the complexes of Rad53-FHA1:Rad9, Dun1-FHA:SCD1, Ki67-FHA:hNIFK, BRCA1-BRCT:BRCH1 and Pin1-WW:CTD systems. We also computed the interaction energy between pThr/pSer and the domains to disclose the driving force of pThr/pSer binding. We propose a model for pThr specificity and potential applications.Saccharomyces. Serevisiae. The target protein of Rad53, Rad9, is phosphorylated in response to DNA damage and interacts with the C-terminal FHA1 domain of Rad53. Two initial structures are from crystallographic coordinates (Protein Data Bank (PDB) code 1G6G) [1K3Q) [2JQL[2AFF[1T2V) of the complex of the BRCT domain in the BRCA1 C-terminus and its target binding partner, BRCH1 [1F8A) [We selected three FHA domains in different families, BRCT repeats and the WW domain. One of the FHA domains is the human Chk2 homolog in yeast, Rad53-FHA1, involved in checkpoint signaling in de 1G6G)  and NMR ) [1K3Q) . Both Ra3Q) [2JQL. The oth2JQL[2AFF. We chosr, BRCH1 . Another1 [1F8A) . Althouget al. [+ was based on the Columbic potential to keep the whole system neutral. Particle Mesh Ewald was used to consider the long-range electrostatic interactions [-1, and the hybrid Nose-Hoover Langevin piston method was used to control the pressure at 1 atm. The SHAKE procedure was used to constrain hydrogen atoms during MD simulations [We performed molecular dynamics (MD) simulations using the Amber10 and NAMD2.6 simulation packages with the ff99sb amber force field -38. Becaet al. . All simet al. . All comet al. . The plaractions . Followiulations .bind, associated with the binding of a domain to its cognate peptide to form a protein-peptide complex is calculated as follows:To quantify the stability of the phosphopeptide binding to the domain, we performed end-point energy calculations, also known as MM-PBSA/MM-GBSA calculations -50. A siThe bracket <E > denotes the average energy computed from a given MD trajectory. The changes in average energy on molecular interactions can be decomposed as follows:PB/ΔWGB and ΔWnp. We note that the binding energy computed here includes the solvation free energy which considers water entropy, and the valance energies cancel out in Eq. 2 due to the single trajectory approach. The solvation free energy can be further decomposed into the polar term, ΔWPB/ΔWGB, and non-polar cavity term, ΔWnp term [representing the changes in valance (v) energy , van der Waal (vdw) interactions, Coulombic (Coul) interactions, and solvation free energy ΔWWnp term ,51. HereWnp term ,46. PB wThe configuration entropy S consisted of phi, psi, omega and sidechain dihedrals, which include both conformational (number of energy wells) and vibrational entropy (width of an energy well) ,48,52. Wwhere p(x) is the probability distribution of dihedral x, and R is the gas constant. T-analyst was used to compute the Gibbs entropy . We conswhere X denotes each dihedral angle, such as phi, psi, omega and sidechain.Ser and Thr have very similar sidechains, so the Ser/Thr protein kinases phosphorylate the OH group of either residue without discriminating between them. Similarly, most pSer/pThr binding modules, such as BRCT and WW domains, can specifically bind to short pSer/pThr-containing motifs equally well. Not unsurprisingly, one more methyl group of Thr has few effects on the overall binding and molecular recognition. However, uniquely, most FHA domains recognize only pThr residues in target proteins, and substitution of pSer for pThr in model peptides severely weakens binding. The pThr specific recognition may function as a filter to further select the protein partner.One of our aims was to investigate how FHA domains can distinguish the tiny difference between Ser and Thr in their binding partner. The work helps gain deeper understanding in molecular recognition and provides valuable insight into strategies of protein engineering. Although we have simulated the entire FHA, WW and BRCT systems, we do not attempt to draw conclusions on properties of the whole system. Instead, we focus on the phosphoresidue binding region, in particular pThr or pSer. Multiple 1 ns MD simulations were performed for each system with different initial velocity so that the simulations evolve independently of each other to reduce potential bias caused by the initial conformation. Because the short phosphopeptides are highly flexible, carrying out short simulations allows the peptide to stay in similar local energy well during different runs ,55. The PB, for electrostatic solvation free energy, and a cavity/surface area term, Wnp, for nonpolar solvation free energy. Because we are particularly interested in studying the local region that contributes significantly to pThr or pSer recognition, we also selected residues within 5 to 7 Å of the phosphate group  and solvation (PB/SA) energy only, and the entropic changes upon binding are not included here. Of interest is knowing which energy term contributes more to weaken the FHA domain-peptide interaction. Because the only difference between pThr and pSer is one non-polar methyl group, the pSer-containing peptide may reasonably result in less favorable van der Waals attraction. However, the trend is not clear, which suggests that replacing pThr by pSer may affect interactions between pThr/pSer and the domain, and the stability of the entire peptide binding to the protein. For example, pSer-containing peptides have weaker van der Waals interactions between Rad53-FHA1 and Ki67-FHA  for wild-type and mutated MD trajectory Table S3: List of residues selected around phosphoresidue Figure S1: RMSD plot of Rad53-FHA1 Figure S2: Detailed illustration of pThr/pSer peptide binding to FHAClick here for file


LGI1) gene encoding the epitempin protein cause autosomal dominant temporal lateral epilepsy (ADTLE), a rare familial partial epileptic syndrome. The LGI1 gene seems to have a role on the transmission of neuronal messages but the exact molecular mechanism remains unclear. In contrast to other genes involved in epileptic disorders, epitempin shows no homology with known ion channel genes but contains two domains, composed of repeated structural units, known to mediate protein-protein interactions.Mutations of human leucine-rich glioma inactivated (in silico model of the two epitempin domains was built to predict the structure-function relationship and propose a functional model integrating previous experimental findings. Conserved and electrostatic charged regions of the model surface suggest a possible arrangement between the two domains and identifies a possible ADAM protein binding site in the β-propeller domain and another protein binding site in the leucine-rich repeat domain. The functional model indicates that epitempin could mediate the interaction between proteins localized to different synaptic sides in a static way, by forming a dimer, or in a dynamic way, by binding proteins at different times.A three dimensional The model was also used to predict effects of known disease-causing missense mutations. Most of the variants are predicted to alter protein folding while several other map to functional surface regions. In agreement with experimental evidence, this suggests that non-secreted LGI1 mutants could be retained within the cell by quality control mechanisms or by altering interactions required for the secretion process. LGI1; GeneID 9211; MIM# 604619) gene has been linked to two different clinical phenotypes: malignant progression of glioma and autosomal dominant lateral temporal epilepsy , a rare familial partial epilepsy syndrome. This gene has been shown to be frequently downregulated in malignant gliomas and to regulate invasiveness of some glioma cell lines LGI1 may serve as a tumor metastasis suppressor gene The human leucine rich, glioma inactivated 1  conformation of LGI1, its binding properties, and ultimately its function(s), we developed an consensus method We employed an integrative bioinformatics approach combining sequence and domain database searches with the consensus from predictions of protein structural features. The LGI1 sequence (accession code: O95970) was downloaded from the SwissProt/TrEMBL database In order to reconstruct the phylogeny of the LGIs, 105 vertebrate and one branchiostomid epitempin sequences have been automatically extracted from the available databases using BLAST http://tree.bio.ed.ac.uk/software/figtree/).A preliminary quartet puzzling analysis has been performed with the Treepuzzle program Structural templates for the two LGI1 domains were found using MANIFOLD URL:http://protein.bio.unipd.it/homer/). The server uses the conserved parts of the structure to generate a raw model, which is then completed by modeling the divergent regions with LOBO, a fast divide and conquer method http://pymol.sourceforge.net/). Position-specific conservation scores for each amino acid were calculated with ConSurf Models for the two LGI1 domains were constructed using the HOMER server  has been included in the analysis. The obtained reconstruction reported in The phylogenetic reconstruction was performed using 105 Vertebrate  sequences. An additional sequence of Ornithorhynchus anatinus protein shares a common node with chicken in group 1 and both are basal to the other mammalians. The phylogeny of LGI1 reveals an early duplication of the gene followed by two other independent duplications as already reported by Gu et al Group 1, 2 and 3 present the fish sequences (blue squares) in a basal position, followed in group 1 and 3 by amphibian and bird sequences (red and green arrows). The mammalian sequences present an apical position in all the groups. The We defined boundaries of each domain in the LGI1 sequence . The firLGI1. The program predicts 4 motifs of 24 amino acids length and the template search selected the structure of the third LRR domain of Drosophila melanogaster SLIT (PDB code:1W8AA) Yersinia