Shigella flexneri is a bacterial pathogen that invades cells of the gastrointestinal tract, causing severe dysentery. Shigella mediates intracellular motility and spreading via actin comet tail formation. This process is dependent on the surface-exposed, membrane-embedded virulence factor IcsA, which recruits the host actin regulator N-WASP. Although it is clear that Shigella requires N-WASP for this process, the molecular details of this interaction and the mechanism of N-WASP activation remain poorly understood. Here, we show that co-expression of full-length IcsA and the Shigella membrane protease IcsP yields highly pure IcsA passenger domain (residues 53–758). We show that IcsA is monomeric and describe the solution structure of the passenger domain obtained by small-angle X-ray scattering (SAXS) analysis. The SAXS-derived models suggest that IcsA has an elongated shape but, unlike most other autotransporter proteins, possesses a central kink revealing a distinctly curved structure. Pull-down experiments show direct binding of the IcsA passenger domain to both the WASP homology 1 (WH1) domain and the GTPase binding domain (GBD) of N-WASP and no binding to the verprolin homology/cofilin/acidic (VCA) region. Using fluorescence polarization experiments, we demonstrate that IcsA binding to the GBD region displaces the VCA peptide and that this effect is synergistically enhanced upon IcsA binding to the WH1 region. Additionally, domain mapping of the IcsA interaction interface reveals that different regions of IcsA bind to the WH1 and GBD domains of N-WASP. Taken together, our data support a model where IcsA and N-WASP form a tight complex releasing the N-WASP VCA domain to recruit the host cell machinery for actin tail formation. Shigella flexneri is a Gram-negative bacterial pathogen that promotes its own engulfment by epithelial cells and gains access to the host cell cytosol by escaping the phagocytic vacuole. After replication in the cytosol, S. flexneri hijacks the host cell actin polymerization machinery to promote intracellular motility and cell-to-cell spread. The polymerization of actin filaments at the bacterial surface leads to the formation of comet-like actin tails, providing the driving force for bacterial propulsion  that is cleaved upon translocation to the periplasm (A).IcsA is localized to the outer membrane of eriplasm . IcsA treriplasm . The paseriplasm , 8  for F-actin . This woormation –20. The . Co-expression of full-length IcsA with the Shigella protease IcsP releases the 72-kDa passenger domain of IcsA (IcsA(53–758)) into the culture supernatant (B). To isolate large quantities of pure IcsA(53–758), a FLAG epitope tag was inserted into this IcsA construct between the secretion signal and the N terminus of the passenger domain (C). Isolating IcsA(53–758) with FLAG antibody resin resulted in high yields of pure protein (A). Purified IcsA(53–758) was folded as shown by thermal denaturation using differential scanning fluorimetry (B). IcsA(53–758) has a melting temperature of 57 °C in Tris-buffered saline (TBS), and high throughput screening of a range of buffers, pH, and additives revealed only very minor increases in stability (e.g. the addition of 5% glycerol conferred a 1º C increase in the melting temperature). The addition of 300 mm imidazole was found to be destabilizing, resulting in a 6º C decrease in the melting temperature, whereas 6 m guanidinium hydrochloride unfolded IcsA, resulting in the absence of a thermal melt curve. Despite predictions that IcsA may bind calcium, the addition of 10 mm CaCl2 does not seem to affect the stabilization of IcsA.Expression in und IcsA B. Co-expernatant B. To isor domain C. Isolat protein A. Purifiorimetry B. IcsA(5C). The early volume at which IcsA elutes off of the size exclusion column (13.3 ml) suggests that the protein may adopt an elongated structure in solution . This observation is consistent with structural predictions that IcsA will possess a β-helix fold similar to that of other autotransporters. Predictive/hierarchical 3D structural modeling generated by I-TASSER .Using multiangle laser light scattering coupled to size exclusion chromatography (SEC-MALS), IcsA53–758) was shown to be monomeric with an experimentally determined molecular mass of 73 kDa C. The eaI-TASSER –28 produ758 was s-section D.gR) through the monomer elution peak is consistent (3.5–3.8 nm) and has no significant dependence on the forward scattering intensity at zero-angle. The final averaged SAXS profile of monomeric IcsA (53–758) is shown in Fig. 3A, and the Guinier plot of the SAXS data is linear , as expected for aggregate-free, monodisperse systems, yielding an gR of 3.64 ± 0.02 nm. This gR is somewhat large for a protein of its size (72 kDa)  that has an gR of 2.8 nm), suggesting that IcsA(53–758) has a relatively non-globular anisotropic mass distribution. The corresponding p(r) versus r profile  supports this observation because the distribution of vector lengths is skewed, showing asymmetry for r > 3 nm that extends to a maximum particle dimension (Dmax) of ∼13 nm (BSA possesses a Dmax of ∼9 nm). The a priori shape classification of the SAXS data places IcsA(53–758) in the “flat/extended” regime; however, both the shoulder observed on the p(r) versus r profile between 6 < r < 9 nm and the associated shape-topology from automated analysis in terms of shapes  was performed to obtain the primary structural parameters and overall shape characteristics of monomeric IcsA(53–758) in solution. In-line size exclusion chromatography SAXS (SEC-SAXS) was employed based on the results from SEC-MALS indicating the presence of trace higher molecular weight species in a typical IcsA(53–758) sample, thus ensuring that the scattering intensity data were measured from the separated monomeric form. In support of this, the radius of gyration  indicat: dummy atom modeling, where the shape is represented as a collection of dummy atoms (beads) using the program DAMMIN, and a dummy residue approach representing the protein structure by a chainlike ensemble of average residues using GASBOR (available on the EMBL Hamburg website). These models support the