pestis cytotoxin YopM The LRR domain was predicted using MANIFOLD. It presents two terminal variable regions, LRR-NT and LRR-CT, reported to have high similarity to those in Nogo-66 receptor (NgR) Staub and co-workers The LGI1 structural model has been evaluated for both conserved regions and electrostatic surface . Using tLGI1 presents two domains that are known to form multi-protein complexes LGI4 is known to interact with ADAM22 The top surface appears to be a specialized region for LGI members because it is particularly conserved across them. The superimposition of LGI1 and the complex of WDR5 with its ligand (PDB code: 3EMH) allowed us to map the putative binding site of a ligand on the top surface of the EPTP domain . LGI1 haIt is well known that the LRR and EPTP domains in LGI1 are N-glycosylated due to their extracellular localization and Sirerol-Piquer et al. To understand the potential role of LGI1 glycosylation we analyzed their distribution over the domain surfaces. In our model, N192 on the LRR domain and N277 and N422 on the EPTP domain are all solvent exposed, confirming the overall correctness of the model. In the LRR domain, the glycosylation site maps to the N terminal side of the LRR-CT portion, while in the EPTP domain, the glycosylation sites map to the β-strand D of the first and fourth blades on the circumference surface. These findings indicate that, while glycosylation modulates the surface properties of LGI1, the putative ligand binding sites are located in non-glycosylated regions.However, the glycosylation of N192 is supposed to have a mechanistic role. The presence of an oligosaccharide in this position indeed likely interferes with attraction of the charged surfaces present in the two domains, possibly preventing a too close interaction between them. From this point of view, N-linked glycosylation also appears important for correct protein folding.et al., in press), the two p.I122T and p.C179R mutations (submitted) and the unpublished p.T380A mutation. Twelve variants affect amino acid residues located in the LRR domain while nine are in the EPTP domain  were predicted to be destabilizing by at least two of the computational methods used . We alsoInterestingly, other variants  occur at residues located in the DA and BC loops that form the top surface of the β-propeller . MutatioAlthough a single transmembrane domain was initially predicted in its central part However, it has also been suggested that LGI1 is secreted as an oligomer The hypothesis concerning LGI1 can also be reasonably extended to other LGI family members. As supported by our phylogenetic analysis and conserved surface residues, binding of ADAM family proteins by LGI is probably a conserved feature. The main difference between LGI1 and other family members appears to be the precise arrangement between the LRR and EPTP domains, as suggested by the presence of a unique insertion on the bottom surface of EPTP in the LGI1 sequences. The effect of this insertion may be a reduced binding affinity for the LRR domain and thus an increased propensity for interaction with other proteins and/or LGI homodimerization. This adaptation could contribute to explain the unique tissue distribution of LGI1 compared to other family members An important task of this study was to uncover the relationship between amino acid sequence, 3D structure, and putative functions of the LGI1 protein. Evolutionary sequence analysis revealed the presence of peculiar sequence stretches for each LGI protein, e.g. LGI1 contains a unique insertion on the fourth blade facing the bottom surface of the propeller. Using a structure-based sequence profile we identified a pattern among the structural units and obtained the models which validated several underlying assumptions, including the inward orientation of conserved non-polar residues and solvent exposure of N-glycosylated residues.The three-dimensional model of LGI1 domains showed how the N- and C-terminal regions are intimately related, revealing a possible mechanism by which LGI1 mediates the trans-synaptic interactions between ADAM proteins. The LGI1 protein contains two conserved binding sites at the concave face of the LRR domain and a circular region on the top surface of the β-propeller domain.et al., in press). Thus, we could hypothesize that the secretion-defective mutant proteins are either incorrectly folded or have altered electrostatic surfaces, which could affects LGI1 export. This explains why many LGI1 variants could not be secreted and opens a question about the mechanisms involved in the molecular pathogenesis of the disease. On the other hand, the p.R407C mutation is compatible with secretion, but rather may exert its pathogenic effect by disrupting interactions with ADAM proteins. Other functional mutations may have the same extracellular effect.We also evaluated the effect of missense mutations found in ADTLE patients on LGI1 protein and we are able to distinguish between structural and functional mutations, the former potentially causing protein unfolding, while the latter interfere with partner protein interactions. Previously published experiments demonstrated that all but one (p.R407C) tested mutants have a defect on secretion Experimental knowledge suggests interactions between LGI1 and ADAM proteins to be mediated by the EPTP domain. We showed that these interactions likely occur through the EPTP top surface. Furthermore, based on the assumption that two protein families usually interact in a similar way, with the same binding site, we predict all four LGI family members to use this interface to interact with different ADAM proteins, albeit with different affinity, in a time and space dependent manner. Finally, we suggest two alternative molecular mechanisms by which LGI1 connects ADAM receptors across the synaptic cleft.Figure S1QMEAN model quality evaluation. The estimated residue error is visualised using a colour gradient from blue (most reliable regions) to red .(TIF)Click here for additional data file.Figure S2Electrostatic potential changes on the LRR surface induced by the E123K, S145R and R136W mutations.(TIF)Click here for additional data file.Table S1Analysis of LGI mutations with stability change prediction methods. The computational predictions were interpreted as stabilizing (S) or destabilizing (D). Stability change prediction is indicated as a ΔΔG value: I-Mutant2.0 (ΔΔG<0 indicates destabilizing variants), Muprot and PoPMuSiC. The protocol used for Eris contains pre-relaxation before calculating the stability change using the flexible-backbone method (ΔΔG>0 indicates destabilizing variants).(XLS)Click here for additional data file.


Ciona intestinalis voltage sensitive phosphatase exhibit faster kinetics. Here we report new versions of ArcLight, in which the Ciona voltage-sensing domain was replaced with those from chicken, zebrafish, frog, mouse or human. We found that the chicken and zebrafish-based ArcLight exhibit faster kinetics, with a time constant (τ) less than 6ms for a 100 mV depolarization. Although the response amplitude of these two probes (8-9%) is not as large as the Ciona-based ArcLight (~35%), they are better at reporting action potentials from cultured neurons at higher frequency. In contrast, probes based on frog, mouse and human voltage sensing domains were either slower than the Ciona-based ArcLight or had very small signals.We previously reported the discovery of a fluorescent protein voltage probe, ArcLight, and its derivatives that exhibit large changes in fluorescence intensity in response to changes of plasma membrane voltage. ArcLight allows the reliable detection of single action potentials and sub-threshold activities in individual neurons and dendrites. The response kinetics of ArcLight  are comparable with most published genetically-encoded voltage probes. However, probes using voltage-sensing domains other than that from the  Genetically-encoded fluorescence protein (FP) voltage probes convert voltage changes across biological membranes into optical signals . In prinCiona intestinalis voltage-sensing domain with super ecliptic pHluorin A227D, exhibited significantly improved response amplitudes (-30 to -40% ΔF/F in response to a 100mV depolarization) in mammalian cells [in vivo in the mouse olfactory bulb , mouse barrel cortex , individual Drosophila neurons [C. elegans . The recently published FP voltage probe, ArcLight, based on fusion of the an cells . Linker  neurons  and C. eCiona-based ArcLight is relatively slow compared with small molecule voltage sensitive dyes [Danio rerio) voltage-sensing domain, has fast kinetics, although its signal to noise characteristics are likely too small for use in vivo [Ciona voltage-sensing domain and Kv3.1 also exhibit fast kinetics [In some experimental situations, a voltage sensing probe with fast response kinetics would be useful. Unfortunately, the response kinetics of the ive dyes  or some ive dyes . Baker e in vivo . Two othkinetics ,18, but Ciona voltage-sensing domain in ArcLight with voltage-sensing domain orthologs from five species: chicken, frog, zebrafish, mouse and human. We found that probes utilizing the chicken or zebrafish voltage-sensing domain exhibited fast responses, and a larger response amplitude than Zahra. We demonstrated that chicken ArcLight can resolve individual action potentials in cultured neurons as well as trains of high frequency voltage steps in the HEK293 cells. The increase in response amplitude afforded by the ArcLight’s super ecliptic pHluorin A227D can be transferred to probes utilizing other voltage sensors and is capable of capturing rapid movements of the voltage sensing domain. These new probes should be useful for detecting fast and high frequency voltage changes in excitable cells.In this study, we sought to combine the large signal size afforded by super ecliptic pHluorin A227D with the speed improvements seen with voltage sensing domains from other phosphatase orthologs. We replaced the Ciona voltage-sensing domain used in the previously published ArcLight probe with voltage-sensing domain orthologs from five species: chicken , frog , zebrafish , mouse  and human . The fusion sites between the voltage-sensing domain and super ecliptic pHluorin A227D in these probes were: Q174 for the frog, Q175 for the chicken and zebrafish, Q331 for mouse and Q193 for human. The fusion sites were chosen to correspond to the fusion site of the Ciona-based ArcLight-Q239 probe , we could detect evoked action potentials in single trials in neurons expressing this probe . It is unclear why the voltage sensing domain of different species would result in reporters with different kinetics. It appears that the coupling between the voltage sensor and the fluorescent protein favors faster kinetics in some species. We previously reported that voltage sensing domain orthologs from different species have faster response kinetics . The curThe chicken and zebrafish ArcLight have similar response kinetics as Zahra, but the response amplitude is larger (-9% for chicken and -8% for zebrafish ArcLight compared with -2% for Zahra). While the mechanism of the superior signal size of the ArcLight voltage probe is unknown, the current study demonstrates that the relatively large signal resulting from the use of super-ecliptic pHluorin A227D is transferrable to other voltage probes containing voltage sensing domain orthologs from different species. Probes containing super-ecliptic pHluorin A227D produce larger signals when compared to similar probes using other FPs . In addition it demonstrates that this enhanced modulatory behavior can respond rapidly. These finding indicate the portability of this fluorescent protein and its mechanism to other probe designs. Ciona-based voltage sensitive probes have a point mutation in the S4 transmembrane region that changes arginine to glutamine (R217Q) and causes a shift of the probe’s voltage dependent fluorescence change to a more physiological range [X. laevis, R309Q for the mouse and R171Q for the human) with a similar effect. Although we cannot be sure whether this effect is due to a change in sensing per se or in the coupling between the voltage-sensing domain and the fluorescent protein [All previously published al range ,19. The  protein , a similX. laevis [The frog ArcLight used for this study is based on Xl-VSP1, one of the two voltage sensitive phosphatase isoforms found in . laevis . This prThe human ArcLight used for this study is based on Hs-TPTE, one of the two voltage sensitive phosphatase isoforms found in humans ,23. Hs-TA single FP, super ecliptic pHluorin A227D, is used in ArcLight and in the newly developed chicken ArcLight. Although FRET based FP sensors enable ratiometric processing of signals and cancelation of bleaching or movement artifacts, there are also advantages for using a single FP as the reporter. One advantage is that it reduces the size of constructs needed for the viral vector delivery . -4 and 9x10-4, respectively [Arch (Addgene Plasmid 22217) and Arch D95N (Addgene Plasmid 34616) are microbial rhodopsin based fluorescence voltage probes and feature fast response to membrane voltage changes and slow bleaching rates . Howeverectively . As a reIn the future, it may be necessary to exploit the voltage-sensing domain from more species in designing improved FP voltage sensors. It may also be useful to introduce mutations to the voltage sensitive domain, especially to the transmembrane domains in screening for probes with fast kinetics. Ciona voltage-sensing domain (R217Q) in ArcLight with voltage sensitive domains from chicken, X, laevis, zebrafish, mouse or human. The coding sequence of the voltage sensitive domains were amplified with the polymerase chain reaction (PCR) using the pfu DNA polymerase , digested with restriction enzymes  and inserted between the HindIII and BamHI sites of the ArcLight construct. An extra codon GAG was introduced immediately after the starting ATG to create an optimal Kozak consensus sequence. Primers used for amplification of chicken voltage-sensing domain were:ArcLight derivatives based on the voltage sensing domains from voltage sensitive phosphatases from three species were generated by replacing the GCGAAGCTTCGCCACCATGGAGACGACTGTGAGGTATGAACAG-3 and 5: CCGGGATCCCCAGAGACCATTCTTCTGGTTAC-3. Primers used for amplification of X. laevis voltage-sensing domain were: 5-GCCGCTAGCGCCACCATGGCCTCAAGTGTAACAGACGAGCAG-3 and 5-CCGGGATCCCCAGAGACCAGCCTCCTTGTTAC-3. Primers used for amplification of zebrafish voltage-sensing domain were: 5-GCCGCTAGCGCCACCATGGCCACGTCTGTGCATTTTAACCCTG-3 and 5-CCGAGATCTCCTGAAACCATTCTCCTGGTGAC-3. Primers used for amplification of mouse voltage-sensing domain were: 5-GCGAAGCTTCGCCACCATGTATGGAGAAAAGAAGAGCCATTTGTATCTC-3 and 5-CGCGGATCCCCTTGATGAGCCAGTTGCAGAATCC-3. Primers used for amplification of human voltage-sensing domain were: 5-GCGAAGCTTCGCCACCATGGAGAATGAAAGTCCTGATCC-3 and 5-CGCGGATCCCCATGAAACAGATGAAAAATTCTTAACAG-3. An arginine to glutamine mutation was introduced to the S4 of the voltage-sensing domains , corresponding to the location of R217Q in ArcLight. Point mutation and modification to the linker sequences between the chicken voltage-sensing domain and super ecliptic pHluorin were introduced by using the QuickChange II XL site-directed mutagenesis kit . All DNA constructs were verified by sequencing using the dye-termination method 5- HEK 293 cells  were maintained in Dulbecco’s Modified Eagle Medium  supplemented with 8% fetal bovine serum . Cortical neurons were isolated from E18 mouse embryos and maintained in Neurobasal medium with 0.5 mM Glutamax-I and 1 ml of B-27 supplement  per 50 ml of cultured medium. Cells were plated on coverslips coated with poly-D-lysine hydrobromide  and kept in an incubator at 37°C with 5% CO2. Transient transfection was accomplished by using half of the manufacturer’s recommended amount of DNA (2 μg per 35 mm dish or 0.4 μg per 12 mm coverslip in 24-well dish) and Lipofectamine2000 .Microelectrode recordings were performed in a perfused chamber with the bath temperature kept at 33°C by a temperature controller and bath solution containing: 150 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM D-glucose, and 5 mM HEPES, pH7.4. We used a 3-5 MΩ glass patch pipettes  that were pulled on a P-97 Flaming/Brown type micropipette puller . The pipette solution contained 120 mM K-aspartate, 4 mM NaCl, 4 mM MgCl2, 1mM CaCl2, 10 mM EGTA, 3 mM Na2ATP and 5 mM HEPES, pH 7.2. Voltage-clamp recordings in the whole-cell configuration were performed using a Patch Clamp PC-505B amplifier  with a holding potential of -70 mV. Cultured cortical neurons were recorded in current clamp mode. Action potentials were evoked by current injections. The pipette solution for neuron recordings contained: 120 mM K-gluconate, 3 mMKCl, 7 mM NaCl, 4 mM Mg-ATP, 0.3 mM Na-GTP, 20 mM HEPES and 14 mM Trisphosphocreatin, pH adjusted with KOH to pH 7.3. The senWhole-cell patch clamped cells were imaged with a Nikon Eclipse E6000FN upright microscope with a water immersion objective, Nikon Fluor 60×/1.00 N.A. A MLL-FN-473 nm 50 mW  was used as the excitation light source. The laser light was transmitted into the microscope by a multi-mode fiber coupler , a quartz light guide and an Achromatic EPI-Fluorescence Condenser . The filter cube contains a dichroic mirror 505DCXR and an emission filter HQ510LP . The fluorescence image was demagnified by an Optem® zoom system, A45699  and projected onto the 80×80 pixel chip of a NeuroCCD-SM camera controlled by NeuroPlex software . The images were recorded at a frame rate of 1000 fps. Confocal images were obtained with a Zeiss 780 LSM  confocal laser scanning microscope using a Plan-Apochromat 63x/1.40 Oil DIC M27 objective. A 488 nm wavelength Argon laser was used for chromophore excitation. The dichroic beam splitter was a MBS 488. The emission filter was 493 - 598nm. Zeiss Zen 2009 software was used for image acquisition and processing.NeuroPlex software  was used to view the image sequences and output optical and electrophysiological recordings. The % ΔF/F was calculated by first subtracting the dark image from all frames, then the average of a region of interest in each frame (F) is subtracted from the average of the region taken from one hundred frames prior to the event of interest (F0) and this value is then divided by F0, i.e. % ΔF/F = ((F-F0) / F0)*100. The data were further processed and statistically analyzed with Origin8.1  and Excel .The probe dynamics are fitted with either a single exponential equation: or a double exponential equation:vs V plot was analyzed with the Boltzmann equation:The ΔF/F 0.5 is the membrane potential in mV at half maximal ΔF/F, and dx is the slope.where xvs V plot was calculated from the Boltzmann fit: The normalized ΔF/F vs V plot of a probe based on the chicken voltage-sensing domain and the Mermaid FRET pair was calculated with the area version of the Gaussian function:The ΔF/F Figure S1Protein sequence alignment of N-terminal regions of the voltage sensitive phosphatase from six species. Conserved residues are shaded. Transmembrane domains are indicated with orange and the S#. The arginine to glutamine point mutation that shifts the F-V curve of the probes to more physiological range is marked in red. The fusion site corresponding to that of ArcLight-Q239 is highlighted with yellow.(TIF)Click here for additional data file.Figure S2Expression of human or mouse ArcLight in transfected HEK 293 cells. Representative confocal images of human ArcLight-Q193 or mouse ArcLight Q331 expression in the HEK 293 cells. The probes carry either an arginine to glutamine mutation or not in the S4 domain.(TIF)Click here for additional data file.