conclusions reached from the primary SAXS data analysis. The individual models representing the low resolution structure of IcsA(53–758) fit the SAXS data (χ2 = 1.9) with no systematic deviations observed, as assessed using Correlation Map ) (–GASBOR and DAMMIN models show a characteristic ∼90° “bend” in the IcsA(53–758) monomer that occurs approximately halfway along the length of the protein so that the overall structure of the protein adopts an L-shaped conformation. When compared with the highest scoring I-TASSER model of IcsA(53–758), which otherwise has an almost identical gR (3.7 nm) and Dmax (13 nm) as that determined from SAXS, the I-TASSER model does not fit the data . The major difference between the predicted I-TASSER model and SAXS-based ab initio model is that the β-helix of the predicted structure is not discernably bent. Using an independent approach to support the conclusions reached from ab initio modeling, the highest scoring I-TASSER model underwent normal mode rigid body refinement directly against the SAXS data  versus r profile centered around 7 nm . Bent, or L-shaped, β-helices are not unprecedented for autotransporters  autotransporter protein from E. coli)  (Two approaches were used for  and E)) –32. Both(SREFLEX ). It is ry 4KH3) .A). Pull-down experiments with FLAG-tagged IcsA(53–758) immobilized on beads identified that the WH1 domain (residues 30–142) alone and the extended GBD (residues 156–274) alone are capable of binding IcsA(53–758) (B). An extended N-WASP construct spanning the WH1 to the end of the GBD also binds IcsA but is less stable and prone to some degradation (B). The N-WASP VCA (residues 393–505) domain is not pulled down by IcsA(53–758).The purified and characterized IcsA passenger domain was tested for its ability to interact with N-WASP. IcsA(53–758) bound to FLAG antibody resin was incubated with HeLa cell extracts, and isolated proteins were identified by mass spectrometry. The interaction partner N-WASP was identified in the pull-down using IcsA(53–758) (three unique peptides being identified), confirming that the expressed and purified passenger domain was functional to interact with N-WASP from human cell extracts. The N-WASP protein can be divided into four main regions: an N-terminal WH1 domain, a central GBD, a proline-rich region, and a C-terminal VCA domain. GST fusion constructs encompassing different domains of N-WASP were expressed and purified for binding studies with IcsA(53–758) A. Pull-d(53–758) B. An extradation B. The N-1MKE (A and B). Four regions of the N-WASP WH1 domain were individually mutated to the equivalent WASP sequence: mutant 1, DRNCM → IPPGAFH; mutant 2, DIKD → GIQA; mutant 3, NSPRG → STPTP; and mutant 4, KKFRK → QAFRA. Pull-down experiments with these chimeric versions of the N-WASP WH1 domain reveal that mutations 1, 2, and 4 abrogate binding to IcsA(53–758) (C). These WH1 chimeras are not severely altered in their folding or stability (C) except for mutant 2, which, when not expressed as a GST fusion, could not be purified from E. coli, suggesting that this mutation has a severe detrimental effect on WH1 folding. The lack of binding to IcsA of chimeras 1 and 4 identifies these regions as determinants of the specificity of IcsA binding to N-WASP and not to WASP.Previous work has shown that IcsA specifically binds N-WASP and not WASP . Sequenc1MKE ) reveals1MKE , A and B(53–758) C. These tability C except 1EJ5 (1CEE (D). This result suggests that IcsA does not mimic the mechanism used by the normal activator Cdc42. Overall, these data identify that IcsA binds directly to two regions of N-WASP: the WH1 domain and the VCA-binding region of the GBD.Structures of autoinhibited WASP (PDB entry 1EJ5 ) reveal 1EJ5 . A strucJ5 (1CEE ) revealeJ5 . Increasing concentrations of IcsA were then added to this complex, and the decrease in fluorescence anisotropy indicates release of the VCA peptide upon IcsA binding (B). IcsA efficiently competed with the VCA peptide for binding to the GBD and possessed a much higher affinity than the VCA peptide for this domain, with a dk of 0.32 ± 0.06 μm.To test whether IcsA binding to N-WASP may relieve autoinhibition by displacing the VCA domain, fluorescence polarization experiments were carried out using a labeled peptide encoding the region of the VCA (residues 462–481) responsible for binding to the GBD. Fluorescence polarization was monitored upon binding of this peptide to the GBD (residues 156–274), and an affinity of 28.8 ± 2.0 μlculated A. Increa binding B. IcsA em (C). The slightly reduced affinity of this construct when compared with the shorter GBD is probably due to the minor destabilization of this longer construct, identified previously (B), rather than to the WH1 domain interfering with binding of the GBD to the VCA peptide. Competition binding experiments revealed that IcsA was now capable of competing off the VCA peptide at a lower concentration, revealing a significantly higher binding affinity of 0.05 ± 0.01 μm (D). These data identify that binding of IcsA to the WH1 domain enhances the affinity for the GBD and allows for more efficient displacement of the VCA peptide.As shown by our pull-down experiments, the WH1 domain also contributes to binding of N-WASP to IcsA, so the longer construct of N-WASP encompassing both the GBD and WH1 domains (residues 30–274) was also used in fluorescence polarization experiments. The VCA peptide bound this construct of N-WASP with an affinity similar to that of the GBD alone, 52.4 ± 6.5 μm C. The sleviously B, rather 0.01 μm D. These B) and the prediction of a β-helix type fold similar to that of other autotransporters (D) suggest that linear truncations from the N and C termini may not drastically disrupt protein folding to the extent they may with a globular protein. In support of this, the only currently available high resolution structural data for IcsA are for a portion at the C terminus of the passenger domain known as the autochaperone domain (residues 591–758) (The elongated shape of IcsA B and thesporters D suggest591–758) .E. coli with IcsP. This expression system is not amenable to truncations because these modified forms of