Lymnaea palustris and L. fuscus are members of the European stagnicolines (Gastropoda: Lymnaeidae). The role of stagnicolines in transmission of Fasciola hepatica has been often proposed. To assess the possible relationship between these two stagnicolines and F. hepatica in Sweden, field monitoring in parallel with experimental infections of L. palustris and L. fuscus were conducted.L. palustris and L. fuscus were examined for F. hepatica DNA by PCR. In the laboratory, different age groups of L. palustris, L. fuscus and G. truncatula were each exposed to two F. hepatica miracidia and main infection characteristics were obtained.Stagnicoline snails were collected and identified on pastures grazed by either sheep or cattle on four farms suffering from fasciolosis in Sweden during 2011–2012. Field-collected L. palustris (out of n = 668) contained F. hepatica as determined by PCR. On the other hand, stagnicolines artificially exposed to F. hepatica miracidia resulted in successful infection with fully differentiated cercariae, but only in juvenile snails  and with a prevalence of 51% and 13% in L. palustris and L. fuscus, respectively. In contrast, 90% of juvenile  and 92% of preadult G. truncatula , respectively, were successfully infected. Delayed, reduced and/or no spontaneous cercarial shedding was observed in the two stagnicolines when compared to G. truncatula. However, at snail dissection most cercariae from L. fuscus and L. palustris were able to encyst similarly to those from G. truncatula.One field-collected L. fuscus and L. palustris can sustain larval development of F. hepatica but with an apparent level of age resistance. The finding of a single F. hepatica positive specimen of L. palustris, together with infection characteristics from the experimental infection, suggest that L. palustris is a more suitable snail vector of F. hepatica than L. fuscus. The reduced growth observed in both stagnicolines was contrary to the ‘parasitic gigantism’ theory. Overall, it seems that the epidemiological role of L. palustris in transmission of F. hepatica in Sweden is likely to be much lower than for G. truncatula.Both  Fasciola hepatica is a worldwide distributed generalistic parasite with a di-heteroxenous life cycle including both mammalian (mainly ruminants) and certain gastropod hosts  and Stagnicola fuscus . Furthermore, the snail isolates collected from Norrköping, Linköping and Kristianstad were morphologically identical to L. palustris. The sequence of the Uppsala isolate of G. truncatula [GenBank: KC248372] used in the experimental study had 100% identity to French and Venezuelan isolates of G. truncatula   from all three sampling sites  during 2011–2012 were examined for liver fluke DNA by PCR. Only one L. palustris collected on the beef farm in Linköping was F. hepatica positive. The species identity of this positive specimen was confirmed by morphology and ITS-2 sequencing [GenBank: KC248374]. All L. fuscus snails (n = 130) were negative.A total of 668 snails determined as L. fuscus and L. palustris no infected snail was observed in the ≥ 2–4 mm or ≥ 4–6 mm groups, and only juvenile snails (1–2 mm) were infected, with respective prevalences of 13% and 51% . In addition, seven snails were successfully infected but they did not produce any cercariae until dissection. In juvenile L. palustris, 17 (of 40) F. hepatica positive snails spontaneously shed cercariae from 61 days PE. Most snails in the 1–2 mm and ≥ 2-4 mm groups of G. truncatula were infected , and more than 50% also spontaneously shed cercariae from day 43 PE. Significant differences between the stagnicoline groups and G. truncatula were noted for the spontaneous cercarial shedding, and also for metacercarial counts obtained from NCS snails, with the highest values in preadult (≥ 2-4 mm) G. truncatula and the lowest in juvenile L. fuscus.In both G. truncatula , but not in the stagnicoline species. However, shell size at the end of the experiment was significantly (p < 0.0001) lower in infected snails than in the negative group for both stagnicoline species, whereas it did not differ between infected and uninfected G. truncatula  also by using the molecular methods . Several studies refereed on susceptibility of lymnaeid snail hosts to F. hepatica[Radix balthica and R. labiata[F. hepatica.Evaluation of the susceptibility of potential intermediate hosts to  hepatica,18,19,27. labiata. Our stuL. fuscus and L. palustris were previously considered to be low susceptible  or resistant (the older snails) to F. hepatica infection [F. hepatica relationship both by detailed investigation of snails collected from the field and by confirmatory experimental infections, including comparison to parallel infection of the most typical intermediate host for F. hepatica in Europe, i.e. G. truncatula. In our study we compared the intramolluscan development of F. hepatica in the two stagnicolines with that of G. truncatula; it can be concluded that although the presence of parasite DNA in the snail bodies was confirmed, these stagnicolines must be regarded as accidental hosts of F. hepatica, and their ecological role in the transmission of the parasite is probably minor. On the other hand, as it has been suggested for Radix spp. [L. palustris as a vector might be more important in G. truncatula free areas.Although nfection , both ofnfection ,29. In tdix spp. ,5 the roG. truncatula can easily be mistaken with pre-adult L. palustris or L. fuscus if the determination is based only on shell morphology. The agreement between ITS-2 sequences and morphological determination  further confirms that the assessment of snail internal anatomy is an applicable method for lymnaeid species identification [Owing to inherent taxonomic problems with the morphological identification of stagnicoline snails ,11, molefication ,30.F. hepatica in snails by dissection and morphological determination of rediae and cercariae constitute the classical screening method [F. hepatica in snails was proposed from the 1990s [F. magna[F. magna were tested. In contrast, amplification of cox-1[F. hepatica and F. magna  was found to be F. hepatica positive. Although this finding is insignificant from an epidemiologic point of view, it demonstrates that L. palustris can be infected with F. hepatica under natural field conditions. However, since snails were examined only by PCR, we cannot say whether the infection was patent or not. To our knowledge, the only cases of natural infection of L. palustris with F. hepatica were reported from Central France [L. fuscus was found only at one farm and the total number of examined snails was lower than that of L. palustris, we cannot fully exclude natural infection of this species elsewhere.With this PCR-based method only one snail , where snails were exposed but remained uninfected.Data from the experimental study clearly show that both  species ,36-38. I species , and is  species . Suppres species . This efF. hepatica infection in both stagnicoline snails with shell size greater than 2 mm at exposure and high prevalence in both age groups of G. truncatula suggest that 2 mm constitutes the onset of age resistance in L. palustris and L. fuscus to F. hepatica. The age resistance in snails has already been reported for L. palustris[L. fuscus[F. hepatica infection [L. palustris[. Thus, natural resistance of L. palustris against F. hepatica was explained by the fact that miracidia are able to penetrate into the snail body but forming sporocysts are immediately encapsulated and removed by the snail defense mechanisms [The absence of palustris,42 and LL. fuscus,21, and nfection . A possichanisms ,42, but G. truncatula was 1789 metacercariae in a snail exposed to 5 miracidia [G. truncatula in the present study. In both stagnicoline species, metacercarial production was significantly lower and also more delayed than in G. truncatula. This suggests that despite the successful differentiation of cercariae, the larval development process was obviously delayed. Furthermore, only a few specimens of L. palustris and a single L. fuscus shed cercariae spontaneously. Most cercariae from L. fuscus and L. palustris encysted in the Petri dish mainly at dissection (day 95 PE), and with an average production of 157–168 metacercariae per infected snail. This is in agreement with the very low spontaneous cercarial production observed in accidental intermediate hosts of F. magna, and also when fully differentiated cercariae were found at dissection [Metacercarial production and the time of first cercarial shedding uncover important details about the snail-trematode interaction. Whilst the highest reported metacercarial production in iracidia , the aveiracidia . This isssection . This phF. hepatica infection on snail growth is a frequently studied variable concerning snail-trematode interactions. Compared to the shell height of negative controls, a significantly reduced size (stunting) of infected snails was noted for both stagnicoline species. Furthermore, as no difference in growth between infected and uninfected G. truncatula was observed, this contradicts the ‘parasitic gigantism’ hypothesis observed in lymnaeid snails [F. hepatica-infected G. truncatula when compared to uninfected controls [L. palustris[L. fuscus[L. stagnalis[Pseudosuccinea columella[F. magna[G. truncatula probably indicates the level of co-evolution, which gradually will happen when the parasite and snail live in close ecological interaction with one another. Obviously, this controversy in growth effect caused by F. hepatica needs further research. Interestingly, cercarial/metacercarial production in our study positively correlated with shell size of G. truncatula but not with that of stagnicoline snails. The dependency of cercariae amounts on snail volume has been reported in G. truncatula as well as in other lymnaeid species [The effect of d snails . Althougcontrols ,47, an ipalustris, L. fuscL. fuscus, L. stagstagnalis and Pseucolumella. As we a[F. magna, this re species ,50.L. fuscus and L. palustris can sustain larval development of F. hepatica but with an apparent level of age resistance. Nevertheless, when all infection characteristics in both stagnicolines are compared, it was evident that L. palustris is better suited as an intermediate host, and thus more suitable as a vector of F. hepatica in Sweden than is L. fuscus. This is further supported by the finding of a naturally infected L. palustris in the field. In fact, the present finding represents the first case where species identification of both the stagnicoline snail and its parasite was confirmed by a molecular method and sequencing. Spontaneous cercarial shedding, viability of metacercariae in L. palustris, together with a high level of amphibiousity in its natural habitats make this species likely as an intermediate host of F. hepatica in Sweden, along with G. truncatula. However, because of age resistance, the role of L. palustris in transmission of the trematode in natural habitats is likely to be much lower than for G. truncatula. Further, the existence of ‘parasitic gigantism’ was doubted in this study. Thus, we believe that the effect on snail growth may be related to co-adaptation/co-evolution of the trematode and snail.From field investigations and experimental infections of snails, it is clear that both aThese taxonomic names are used throughout the text and the term ‘stagnicoline’ to describe all species with a similar shell morphology, including Lymnaea palustris, L. fuscus, L. corvus, and Catascopia terebra.The authors declare that they have no competing interests.AN designed the study, participated in field collections, performed all laboratory work and wrote main parts of the manuscript. MK helped with writing of the manuscript and participated in molecular identification of snail species. LB carried out morphological identification of snails. DR helped with designing of experimental infections and made comments to the manuscript. JH conceived the study and attracted the funding, as well as contributed to the interpretation of the data and writing of the manuscript. All authors have read and approved the final manuscript.


Mycobacterium smegmatis genome suggests that it codes for several thiolases and thiolase-like proteins. Thiolases are an important family of enzymes that are involved in fatty acid metabolism. They occur as either dimers or tetramers. Thiolases catalyze the Claisen condensation of two acetyl-Coenzyme A molecules in the synthetic direction and the thiolytic cleavage of 3-ketoacyl-Coenzyme A molecules in the degradative direction. Some of the M. smegmatis genes have been annotated as thiolases of the poorly characterized SCP2-thiolase subfamily. The mammalian SCP2-thiolase consists of an N-terminal thiolase domain followed by an additional C-terminal domain called sterol carrier protein-2 or SCP2. The M. smegmatis protein selected in the present study, referred to here as the thiolase-like protein type 1 (MsTLP1), has been biochemically and structurally characterized. Unlike classical thiolases, MsTLP1 is a monomer in solution. Its structure has been determined at 2.7 Å resolution by the single wavelength anomalous dispersion method. The structure of the protomer confirms that the N-terminal domain has the thiolase fold. An extra C-terminal domain is indeed observed. Interestingly, it consists of six β-strands forming an anti-parallel β-barrel which is completely different from the expected SCP2-fold. Detailed sequence and structural comparisons with thiolases show that the residues known to be essential for catalysis are not conserved in MsTLP1. Consistent with this observation, activity measurements show that MsTLP1 does not catalyze the thiolase reaction. This is the first structural report of a monomeric thiolase-like protein from any organism. These studies show that MsTLP1 belongs to a new group of thiolase related proteins of unknown function.An analysis of the  A striking feature in all these genomes is the abundance of genes coding for enzymes involved in fatty acid and lipid metabolism; more than 250 in Mycobacterium tuberculosis compared to only 50 in Escherichia coli. The mycobacterial genome codes for over a hundred enzymes involved in fatty acid degradation The genus Mycobacterium comprises some of the most devastating pathogens that infect both animals and humans. To date, twenty eight genomes of mycobacterial species have been sequenced completely [Zoogloea ramigeraZ. ramigera thiolase). This nucleophilic cysteine participates in the reaction by forming a covalent intermediate with the substrate. There are two other important catalytic residues, either two histidines or an asparagine and a histidine (Asn316 and His348 in Z. ramigera thiolase) that are also conserved in the thiolase superfamily Z. ramigera, a cysteine (Cys378) has been shown to play this role Z. ramigera), is found in the N-terminal half of the protomer. Catalysis in both the synthetic and the degradative directions starts with the acylation of the catalytic, nucleophilic cysteine (Cys89 in Z. ramigera thiolase) Thiolases are a ubiquitous group of enzymes that are involved in biosynthetic and degradative pathways of lipid metabolism. In the last step of the β-oxidation pathway In humans, six different thiolases have been identified  Mycobacterium smegmatis genome revealed the presence of several putative thiolase genes M. smegmatis thiolase-like genes  was over expressed in E. coli, purified and crystallized MsTLP1 is described here. Analysis of the structure revealed that the protein has an N-terminal domain with a typical thiolase fold and an additional C-terminal domain. The C-terminal domain is a six stranded anti-parallel β-barrel which is completely different from the structure of SCP2. A detailed analysis of the structure and biochemical properties of this protein revealed that its fold agrees with that of classical thiolase, but the nucleophilic active site cysteine and other characteristic thiolase sequence fingerprints are not conserved. Accordingly, it is found that the protein does not exhibit thiolase activity. Therefore, this protein can be classified as a new subfamily of the thiolase superfamily.Examination of the MsTLP1 and MsTLP2) were identified in M. smegmatis. The pair wise sequence identity between the two corresponding proteins is 31%. A similar search of the M. tuberculosis genome revealed the presence of only one such gene. The pairwise sequence identities of the protein encoded by this gene with MsTLP1 and MsTLP2 are 33% and 69%, respectively. Subsequent searches revealed the presence of TLP homologs in several other bacterial genomes. However, a protein homologous to TLP was not found in the human genome. A multiple sequence alignment was performed using six human thiolases, several other eukaryotic thiolases and seven TLPs from various bacterial sources including M. smegmatis and M. tuberculosis. The phylogenetic relationships of TLPs with thiolases were analyzed and the results are presented in From a genome database search, two TLP coding genes  (Z. ramigera thiolase) motif of thiolases. This motif is known to be important for the geometry of the oxyanion hole Z. ramigera enzyme) were also not conserved in MsTLP1  finger print in TLPs . The paie: 1DM3)  shows thn MsTLP1 . The seqe is 15% .MsTLP1 protomer as calculated from the sequence is 56 kDa. Static Light Scattering (SLS) and size exclusion chromatography experiments show that, unlike other thiolases, MsTLP1 is a monomer of 56 kDa in solution  (n = 3). The assays were carried out using two different batches of freshly purified enzyme.Surface Plasmon Resonance (SPR) binding assays showed that TLP1 binds CoA . The binMsTLP1 was determined using the single wavelength anomalous dispersion (SAD) method. As the triclinic P1 cell could accommodate six protomers of the polypeptide, rotation functions were computed using reflections in the 10 Å - 4 Å resolution shell and a radius of integration of 30 Å. Rotation functions corresponding to κ = 180° and κ = 120° hemispheres had significant peaks consistent with non-crystallographic 32 symmetry The crystal structure of The structure determination was not straightforward and various strategies as described in the methods section had to be used for obtaining a nearly complete structure. About 63 residues of the thiolase domain distributed over four loops could not be built, indicating that these residues belong to truly disordered segments. Electron density is missing for four segments consisting of residues 17–25, 64–77, 135–170 and 253–254 in the thiolase domain see also  as well MsTLP1 polypeptide fold is illustrated in MsTLP1 exhibits the characteristic thiolase fold. DALI search against the PDB using the N-terminal domain as the template revealed several structural homologs. Functionally, all these proteins are involved in fatty acid metabolism. The N-terminal domain can be further divided into two topologically similar N-terminal and C-terminal halves, both of which exhibit the βαβαβαββ topology characteristic of thiolase domains , indicating that this region of the loop domain is highly flexible. The end of the thiolase loop domain is also disordered in MsTLP1  is made up of 6 β strands and resembles a barrel-like structure with three anti-parallel β-strands on either side of the barrel . There iThe six protomers in the asymmetric unit are related by a nearly perfect non-crystallographic 32 symmetry . The lay2. Due to the 3-fold related interactions, the area that gets buried in the A/B interface is 561 Å2 per protomer. This represents only 3.2% of the total area and is small compared to the buried surface area of tightly associated subunits in protein oligomers. This suggests that the interface between 3-fold related subunits is not strong. On the average, about 20 residues from the N-terminal domain of one protomer and 15 residues from the C-terminal domain of the adjacent protomer participate in the interface. The interface is stabilized by 5 hydrogen bonds  and one salt bridge (Glu 35-Lys 473). The contact between protomers related by the non-crystallographic 2-fold is much weaker. No significant hydrogen bonds or salt bridges were detected between the layers. In contrast, an area of 2546 Å2 representing 16% of the total surface area gets buried in the dimerization of Z. ramigera thiolase. The interface is stabilized by 62 H-bonds and 18 salt bridges and is completely different from the interface between A and A′ subunits of MsTLP1. Therefore, the hexameric state observed in the crystal structure of MsTLP1 is likely to be the result of crystal packing. This observation is consistent with the monomeric state of MsTLP1 in solution suggested by size exclusion chromatography and Static Light Scattering (SLS). Modeling studies suggested that the linker region between the N and C-terminal domains of MsTLP1  of 2.4 Å (MsTLP1 and Z. ramigera thiolase are in the thiolase loop domain (119–249 of Z. ramigera thiolase). In Z. ramigera thiolase, five segments of this domain appear to be important for catalysis and substrate specificity Z. ramigera thiolase and MsTLP1 highlighting the five functionally important segments. The tetramerization loop occurring at the N-terminus of this domain is essential for the tetrameric organization of Z. ramigera thiolase. Residues from this loop also interact with the substrate. The covering loop occurs immediately after the tetramerization loop and covers the active site pocket. The pantetheine loop interacts with the pantetheine part of bound CoA. The covering loop and the pantetheine loop together shape the entrance to the catalytic pocket of Z. ramigera thiolase. The cationic loop is solvent-exposed and is thought to capture the negatively charged substrate. The adenine binding loop promotes binding of the adenosine moiety of CoA. The tetramerization loop and the pantetheine loop that occur at the end of the thiolase loop domain are disordered in MsTLP1. From sequence comparison  of Z. ramigera thiolase makes a hydrogen bond with the catalytic water MsTLP1. The GHP motif , which is involved in the formation of the oxyanion hole, has been replaced by GGL (residues 364–366 of MsTLP1). Finally, Cys379 of the CXG motif , which acts as the base for proton abstraction from the substrate in thiolase, has been replaced by Asn395 of the corresponding NGG motif in MsTLP1 (395–397). The altered motifs YSCF, GGL and NGG are, however, conserved in homologs of MsTLP of other prokaryotes  and the loop region just before the Nβ3 dimer interface β-strand of the other subunit of the hypothetical dimer (residues 73–83), as shown in The C-terminal domain of 2. This constitutes 22% of the C-terminal domain and 4% of the N-terminal domain surface areas, respectively. There are 19 hydrogen bonds and one salt bridge  between the two domains. Interestingly, Arg190 is largely conserved in the homologs of TLP. Asp487 is also conserved in the homologs of TLP. In three of the seven homologs shown in 2) as compared to the N-terminal thiolase domain (41.6 Å2). The role of the C-terminal domain is yet to be elucidated. Deletion studies are underway to shed more light on the functional importance of this domain.The interface area between the N-terminal thiolase domain and the C-terminal domain is 1035 ÅMsTLP1 has the conserved core α/β/α/β/α structure and conforms to the thiolase fold strictly. Although the dimerization is not a conserved feature in MsTLP1, the location of the putative active site is similar to those in other thiolases. The ligand binding groove of MsTLP1, identified by structural superposition with Z. ramigera thiolase, is larger than that of Z. ramigera. The absence of the catalytic cysteine and the consequent lack of thiolase activity suggest that though the protein has the strictly conserved thiolase fold, it might perform an entirely different function. Therefore, the protein appears to belong to a new subfamily in the thiolase superfamily. MsTLP1 is the first monomeric member of this superfamily. The initial sequence annotation suggested correctly that MsTLP1 is a thiolase-like protein, although the sequence identities are not very significant. Interestingly, none of the catalytic residues of the thiolase subfamily is conserved in MsTLP1. Nevertheless, the fold of the thiolase part of MsTLP1 closely resembles the Z. ramigera thiolase structure. The location of the ligand binding pocket for CoA in the two proteins also appears to be conserved. MsTLP1 has a unique extra C-terminal domain of unknown function. The structure of this domain is completely different from that of the SCP2 domain of SCP2-thiolase. Examination of TLP1 homologs suggests that the interactions between