IcsA are not correctly targeted to the bacterial outer membrane, and no truncated protein can be isolated from the culture supernatant. Instead, cell-free expression systems were used to produce small quantities of Myc-tagged IcsA for pull-down experiments. A series of N- and C-terminal truncations of the IcsA passenger domain were produced in wheat germ extracts and used in pull-down experiments against N-WASP domains spanning the WH1 (residues 30–142), extended GBD (residues 156–274), and WH1 + GBD (residues 30–274) (A). The construct of IcsA encoding residues 53–508 is capable of binding all N-WASP constructs tested. This is in agreement with previous studies showing that residues 53–508 are required for F-actin assembly, and deletions within this region abolish this process (B).The IcsA used in this study is expressed as the full-length protein and must be co-expressed in  30–274) A. The co process . Upon tr process B.Previous studies using recombinant IcsA for interaction studies have used fusion proteins and required immunoblotting for protein detection. The quality of the protein being expressed is therefore difficult to evaluate, and interaction studies may be prone to misinterpretation due to misfolded IcsA. In this study, we have used an expression system very closely related to the native expression of IcsA by co-expressing the full-length IcsA with the membrane protease IcsP to isolate the soluble IcsA passenger domain. We have shown that this protein is correctly folded, monodisperse, and competent to bind the relevant host factor N-WASP.Detailed analysis of SAXS data confirms that IcsA possesses an elongated shape consistent with the β-helix folds of other autotransporters. However, the SAXS data also indicate that IcsA is not a straight rod but that it possesses a central kink resulting in an L-shape, potentially similar to that of Ag43a. The kink in Ag43a is thought to help facilitate trans-mediated dimerization as part of its role as an adhesin . BiophysU from enterohemorrhagic E. coli (EHEC), that also hijacks the actin polymerization machinery of the host cell via binding to WASP family proteins, does not possess this specificity and does not bind the WH1 domain  or binding the host activator protein Cdc42 (residues 194–253). These shorter fragments of the GBD were tested for binding to IcsA, and it was found that IcsA binds the short C-terminal region, which is responsible for N-WASP autoinhibition, rather than the region bound by Cdc42. Furthermore, IcsA is capable of displacing the VCA peptide from the GBD domain, suggesting that the mechanism of actin polymerization by IcsA is release of autoinhibition via displacement of the VCA domain. Displacement of the VCA from the GBD is the same mechanism used by the EHEC effector EspFA). Further domain mapping of this interaction identified a region toward the C terminus of the IcsA passenger domain that may be critical for correct folding because C-terminal truncations beyond residue 433 abolished binding to all N-WASP constructs. This may be analogous to the intramolecular interactions in the autotransporter protein, hemoglobin protease, which possesses an extended loop that stretches from a central region of the protein to interact with the N-terminal domain   . Loss ofShigella virulence factor IcsA binds and activates host N-WASP.In conclusion, we have identified that the IcsA passenger domain is a monomeric, elongated protein with a distinct, bent shape that specifically binds the N-WASP WH1 and GBD domains. We show that IcsA binding to the GBD domain efficiently displaces the VCA peptide and that this interaction is further enhanced by IcsA also binding to the WH1 region of N-WASP. Although release of the VCA by binding to the GBD is a mechanism used by other N-WASP activator proteins, IcsA is, as far as we know, the only activator that enhances this interaction by also binding the WH1 domain. Our results provide significant new insight into the mechanism by which the icsA and icsP genes were amplified from the pWR100 virulence plasmid of S. flexneri (gift from Ariel Blocker), and N-WASP constructs were amplified from full-length N-WASP (gift from Bob Robinson). PCR products were amplified using KOD DNA polymerase (Novagen), cloned into pETDuet-2 (for IcsA/P co-expression), pGEX-6P-1 or pGEX-4T-2 (for GST fusions) or pF3AWG (for cell-free expression) and sequence-verified. For site-directed mutagenesis, PCR was carried out using Pfu Turbo® polymerase (Agilent) with primers encoding the mutation (Sigma).Full-length E. coli C43 (DE3) for IcsA and E. coli RosettaTM(DE3) for N-WASP. Cultures of 2× TY medium supplemented with antibiotics were grown until an optical density (600 nm) of 0.6–0.8, when expression was induced with 1 mm isopropyl β-d-1-thiogalactopyranoside. Cultures were incubated overnight at 30 °C for IcsA or 22 °C for N-WASP. Cells were harvested by centrifugation , and pellets of N-WASP were stored frozen (−20 °C). FLAG-tagged, secreted passenger domain IcsA(53–758) was purified from medium using anti-FLAG® M2 magnetic beads. Beads were washed with TBS  before incubation with medium for 1 h at 4 °C on a rotating platform. The protein was eluted in TBS, 10 mm CaCl2 (TBSC) and 100 μg/ml 3× FLAG peptide (Sigma) (pH 7.4). Affinity chromatography was followed by size exclusion chromatography using a Superdex S200 10/300 column  pre-equilibrated in TBSC. GST-tagged N-WASP cell pellets were thawed and resuspended in TBSC with protease inhibitor (Roche Applied Science) and 400 units of DNase I (Sigma)/liter of culture. Cells were lysed using a cell disruptor (Constant Systems), and lysates were cleared by centrifugation . Proteins were purified from cleared lysate using glutathione-Sepharose 4B  washed with TBSC, 1 mm DTT and eluted with the addition of 50 mm reduced glutathione. Affinity chromatography was followed by size exclusion chromatography performed as above.Bacterial strains used for protein expression were E. coli C43 (DE3) as above. 