the thiolase-like domain and the C-terminal additional domains are largely conserved. The physiological function of the full length MsTLP1 protein has not yet been established. Further studies have been initiated to establish the intriguing role of MsTLP1 in the lipid metabolism of mycobacterium and other microorganisms.All enzymes of the thiolase superfamily that have been structurally characterized so far share four features: 1) conservation of the core α/β/α/β/α-layered structure of the thiolase domain, 2) conservation of the extensive dimerization interface, 3) the location of the active site pocket and conservation of key active site residues and 4) the use of a nucleophilic cysteine residue in catalysis. M. smegmatis TLP1 (YP_889758) was cloned, over-expressed in E. coil and purified as described earlier (19).The gene coding for MsTLP1 clone was transformed into BL21 (DE3) Rosetta strain of E. coli. A single colony was picked and grown in 1 ml LB-ampicillin overnight at 37°C. This was then inoculated into 500 ml minimal media with 100 µg/ml ampicillin and grown for 12 hrs at 37°C. Methionine synthesis was inhibited by the addition of an amino acid mixture containing 50 mg/l of leucine, isoleucine, valine, lysine, threonine and phenylalanine. After incubation for half an hour, protein expression was induced with 1.0 mM isopropyl-beta-thio galactopyranoside (IPTG). Selenomethionine was added at the time of induction. Purification of the protein was carried out as described for the native enzyme. Selenomethionine incorporation was confirmed by accurate mass determination using Electrospray ionisation mass spectrometry (ESI-MS).The MsTLP1 crystals were obtained under the same conditions. Three data sets of selenomethionine labeled crystals (Se-MsTLP1) were collected to 2.7 Å resolution at a wavelength of 0.9789 Å (peak) at beam line BM14 of the European Synchrotron Radiation Facility (ESRF), Grenoble, France, using three different crystals. The data were processed using DENZO and SCALEPACK from the HKL-2000 suite 3Da−1−2.0 Å3Da−1, respectively. The final statistics for data collection and processing are summarized in The crystals were obtained as described previously MsTLP1 was determined as follows. Using anomalous differences, 63 selenium positions were identified and refined using the program SHELXD The structure of These calculations were based on only one of the three data sets collected at the peak wavelength. At this stage, all the three peak data sets collected at ESRF were merged and scaled and the resulting data were used to perform Molecular Replacement- Single wavelength Anomalous Dispersion (MR-SAD) using PHASER Z. ramigera thiolase (PDB ID: 1DM3) has been used for all sequence and structural alignments. 1DM3 represents the acetylated Z. ramigera thiolase structure complexed with acetyl CoA.The geometry of the final model was examined using PROCHECK MsTLP1 was used in all sequence analyses. The amino acid sequence of MsTLP1 was used as a starting point in an extensive bioinformatics analysis of the M. smegmatis genome: (i) Proteins homologous to MsTLP1 were identified in the non-redundant protein sequence database of SWISSPROT MsTLP were identified using the six characterized human thiolases as query sequences in the BLAST program. Two TLP homologs, TLP1 and TLP2 (YP_887911.1 and YP_889758.1), were identified in the M. smegmatis genome. The catalytic cysteine is not conserved in either of these proteins although they have been annotated as thiolases. These proteins appear to have an N-terminal thiolase-like domain and an extra domain at the C-terminus. (iii) A BLAST search against the human genome using the MsTLP1 amino acid sequence did not reveal any hits. However, homologs of MsTLP1 could be identified in many prokaryotic genomes. TLP homologs were also observed to be present in all other Mycobacterium spp. Interestingly, only one TLP sequence was found in the M. tuberculosis genome. (iv) A multiple sequence alignment was performed by ClustalW mycobacterial spp. This alignment was used to generate a phylogenetic tree using the neighbor-joining method with 10,000 bootstrap replicates in MEGA5 Only the thiolase domain of MsTLP1 in solution was determined using a static light scattering (SLS) system (Wyatt Minidawn) with its flow cell connected to an ÄKTA purifier operated at 17°C. MsTLP1 (0.5 ml of 4.0 mg/ml) protein solution in 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 10% glycerol, 5 mM 2-mercaptoethanol was loaded on a Superdex 200 (10/300) GL column  attached to the ÄKTA purifier. The elution profiles  as the linker enzyme MsTLP1 to a mixture containing 50 mM Tris-HCl, pH 7.8, 40 mM KCl, 0.2 mM NADH, 1 U of SCHAD (1 U is 1 µmol/(min. mg) of protein), 0.5 mM dithiothreitol (DTT) and 2 mM acetyl CoA. The total reaction volume was 500 µl and the rate of NADH oxidation was monitored at 340 nm for 3 minutes.In the thiolytic direction, the 500 µl reaction cocktail contained 50 mM Tris-HCl pH 7.8, 25 mM MgClMsTLP1 with CoA were performed with a Biacore 3000  optical biosensor at 25°C. 2 µM of MsTLP1 protein was immobilized by amine coupling on the surface of a CM5 sensor chip  to a level of ∼3,000 response units using the GE Healthcare standard immobilization protocol. Binding and dissociation were measured at a flow rate of 20 µl per minute. The ligand solution was run over the sensor surface at five different ligand concentrations  in a running buffer of 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM ethylenediaminetetraacetic acid (EDTA) and 0.005% P-20 The sensor surface was regenerated with borate buffer pH 8.5. The binding curves were corrected for non-specific binding by subtracting the signal obtained for the negative control flow cell. Kinetic constants for association and dissociation were derived from linear transformations of the binding data. The data were fit to the simple 1∶1 Langmuir interaction model using the BIA EVALUATION software.The binding studies between Figure S1The oligomeric state analysis of the recombinantly expressed, purified MsTLP1 has been determined by using static light scattering (SLS) in combination with size-exclusion chromatography. A Superdex 200 10/300 GL column  was used for the size-exclusion chromatography. The elution profile is provided by the UV signal (thin red line). The SLS signal is combined with the RI-signal for the calculation of the molar mass (thick red line) by the ASTRA program.(TIFF)Click here for additional data file.Figure S2Kinetics of binding of CoA to MsTLP1 determined using SPR. The SPR sensogram was obtained by flowing different CoA solutions (see inset) over the TLP1 immobilized sensor chip. The data show that CoA binds to TLP1 with a Kd in the millimolar range.(TIFF)Click here for additional data file.


Botulinum neurotoxins are highly effective therapeutic products. Their therapeutic success results from highly specific and potent inhibition of neurotransmitter release with a duration of action measured in months. These same properties, however, make the botulinum neurotoxins the most potent acute lethal toxins known. Their toxicity and restricted target cell activity severely limits their clinical utility. Understanding the structure-function relationship of the neurotoxins has enabled the development of recombinant proteins selectively incorporating specific aspects of their pharmacology. The resulting proteins are not neurotoxins, but a new class of biopharmaceuticals, Targeted Secretion Inhibitors (TSI), suitable for the treatment of a wide range of diseases where secretion plays a major role. TSI proteins inhibit secretion for a prolonged period following a single application, making them particularly suited to the treatment of chronic diseases. A TSI for the treatment of chronic pain is in clinical development. Botulinum neurotoxins (BoNTs) are clinically valuable for treating various neuromuscular and autonomic conditions, and, as clinical products, have sales of more than a billion U.S. dollars . NeurotoClostridium, specifically by several strains of C. botulinum, C. butyricum and C. argentinense. To date, seven immunologically-distinct forms of BoNT have been identified based upon serological cross-reactivity: serotypes A, B, C1, D, E, F and G. Within the serotypes, multiple sub-serotypes have been defined based upon sequence homology [Clostridium tetani. All BoNTs and TeNT share similar structures and modes of action and are collectively known as clostridial neurotoxins (CNTs) [BoNTs are produced by anaerobic, spore-forming, gram-positive bacteria of the genus homology . A closes (CNTs) . CNTs arBoNTs inhibit ACh release from the pre-synaptic terminals of peripheral cholinergic neurons, particularly the motor-end plates of motor neurons. This results in flaccid paralysis. By contrast, TeNT inhibits the release of glycine from inhibitory Renshaw cells in the ventral motor horn of the spinal cord. This results in a dis-inhibition of motor-neuron activity and the characteristic spastic paralysis of tetanus. Despite the contrasting symptoms of botulism and tetanus at the whole animal level, at the cellular and molecular level the CNTs have very similar mechanistic and biochemical actions—inhibition of neurotransmitter release from their respective target neurons . NotablyThe lethal dose of BoNT for humans is not known, but can be estimated from primate studies. The potency varies between serotypes, with BoNT/A being the most potent in humans. By extrapolation, the lethal amounts of crystalline BoNT/A for a 70 kg human would be approximately 0.09–0.15 µg intravenously and 70 µg orally. This makes it the most potent acute lethal toxin known. Given its extreme potency, and routes of intoxication, BoNT is a potential bio-warfare agent and bioterrorist threat agent [Despite the inherent toxicity of BoNTs and the severe and often fatal nature of botulism, BoNT has become a blockbuster therapeutic and a mainstay for treating many debilitating conditions caused by neuronal hyperactivity. Christian Andreas Julius Kerner published the first clinical account of a food-borne botulism outbreak in 1817. He extracted the active substance from contaminated food and studied its effects, both in animals and on himself, and in 1822 suggested that the physiological effects of BoNT could be put to therapeutic use . He predIt was more than 150 years, however, before this prophetic proposal was realized. In 1980, Alan Scott of the Smith Kettlewell Eye Research Institute, San Francisco, published a report on the local injection of BoNT/A into ocular muscles to correct strabismus in patients ,11. Foll®. In 1997, Allergan introduced a new Botox® formulation with reduced ancillary proteins and lower immunogenic potential [® by Ipsen Ltd., was developed [The U.S. Food and Drug Administration (FDA) first approved BoNT/A in 1989 as an orphan drug called “Oculinum”, later marketed by Allergan Inc. as Botoxotential . In the eveloped .® (onabotulinumtoxinA) and Dysport® (abobotulinumtoxinA) consist of a purified neurotoxin complex, which includes the neurotoxin protein and associated non-toxic proteins. The non-toxic proteins are believed to help protect the neurotoxin from the harsh environment of the gut during natural intoxication. Xeomin® (incobotulinumtoxinA) is a recently developed formulation of purified BoNT/A without the non-toxic proteins [®, is in development. Other BoNT/A products, available in parts of Asia, include CBTX-A and Neuronox® [® in Europe and MyoblocTM in the United States [Botoxproteins . A seconeuronox® . While td States . There ad States ,23,24,25®/MyoblocTM, all the commercially-available BoNT products are type-A based, they are not directly interchangeable [50 . Due to differences in the method of production, formulation and performance of the LD50 assay, the units for a given product are not clinically equivalent to those of another product. It is therefore critically important to recognize that each product requires different unit doses to achieve an equivalent, safe clinical effect. The various BoNT/A products are distinct and unique, and should not be considered as generically equivalent products. Although, with the exception of Neuroblocangeable ,28,29,30® when she observed during treatment of blepharospasm that vertical glabellar creases (frown lines) disappeared [Treatment of muscle hyperactivity continues to be the main use of BoNT and the basis of its regulatory approval. Following Scott’s pioneering studies in strabismus, BoNT/A was shown to relieve the muscle spasm involved in various focal dystonias. In 1987, ophthalmologist Jean Carruthers initiated the cosmetic use of Botoxappeared . In addiAs well as involuntary movement disorders, BoNT has been used to treat muscle tone disorders, including spasticity associated with cerebral palsy, stroke, brain trauma and multiple sclerosis ,33,34. T® as a preventive treatment for chronic migraine headaches. Data supporting the clinical studies have been reported [Several studies have reported that following BoNT application to treat muscle spasm, associated pain is relieved ,37. Amonreported . The FDAreported ,40, therreported . Alteratreported . Whatevereported ,43. Given that BoNT inhibits ACh release at cholinergic parasympathetic and post-ganglionic sympathetic nerves, it is an effective treatment for several disorders of the autonomic nervous system ,43,44. IThe ability of BoNT injections to relax or inhibit smooth muscle contractility and treat several gastrointestinal disorders has also been investigated . DysphagCurrently, one of the most active and exciting areas of development for the clinical utility of BoNT outside of musculoskeletal applications is in urology . PatientThere has been a huge increase in the range of medical disorders for which therapeutic use of BoNT has been explored and demonstrated since 1980, and there continue to be reports of new applications ,48. The Improvements in the understanding of the toxin serotypes and subtypes promise to further expand the clinical value of BoNT as a therapeutic agent. They will not, however, address fundamental limitations inherent in the neurotoxin. These limitations derive from the properties that make BoNT such a successful product, namely its potent and prolonged inhibition of peripheral cholinergic neurotransmission . The potsoluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins [2+-dependent metallo-proteases that have evolved to selectively cleave SNARE proteins involved in the docking and fusion of synaptic vesicles with the pre-synaptic membrane in their target neurons. Each CNT cleaves one or other of the SNARE proteins at a single peptide bond [Inhibition of neurotransmitter release by the CNTs is driven by the proteolytic cleavage of SNARE  of approximately 50 kDa and a heavy chain (HC) of approximately 100 kDa . The LC The binding event underpins both the highly selective neuronal targeting and the potency of the neurotoxins. A dual receptor model of interaction has been proposed, in which the neurotoxins initially interact with polysialogangliosides, particularly GD1b and GT1b, at the pre-synaptic terminal . The neuN domain of HC forms a pore in the vesicle membrane, allowing entry of the LC into the cytosol [Following acidification, the H cytosol . A key f cytosol . Althoug cytosol , surviva cytosol ,80 and i cytosol ,82 withiThe first reported crystal structure for a CNT was for BoNT/A . SubsequC, and the amino-terminal half, HN. HC further comprises two sub-domains, each of approximately 25 kDa: the extreme carboxy-sub-domain, HCC, and the HCN sub-domain. The HCC sub-domain is where the ganglioside and protein binding sites are located [CN sub-domain is currently unknown, although it may help orientate the binding domain relative to the translocation domain and enable efficient insertion of the translocation domain [The HC consists of two distinct domains: the carboxy terminal half, H located . The pren domain . In BoNTn domain . The arrn domain .N domain is the translocation domain and consists of two long α-helical regions. Acidification of the endosomal compartment following internalization of the bound toxin triggers the HN domain to undergo a conformational change enabling insertion into the vesicle membrane [N domain is a long loop, aptly referred to as the belt, which wraps around the LC domain. It has been proposed that this belt region acts as a surrogate pseudosubstrate LC inhibitor, protecting the active site and acting as a chaperone during the translocation process [N channel of BoNT/A has an estimated diameter of 15 Å, which is too small for the LC to pass through without unfolding, and there is evidence that under the low pH conditions prevailing in the endosome, the LC adopts a molten globule-like structure [N channel enables the protease to access its substrate SNARE protein.The Hmembrane . An unus process ,87. The tructure  and trantructure . Translo2+-metallo-proteases, such as thermolysin, and contains the characteristic HExxH zinc protease consensus motif [The LC itself comprises a mixture of α-helices, β-sheets and strands that form a compact globular-like protein structure. There is a large, open cavity, which contains the zinc atom, coordinated by two histidine residues (229 and 233), a glutamate residue (267), and a water-mediated coordination through an additional glutamic acid. This region shares structural similarity to the active site of Znus motif ,90. Thesus motif ,92,93.Understanding CNT structure and its relation to function has opened up the possibility of developing recombinant proteins based on the specific pharmacology of the different domains . The invN translocation domain to enable cytosolic delivery, the LHN fragment, together with a binding domain that binds to a cell surface receptor on the proposed target cell  has been reported [A fully recombinant fusion protein consisting of the LHreported . The proN domains of the neurotoxin [N/A that was able to deliver the type A endopeptidase into PC12 cells resulting in cleavage of SNAP-25 and inhibition of noradrenaline release [N/A fragment was reported to deliver the endopeptidase into both neuronal and non-neuronal cell types with a consequent cleavage of SNAP-25 and inhibition of secretion [N domain to function in the new target cell following binding and endocytosis via the new binding domain.The first description of modifying a CNT’s cell-binding domain was by Bizzini, who, as part of a study into the mechanism of tetanus toxicity, designed conjugates coupling either ricin toxin B-chain or wheat germ agglutinin to a proteolytically generated fragment of TeNT that retained functional LC and Hurotoxin . The fir release . Followiecretion . One of Erythrina cristagalli lectin and LHN/A (ECL-LHN/A) to nociceptive afferents, a therapeutically-relevant target cell, via galactose-containing carbohydrates specific to those particular neurons [N/A inhibited release of both substance P and glutamate from embryonic dorsal root ganglion neurons in culture. Importantly, no effect was seen at equivalent concentrations on cultures of embryonic spinal cord neurons from an anatomically adjacent region, thereby demonstrating selectivity of action between neuronal populations [N/A significantly reduced the nociceptive inputs to convergent dorsal horn neurons by primary sensory afferents of the C-fiber and Aδ types, whereas there was little or no effect on sensory inputs from Aβ-fibers [N/A also resulted in prolonged withdrawal latency in a ‘hotplate’ model of acute thermal pain. This effect was sustained for more than 30 days post-administration of the conjugate, confirming the in vitro finding that the conjugate retained the duration of effect properties associated with the native neurotoxin [An exciting development of this approach was targeting a conjugate of  neurons . ECL-LHNulations . The effβ-fibers . Intrathurotoxin .C domain of one serotype was expressed recombinantly fused to the LHN of the other serotype have also been reported [N domains, whilst the neuronal specificity was influenced by the identity of the HC. The therapeutic potential of creating a hybrid neurotoxin TSI in this manner was demonstrated by showing that the LHN/E-HC/A hybrid protein was effective at blocking the responsiveness of nociceptive neurons to inflammatory or pain stimuli, whereas BoNT/A was ineffective [Another approach to generating TSI in which the targeting properties of CNT are modified in order to create proteins with differentiated properties and therapeutic potential from native neurotoxins is the creation of hybrids between CNTs that combine the properties of the component domains. Delivery of TeNT LC to peripheral motor-neurones using BoNT HC has been reported, resulting in a spastic paralysis reminiscent of botulism . The crereported . The traffective .C domain and the availability of co-crystal structures for many of the interactions with both ganglioside and protein receptors, it has been possible to begin to define key residues involved in those interactions [CC domain of BoNT/A and /B defined by the conserved motif H…SXWY…G has been identified [With the structural definition of the binding loops within the CNT Hractions . For exaentified . Site dientified . This beIn addition to engineering the binding domains of CNT to modify functionality and enable creation of designed TSI proteins, attention has also been given to the endopeptidase domain and its substrate specificity. A major limitation to using clostridial endopeptidases to cleave SNARE proteins and thereby inhibit vesicle trafficking in non-neuronal cells is in relation to the SNAP-25 cleaving serotypes, BoNT/A, /C and /E. SNAP-25 is restricted in its expression to neurons and the ubiquitously expressed homologue, SNAP-23 , is not,The potential to create novel proteins that enable delivery of the endopeptidase component of a CNT to a diverse range of cell types is now well established. The ability of such proteins to produce pharmacological effects in disease-relevant animal models, has also established the therapeutic potential of this approach. The various studies have demonstrated that the retargeted endopeptidase proteins retain the prolonged duration of action that is the hallmark of CNTs. This means that recombinant proteins based upon this approach are particularly suitable for treating chronic diseases.In terms of pre-clinical and clinical exemplification of the TSI platform, a novel targeted BOTOX has recently been reported to have completed Phase I studies and to have entered Phase II studies in Post-herpetic neuralgia (PHN) . By targin vivo? Pre-clinical observations indicate that the receptor binding domain engineered into TSIs is capable of directing the novel protein to the target cell of choice and effecting inhibition of secretory processes. As TSI proteins enter clinical trials in man data will begin to become available that will address tolerability and safety while defining the dose interval and minimal effective clinical dose for clinical efficacy for the TSI platform. With the emergence of this information, together with data from further pre-clinical studies spanning a range of indications, many of these important ‘drugability’ questions will begin to be answered for this novel therapeutic platform. With such broad clinical potential, TSIs are an exciting application of innovative science. As an innovative platform, however, it is inevitable that not all of the answers to classic drug development questions are yet established in the literature. For example, what is the bioavailability, how stable are the fusion proteins, how well do they reach their specific target BoNT/A is a major therapeutic product that can be widely used to treat various neurological and neuromuscular conditions. The therapeutic success of the BoNTs results from their specific and potent inhibition of neurotransmitter release from peripheral cholinergic neurons combined with a duration of action measured in months. The clinical utility of the neurotoxins is, however, severely constrained, both by their limited range of target cells and narrow therapeutic window. Advances over the last twenty years in understanding the structure and biology of CNTs, combined with developments in recombinant protein engineering is opening up opportunities to engineer novel therapeutic proteins based upon the unique pharmacological properties of the CNTs. One such opportunity, Targeted Secretion Inhibitors, is already progressing in the clinical setting and pre-clinically for a range of indications not treatable with neurotoxin products. These proteins will increase the medical benefits achieved through clinical application of the native neurotoxins, particularly BoNT/A, while removing the inherent toxicity and providing proteins with a much-improved therapeutic window. Harnessing the properties of the neurotoxins’ protein domains in novel recombinant proteins will lead to the creation of a completely new class of biologics. By inhibiting secretion, these will treat chronic conditions like chronic pain, which currently have few effective treatments.