10-ml cultures were harvested by centrifugation , and pellets were diluted 1:10 in TBS. The supernatant was incubated with 10 μl of StrataCleanTM resin (Agilent) for 5 min, and beads were collected by centrifugation . Samples were boiled at 95 °C for 5 min in SDS loading dye and resolved on a 12% SDS-polyacrylamide gel followed by immunoblotting.Full-length IcsA was expressed alone or co-expressed with full-length IcsP in mT) was the inflection point of the sigmoidal curve obtained by curve fitting to a Boltzmann-sigmoidal model using differential scanning fluorimetry analysis scripts and Prism version 5 (GraphPad Software)  and different additives in a final volume of 20 μl before thermal denaturation was performed using an Applied Biosystems Viia7 quantitative PCR machine. Samples were heated from 25 to 95 °C at 1 °C/20 s, and fluorescence was monitored at 1 °C increments. The melting temperature (oftware) . Melt cuDetermination of molecular masses of IcsA was carried out using MALS performed at room temperature immediately following SEC on a 1260 Infinity FPLC (Agilent) by in-line measurement of static light scattering , the differential refractive index , and UV absorbance at 280 nm (Agilent). IcsA was injected onto an analytical Superdex 200 Increase 10/300 gel filtration column  in TBSC at a flow rate of 0.5 ml/min. Molecular masses were calculated using the ASTRA6 software package (Wyatt Technology).I-TASSER, online server (The amino acid sequence of IcsA53–758) was used as input for predictive/hierarchical 3D structural modeling using the Iterative Threading ASSEmbly Refinement, or e server –28. Full758 was uI(s) versus s, where s = 4πsinθ/λ nm−1, 2θ is the scattering angle, and λ is the X-ray wavelength, 0.124 nm) were collected at room temperature using 1-s sample exposure times for a total of 3,600 1-s data frames spanning the entire course of the SEC separation monitored at 280 nm (Agilent). The IcsA(53–758) sample (75 μl at 8 mg/ml) was injected onto an analytical Superdex 200 Increase 10/300 gel filtration column equilibrated in TBSC, 3% (v/v) glycerol at a flow rate of 0.4 ml/min. The SAXS data were recorded from macromolecular free fractions corresponding to the matched solvent blank  and for the separated IcsA(53–758) monomers . Data reduction to produce the final scattering profile of monomeric IcsA(53–758) was performed using standard methods. Briefly, 2D-to-1D radial averaging was performed using the SASFLOW pipeline  of 0.01  versus r profile) of IcsA(53–758) were calculated using GNOM (gR and Dmax were determined. The p(r) versus r profile was also used for volume and subsequent molecular weight estimates of the IcsA(53–758) monomers, as evaluated by the datporod (Porod volume) (datmow (datvc (a priori assessment of the non-uniqueness of the IcsA(53–758) scattering data was performed using AMBIMETER (ab initio bead modeling of IcsA(53–758) was performed using GASBOR , both GASBOR and DAMMIN were run 10 times, and the consistency of the individual models was evaluated using the normalized spatial discrepancy (NSD) metric   was calculated using the DAMAVER set of programs , the latter being independent from correct error estimation/propagation through the data reduction process (ab initio bead models of IcsA(53–758) have been deposited into the Small-Angle Scattering Biological Data Bank (SASBDB)  , 45, equpipeline . The buf of 0.01  before bximation ) were scximation . The inding GNOM , from wh volume) , datmow w  . A finalprograms . Normal  SREFLEX . All ind process . The SAX The IcsA–758 sampm/z 400) between m/z 300 and 2,000 with MS/MS switching performed in a Top6 DDA fashion. Raw files were converted to mzXML using MSCovert (ProteoWizard) and searched against a SwissProt database with no species restrictions  using Mascot version 2.3. Carbamidomethylation (C) was defined as a fixed modification, and deamidation (N/Q) and oxidation (M) were defined as potential variable modifications. Peptide and protein validation was performed in Scaffold version 4.3.Samples were resolved ∼1 cm into a precast SDS-polyacrylamide gel. The entire lane was excised and cut into four equal slices. Proteins were reduced, alkylated, and digested in-gel using trypsin. The resulting peptides were dried in a vacuum concentrator (Eppendorf) and resuspended in 8.0 μl of MS solvent . LC-MS/MS was performed on an Orbitrap XL (Thermo Fisher Scientific) coupled to a nanoAcquity UHPLC (Waters). MS was carried out at 60,000 resolution  were added to a microcentrifuge tube and washed twice with TBS, 0.1% Nonidet P-40 (TBSN). 50 μg of purified FLAG-tagged IcsA were incubated with washed anti-FLAG® M2 magnetic beads with shaking for 10 min at 4 °C. Beads were immobilized on a magnet, and supernatant was removed before the beads were washed twice with TBSN before transferring to a flat-bottomed 96-well plate. 50 μg of GST-N-WASP in 200 μl of TBSN were incubated with the FLAG beads for 1 h at 4 °C with shaking. Beads were washed four times in TBSN with shaking for 1 min. 48 μl of TBSN supplemented with 20× FLAG peptide were added to the beads and incubated with shaking for 1 min. The eluted sample was boiled in SDS-loading dye at 95 °C for 5 min and resolved on 12% SDS-polyacrylamide gel.m DTT to a final concentration of 20 nm. For binding experiments, duplicate serial dilutions of GST-GBD and GST-WH1 + GBD  were loaded onto a black polystyrene low bind half-area 96-well plate (Corning) and mixed with the peptide . For competition experiments, duplicate samples of GST-GBD and GST-WH1 + GBD  were mixed with the peptide , followed by the addition of serially diluted IcsA . The anisotropy was measured continuously, and values were recorded after the readings had stabilized. Binding constants (dk) were calculated by fitting data to a one-site equilibrium binding model in Prism version 5.01 (GraphPad). Each binding experiment was repeated three times, and the mean and S.D. are reported for the three independent fits to the data.Fluorescence anisotropy experiments were performed at 25 °C in a Clariostar® (BMG Labtech) with an emission wavelength of 525 nm and slit width of 20 nm and with an excitation wavelength of 480 nm and slit width of 15 nm. Binding and competition experiments were performed as described previously . Brieflym Tris, pH 7.4, 50 mm NaCl, 0.1% Nonidet P-40) and