The catalytic subunits of all class IA phosphoinositide 3-kinases (PI3Ks) associate with identical p85-related subunits and phosphorylate PIP2 yielding PIP3, but they can vary greatly in the signaling pathways in which they participate. The binding of the p85 subunit to the p110 catalytic subunits is constitutive, and this inhibits activity, but some of the inhibitory contacts are reversible and subject to regulation. Interaction with phosphotyrosine-containing peptides (RTK-pY) releases a subset of these inhibitory contacts. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) provides a map of the dynamic interactions unique to each of the isotypes. RTK-pY binding exposes the p110 helical domains for all class IA enzymes (due to release of the nSH2 contact) and exposes the C-lobe of the kinase domains of p110β and p110δ (resulting from release of the cSH2 contact). Consistent with this, our in vitro assays show that all class IA isoforms are inhibited by the nSH2, but only p110β and p110δ are inhibited by the cSH2. While a C2/iSH2 inhibitory contact exists in all isoforms, HDX indicates that p110β releases this contact most readily. The unique dynamic relationships of the different p110 isozymes to the p85 subunit may facilitate new strategies for specific inhibitors of the PI3Ks. The 3-phosphorylated phosphoinositide second messengers have widespread roles in cell signaling. Their involvement in human diseases such as cancer, diabetes, auto-immunity and inflammation has made the phosphatidylinositol 3-kinases (PI3Ks) that produce these lipid second messengers the target of intense efforts at inhibitor development. In mammals, the PI3Ks consist of eight enzymes . HoweverThe class IA PI3Ks consist of p110α, p110β and p110δ catalytic subunits, which associate tightly with a p85-related regulatory subunit. The domain organizations (from N- to C-terminus) of the p110 catalytic subunits are identical: adaptor-binding domain (ABD), Ras-binding domain (RBD), C2 domain, helical domain and kinase domain . The p85The minimal p85 construct capable of fully down-regulating the basal activity of all three class IA isozymes  consists of the nSH2, iSH2 and cSH2 domains . SH2 domHydrogen deuterium exchange mass spectrometry (HDX-MS) has become a powerful method to determine dynamic structural perturbations in proteins . The tec2 fluorescence polarization assay (Bellbrook laboratories). Reactions were performed in 10 μL in 384 well black plates (Corning 3676). The reactions contained 100 μM ATP, and a dilution series of protein concentrations varying from 1 μM to 1 nM. The protein was serially diluted in a buffer containing 50 mM Hepes pH-7.4, 100 mM NaCl, and 2 mM TCEP. Experiments were carried out in the presence and absence of a pY phosphopeptide . Reactions were started by the addition of ATP  in a buffer that gave a final concentration of 50 mM Hepes pH-7.4, 100 mM NaCl, 3 mM MgCl2, 1 mM EGTA, and 2 mM TCEP. Reactions were allowed to run for 60 min with shaking at 500 rpm. Reactions were stopped using 10 μl of the transcreener stop buffer . This was allowed to equilibrate for 30 min and then the plate was read using a PHERAStar plus HTS microplate reader (BMG Labtech), using a fluorescence polarization module with excitation centered at 633 nm and emission at 650 nm.ATPase assays were carried out using the transcreener ADPThe lipid kinase activity was determined using a modified membrane capture assay measuring production of 32P-labeled PIP3 . Lipid v2O solution , resulting in a final concentration of 78% D2O. “On exchange” reactions were carried out for 3, 30, 300, and 3000 s at 23 °C, followed by addition of a quench buffer, giving a final concentration 0.8% formic acid and 0.5 M Guanidine–HCl. Samples were frozen in liquid nitrogen, and stored at −80 °C until mass analysis.HDX reactions were initiated in HDX buffer  by the addition of proteins  to either pY solution  or just pY buffer. This was allowed to preincubate for at least 15 min followed by the addition of 88% DProteins were expressed and purified as described previously .Lipid vesicles were prepared as described previously .®, 2-3131-00) at 130 μL/min, and onto a particle van-guard trap (Waters) for 3 min. The trap was then eluted onto an Acquity® 1.7 μm particle, 100 mm × 1 mm C18 UPLC column (Waters) using a 5–45% gradient (buffer A 0.1% formic acid and buffer B 100% acetonitrile) over 20 min and injected into an LTQ Orbitrap XL (Thermo Scientific) acquiring from 350 to 1500 m/z, with an ESI source operated at a capillary temperature of 225 °C, and a spray voltage of 3.5 kV.Samples were rapidly thawed on ice and injected onto a UPLC system immersed in ice as described previously . The prom/z state and to check for the presence of overlapping peptides. Ambiguous peptides were excluded from the analysis. The list of all p110 and p85 peptides is shown in Peptide identification and mass analysis of the peptide centroids was performed as described previously, using the Mascot search within Proteome Discoverer (Thermo Scientific) and HD-Examiner  . Mascot Selected peptides were manually examined for deuterium incorporation and accurate identification. Results are presented as relative levels of deuteration with no correction for back exchange, since no fully deuterated protein sample could be obtained. A correction was applied to compensate for differences in the level of deuterium in the exchange buffer (78%). The average error was ≤0.2 Da for corrected data of two replicates. Changes between conditions where the mass difference for a peptide was greater than 6% and greater than 0.5 Da were considered significant. The relative HDX level for every peptide analyzed for every mutant and time point is shown in Although all class IA PI3Ks associate with p85-related regulatory subunits, the effect of this association varies among the isotypes. While both p110α and p110β are expressed in most cells, several observations suggest that association with phosphorylated RTKs activates p110α, but has less influence on p110β . One of In an effort to understand to what extent the differential signaling roles of the class IA PI3Ks downstream of tyrosine kinases are intrinsic to the enzymes themselves rather than the interactions that they make with signaling components other than phosphorylated tyrosine-containing peptides (pY), we examined the mechanisms of pY-mediated activation of all class IA PI3Ks in vitro. As shown in Titration with the pY  shows that the lipid kinase activity of p110α/p85α complex is stimulated at a lower concentration of the peptide than p110β/p85α or p110δ/p85α heterodimers  in the presence of the p110α catalytic subunit compared to p110β and p110δ A,B. ThisIn contrast to the cSH2, interaction with p110α results in decreased HDX in the nSH2 and iSH2 compared with either p110β or p110δ . The oneAll three class IA PI3Ks show an increase in exchange in the helical domain of the p110 subunit in the presence of 15 μM bis-phosphorylated PDGFR-derived peptide . This exThere is an inhibitory interaction between the C2 domain and the second helix of the iSH2, and mutants that disrupt this interaction, such as mutants of D560 and N564 in the iSH2 domain are oncogenic for all class IA PI3Ks . MutatioIt has been suggested that p110β represents an activated PI3K relative to p110α based on the observation that p110α undergoes numerous spontaneous gain-of-function mutations in human cancers , and traOnly p110β and p110δ show an exposure in the C-lobe of the kinase domain in response to pY binding . ConcomiFor the three class IA PI3Ks, there is an increase in exposure of the BH domain on pY binding , suggestAlthough the HDX-MS results can be readily used to estimate the amplitudes of structural fluctuation upon pY binding, there is no information about the direction of the movement. Normal mode analysis of protein structures can be used to computationally estimate conformational fluctuation. The elastic network model of normal mode analysis  uses an Movie 1. An illustration of the fluctuation dictated by the lowest frequency normal mode for the p110α/iSH2 and p110β/iSH2 complexes . The strThe following are the Supplementary data related to this article:In Movie 1, we illustrate the lowest frequency normal modes for p110α and for p110β. For the purpose of these calculations, we have used only the p110/iSH2 complex, since the iSH2 domain is the only domain of p85 that is in common between the p110α and p110β crystal structures. The extremely simplified potential function used for these calculations means that the normal mode is dominated by the shape of the complex . This anThe observation that p110α is involved in signaling downstream of receptor tyrosine kinases in preference to p110β may reflect the enhanced sensitivity to pY activation that we see in vitro. This would also be consistent with a model proposed for prostate epithelial cells in which p110α activity is stimulated by insulin whereas p110β provides a baseline level of PIP3 production . The rol