added to GST-N-WASP proteins (50 μg) bound to magnetic GST beads. Reactions were incubated with shaking for 1 h at 4 °C. Beads were then washed four times with wash buffer with shaking for 1 min, and 48 μl of GST elution buffer (wash buffer supplemented with 50 mm reduced glutathione) were added to the washed beads and incubated with shaking for 1 min at 4 °C. SDS loading dye was then added to the supernatant, which was boiled at 95 °C for 5 min and resolved on a 4–12% BisTris gradient gel (Sigma) and visualized by Western blotting.IcsA constructs for WG expression were cloned into a modified pF3A WG vector encoding an N-terminal Myc epitope tag. 3 μg of DNA were made up to a volume of 20 μl with ultrapure water and kept on ice. 30 μl of TNT® SP6 high yield wheat germ reaction mix (Promega) were added to the DNA and incubated for 2 h at 25 °C. Cell-free reactions were made up to a final volume of 200 μl with wash buffer  + PBST for 30 min followed by incubation with the primary antibody (diluted in blocking solution) overnight at 4 °C rocking. After washing, infrared fluorophore-conjugated secondary antibody was added to the membranes and incubated for 45 min. Membranes were visualized using the Odyssey infrared imager (LI-COR Biosciences).For immunoblotting, the following were used: monoclonal mouse anti-Myc antibody clone 4A6 (Merck Millipore), polyclonal rabbit anti-IcsA (gift from Ariel Blocker), polyclonal goat IRdye800-conjugated anti-mouse IgG (LI-COR Biosciences), and polyclonal goat IRdye680-conjugated anti-rabbit IgG (LI-COR Biosciences).J. E. D. conceived and coordinated the study. C .M. J. and D. I. S. designed, performed and analyzed the SAXS experiments. R. P. M. M. designed, performed, and analyzed all other experiments. J. E. D., R. P. M. M., D. I. S., and C. M. J. wrote the paper and prepared the figures. All authors reviewed the results and approved the final version of the manuscript.


TRIF is a key protein in antiviral innate immunity, operating downstream of TLRs. TRIF activation leads to the production of interferon-β and pro-inflammatory cytokines. There is evidence from experiments to suggest that the N-terminal domain of TRIF binds to its TIR domain to avoid constitutive activation. However, no structure of a complex between the N-terminal domain and the TIR domain exists till date. The disordered nature of the region connecting the N-terminal domain and the TIR domain compounds the issue of elucidating the mechanism of autoinhibition of TRIF. In this study, we have employed an integrative approach consisting of mutual information analysis, docking, molecular dynamics simulations and residue network analysis, in combination with existing experimental data to provide a glimpse of TRIF in its autoinhibited state.Our extensive docking approach reveals that the N-terminal domain binds to the BB loop-B helix region of the TIR domain, consistent with experimental observations. Long length molecular dynamics simulations of 1 microsecond performed on the docked model highlights residues participating in hydrogen bonding and hydrophobic interactions at the interface. A pair of residues present in the vicinity of the interface is also predicted by mutual information analysis, to co-evolve. Residues mediating long-range interactions within the TIR domain of TRIF were identified using residue network analysis.Based on the results of the modelling and residue network analysis, we propose that the N-terminal domain binds to the BB loop region of the TIR domain, thereby preventing its homodimersation. The binding of TRIF to TLR3 or TRAM could induce a slight conformational change, causing the interactions between the N-terminal domain and TIR domain to disrupt, thereby exposing the BB loop and rendering it amenable for higher-order oligomerisation.This article was reviewed by Michael Gromiha, Srikrishna Subramaniam and Peter Bond (nominated by Chandra Verma).The online version of this article (doi:10.1186/s13062-017-0179-0) contains supplementary material, which is available to authorized users. The Toll/Interleukin-1 receptor-like (TIR) domain-containing adaptor molecule-1 (TICAM-1) or TIR domain-containing adaptor molecule inducing interferon-β (TRIF) belongs to the Toll-like receptor (TLR) family of pathogen-recognition receptors (PRRs) and associated downstream adaptor proteins . These pThe other TIR domain-containing adaptor proteins include MyD88 (Myeloid differentiation primary response gene 88), MAL/TIRAP (MyD88 adaptor-like/TIR domain-containing adaptor protein), TRIF/TICAM-1 (TIR domain-containing adaptor molecule-inducing interferon-β/TIR domain-containing molecule 1), TRAM/TICAM-2 (TRIF-related adaptor molecule/TIR domain-containing adaptor molecule 2) and SARM (sterile-α and HEAT-Armadillo motifs containing protein). MyD88 is utilized by all the receptors except Toll-like receptor 3 (TLR3) while TRIF is involved in TLR3 and Toll-like receptor 4 (TLR4) signalling , 7. EmerThe TRIF protein is 712 amino acids long, comprising of an N-terminal protease resistant domain, an intermediate long disordered proline-rich region, a TIR domain and a C-terminal disordered region (containing a RIP homotypic interaction motif (RHIM) domain)   algorithq(xi) :probability of amino acid x being present at position iq(yj):probability of amino acid y being present at position jPPP: Joint probability taking profile-based pseudocount into accountPobs: Joint probability without pseudocountNeff: Effective number of sequences aligned at both positions i and ja,b: positive constantsP: Joint probability of amino acid x being at position i and amino acid y being at position j.MIp = MI – MIMIMIMI: MI of i averaged over all other positions in the alignmentMI: MI of j averaged over all other positions in the alignmentMI: MI averaged over all positionsA total of 97 sequences, corresponding to the N-terminal domain and the TIR domain of TRIF, were collected from different organisms. The list of these organisms is listed in Additional file The structures of the TIR domain of TRIF PDB ID: 2M1X) and the N-terminal protease-resistant domain PDB ID: 4BSX) were used for structural analysis and docking , 25. TheBSX were M1X and tA model of the interaction between N-terminal domain and the TIR domain of TRIF was constructed through multiple docking schemes wherein different regions of each domain was used to guide docking. Analysis of all the top poses showed that the BB loop was indeed present in all the interfaces. A summary of the residues predicted to be in the interface by Cluspro for each case is listed in Additional file Three independent molecular dynamics simulations, to a timescale of 1 microsecond each, were performed on the N-TIR complex to assess its stability . The variation in the backbone RMSD and the radius of gyration along the trajectory are shown in Fig. Additional file 8: Video S1.Molecular dynamics simulations of the TRIF N-TIR docked complex. The N-terminal domain is coloured in violet while the TIR domain in green. Residues of the N-terminal domain participating in either H-bonding or hydrophobic interactions are represented as blue sticks while those from the TIR domain are represented as red sticks. (MPG 108645 kb)Since the BB loop and 522R/523 K site are known to be important for the function of TRIF  and mighLong-range interactions have also been studied in other proteins using residue network analysis. The number of residues mediating these long-range interactions is variable. For example, in the MetRS-tRNA complex, different communication paths were identified between the active site and the anticodon binding region and the average number of residues mediating the interactions was between 10 and 17 . Hence, Among the wide repertoire of proteins involved in innate immune signalling, TRIF is a key protein whose function is of major significance to the proper regulation of the innate immune system. The disordered proline-rich region between the N-terminal domain and the TIR domain is a major signalling hub for various downstream proteins leading to different signalling outcomes. Despite the critical role played by TRIF in antiviral innate immunity, the mechanism of its autoinhibition remains to be elucidated. It has been established that its N-terminal domain binds to the TIR domain to avoid constitutive activation, yet no structure of this complex is available. In this study, we have attempted to construct a feasible model of the interaction between N-terminal domain and the TIR domain of TRIF, by combining available experimental information with docking and molecular dynamics simulations studies. In our model, the N-terminal domain binds to the BB loop-B helix region of the TIR domain and this agrees with experimental observations reporting that the N-terminal domain binds to the N-terminal region of the TIR domain.The results of our modelling and residue network analysis enables us to hypothesize that, upon TLR3 activation, the RK site on the TRIF TIR domain interacts with the TIR domain of TLR3. This RK site has also been shown to be required for binding to the TIR domain of TRAM . This coMAVS is an adaptor protein associated with the innate immune receptor RIG-I (retinoic acid- inducible gene-like receptor-I) that also senses viral double stranded RNA, similar in function to TLR3, but localised in the cytoplasm instead. MAVS contains a N-terminal CARD domain for binding to the CARD domain of RIG-I , an intermediate disordered region and a C-terminal transmembrane domain for targeting it to the mitochondria . Both MAMutual information was used to predict co-evolving residues between the N-terminal domain and the TIR domain of TRIF. Homologous sequences corresponding to these regions were extracted from both the Uniprot database and non-redundant database hosted at the National Centre for Biotechnology Information. Redundant sequences were removed and domain boundaries delineated using the Pfam domain database. Subsequently, sequences corresponding to a particular domain were aligned using MUSCLE and manually edited. Columns with >30% gaps were removed. The multiple sequence alignments of both domains were concatenated. Residue pairs that are predicted to co-evolve were identified using the CMAT  . This alA series of dockings were attempted to identify a putative docked pose between the N-terminal domain and the TIR domain and to assess if the same docking pose was achieved through these different strategies and thus reduce the chances of false positives. The structure of N-terminal domain (PDB ID: 4BSX) and the representative NMR structure of the TIR domain (PDB ID: 2M1X) were used in this study. Prior to docking, the His 434 residue in the TIR domain was modified back to Proline and subsequently energy minimized in SYBYL. Two docking programs- Cluspro  and our g_hbond tool implemented in GROMACS. The occupancy of each hydrogen bond was calculated by using a Python script, readHBmap.py, supplied for use with GROMACS trajectories. For analyses of interaction energies at the interface of the N-TIR complex, snapshots were extracted at every 1 ns, and provided as input to the PPCheck algorithm and the results of all snapshots were consolidated using short python scripts developed in the lab.To assess the stability of the docked pose, molecular dynamics simulations were carried out using the GROMACS 54a7 force field. The complex of the N-terminal domain and TIR domain was first processed and then solvated in a rhombic dodecahedron box using the SPCE water model. The distance between the edge of the box and the protein complex was set to 10 nm. The system was energy minimized using 50,000 steps of steepest descent with a time step of 1 femtosecond and no position restraints applied. The protein complex was then restrained while the solvent around it was equilibrated for 200 ps with a time step of 2 femtoseconds in order for the system to attain a temperature of 300 K. The V-rescale thermostat  was usedmin value) of 2.5 was chosen after plotting the size of the largest cluster versus different values of interaction strength. The value at which the cluster size undergoes a transition is taken as the Imin value. MD snapshots were extracted every 1 ns from the trajectory and the cut-off for dynamic stability was 50%. For each snapshot, the shortest path of communication between select residues on the BB loop and residue Arginine 522 of the RK site was determined. The average shortest path between a pair of source and sink residue was obtained by calculating the shortest path in each of the MD snapshots and taking their average.The possible path of communication between the residues of the BB loop and the RK site on the TIR domain of TRIF were investigated using PSN-Ensemble . The proIn this work, the authors utilized computational methods and experimental data to understand the auto inhibited state of TRIF. They have performed extensive computations including mutational analysis, docking, molecular dynamics simulations and normal mode analysis. Using the results obtained from the study the authors proposed that the N-terminal domain binds to the BB loop region of the TIR domain and binding of TRIF induce conformational changes, which disrupt the interactions between the N-terminal and TIR domain thereby exposing the BB loop and rendering it amenable for higher-order oligomerisation. The work is interesting and exhaustive to derive the conclusions. The manuscript is well written and necessary details are provided.1. Different docking algorithms are used in the manuscript (say Cluspro and capsdock etc.). The consensus binding sites obtained with these programs could be discussed.Thank you for the suggestions. We have now provided the consensus binding sites obtained with each program. They are listed in Table S2.Author response: 2. Likewise several modelling software are used to predict the proline-rich region between the N-terminal and TIR domain of TRIF, which utilize different techniques. The reliable models may be discussed along with available experimental data.We have included additional details of the modelling softwares used to predict the model of the proline-rich region between the N-terminal domain and the TIR domain of TRIF, in this revised version of the manuscript.Author response: 3. References should be properly formatted. Volume numbers are missing in several references.Thanks for this. The references have been formatted, corrected and volume numbers included in the references.Author response: In their manuscript entitled “Integrative modelling of TIR domain-containing adaptor molecule inducing interferon-β; (TRIF) provides insights into its auto-inhibited state” by Mahita and Sowdhamini, the authors present a plausible explanation for the auto-inhibition of TRIF via interaction with the TIR domain. The authors have used extensive docking followed by molecular dynamics of the N-terminal domain to the BB loop-B helix region of the TIR domain. Taken together the proposed mechanism seem convincing.Minor issuesPlease detail any minor comments for the authors attention  so that, once addressed, the authors may remove them from the review.I have some minor comments that I hope the authors will address.1. The authors predict eight inter-domain residue pairs to be co-evolving. I suggest that they provide names of residues in addition to their position in Table 1. Do these residue pairs interact in both the predicted docked complexes as well as the MD simulated complexes?Thank you for the suggestions. We have now provided the names of the residues along with their positions in Table S1.Author response: These residue pairs do not interact in either the docked complexes or the MD simulated complexes. However, in some of these inter-domain residue pairs, one of the residues making up the co-evolving residue pair, is present at the interface. This could be due to the fact that we have fewer homologous sequences, which are not sufficient for predictions on the basis of residue pair correlations. We have specifically mentioned that the results of the co-evolution analysis needs to be interpreted with caution due to the lower number of sequences considered.2. The docking study, as well as prior literature suggests an important structural role of the BB loop (residues 428-439) of the TIR domain for its interaction with the N-terminal domain.a. The BB loop is highly flexible as observed in the 20 NMR models of TIR domain (PDBid: 2M1X). Have the authors accounted for the inherent flexibility of this loop during the docking experiments?This is a good point. We had performed docking using Cluspro, CAPSDOCK and HADDOCK. HADDOCK performs a short MD simulation on the binding site which includes the BB loop. Hence the flexibility of the BB loop has been taken into account during HADDOCK docking. Furthermore, following docking, molecular dynamics simulations of 1 microsecond have been performed to assess the stability of the complex. The flexibility of the BB loop is therefore considered during the MD simulations as well.Author response: b. The Methodology section says that a representative structure of the TIR (PDBid: 2M1X) was selected from the set of NMR structures. What criteria were considered to select the representative structure?According to the details provided in the PDB file (2M1X), the first NMR model is considered as the best representative conformer among all conformations in the NMR ensemble. Hence, we chose the first model for our analysis.Author response: c. The NMR structures of TIR domain (PDBid: 2M1X) is a Pro434His mutant structure. On what basis the phi-psi angles of the Proline have been taken in the modeled structure before docking? Kink forming residue like Proline is likely to induce a change in the backbone conformation. Would energy minimization of the protein be sufficient for attaining a local minimum conformation?The phi and psi angles of the corresponding proline in the BB loop of available TIR domain structures-TLR1,2,6 ,10 as well as MyD88 were first checked. Based on this information, the phi and psi values of the Pro 434 residue in the BB loop of TRIF were set. Energy minimization of the protein reduces steric clashes among the atoms and is sufficient to attain a local minimum if the minimization has converged. Further to energy minimisation, we carefully checked the local environment to ensure that there is no strain attributed to the fold.Author response: 3. Structural modeling of the N-terminal domain-intermediate region-TIR domain does not add new insights. I suggest deleting this section.Structural information is available for both N-terminal domain and TIR domain with high reliability since there are homologous protein domains with known structure. It is true that the intermediate region of TRIF is not relatively easy to predict structure. Further, this region is known to contain low-complexity and fairly unstructured regions further complicating the structure prediction. However, our fold prediction results are compelling and relates to the known fold of a viral protein with high confidence. Therefore, we went ahead and attempted modelling of this region as well. As is true with other viral proteins, like the coat proteins, this region – even in the template – has large structural embellishments. Therefore, the intermediate region might appear floppy. On the whole, we are also trying to obtain full-length-model of the TRIF protein, where two domains could be modelled with high reliability and the intermediate region is of relatively low accuracy (derived from fold prediction and contains few floppy regions). But, we trust it is still better than *not* having any structure of the full-length TRIF. The availability of full-length model of TRIF has also enabled us to examine the conformational flexibility through normal mode analyses. For these reasons, we would request that the intermediate region modelling be retained.Author response: Whilst the authors use an original attempt to incorporate data from diverse sources, to come up with a reasonable working model for an important component of the innate immune system, the methodology used ranges from out-of-date to out-right disastrous. Thus, the validity of the results cannot be trusted, and the significance is minimal.Reviewer recommendations to authorsPlease make your report as constructive as possible, if necessary, recommending specific improvements so that the authors have the opportunity to overcome any serious deficiencies that you find. Please divide your comments into major and minor recommendations.This article sets out to provide novel insights into the mechanism of autoinhibition of TRIF, by predicting the complex between its TIR domain and N terminal domain. This is potentially very worthwhile, and could be useful for the innate immunity community. However, I have some major concerns about the methodology that prevent this manuscript from being published, unfortunately…1. The authors do a reasonable job of combining sources of data from evolutionary/mutational analysis of potentially interacting sites in each domain, docking, and comparison with available experimental data, leading them to propose that the N-terminal domain binds to the key BB loop of the TIR domain to prevent homodimerization, which seems reasonable. However, given the sparse data utilised to build this model, and the low result confidence, we need either new experiments to be performed to test the model, or EXTENSIVE simulations. Unfortunately, only one 50 ns and one 100 ns simulation has been used to test the stability of the model. This is far too limited sampling, by today’s standards. I would expect to see minimum of 1 microsecond of sampling, and at least 3-5 repeats of each system, to confirm the stability of the model and to check for disassembly or even unfolding, and to ensure the significance and reproducibility of the data.Thank you for your suggestions. We have now performed three replicates of MD simulations, each of 1 microsecond on the docked N-TIR complex and we find that in all the three replicates, the complex is stable and does not undergo disassembly or unfolding during the length of the entire simulations. The hydrogen bonds present at the interface are also conserved between replicates.Author response: 2. Even for the simulations that have been performed, there is extremely limited quantitative analysis presenting with regards the stability. I would expect to see basic conformational/structural analysis, such as time-dependent RMSDs, radii of gyration, solvent-accessible and buried areas, etc….. do we simply have to trust the conclusions without seeing any evidence??Thanks for this suggestion. The basic analyses  performed on the MD simulations has now been included in the Supplementary Materials section of the revised version.Author response: 3. If I understand the manuscript correctly, the second half is concerned with modelling the linker between the N-termianl domain and TIR domain. This spans residues 154-391… i.e. over 200 residues! Because no protein of known structure with similar sequence is available, fold-recognition approaches were used to identify a possible protein to serve as a template for homology modelling. Frankly, this is pure fantasy. Even for a template with high sequence identity (e.g. >30%), it can be tough to correctly predict its structure. To do so for a template without identity, and an intrinsically disordered region at that, is utterly impossible. All the subsequent results based on this model are likely to be junk, and should be discarded.Structural information is available for both N-terminal domain and TIR domain with high reliablility since there are homologous protein domains with known structure. It is true that the intermediate region of TRIF is not relatively easy to predict structure. Further, this region is known to contain low-complexity and fairly unstructured regions, thereby complicating the structure prediction. However, our fold prediction results are compelling and relates to the known fold of a viral protein with high confidence. Therefore, we went ahead and attempted modelling of this region as well. As is true with other viral proteins, like the coat proteins, this region – even in the template – has large structural embellishments. The intermediate region might thus appear floppy. On the whole, we are also trying to obtain this full-length-model of the TRIF protein, where two domains could be modelled with high reliability and the intermediate region is of relatively low accuracy (derived from fold prediction and contains few floppy regions). But, we trust it is still better than *not* having any structure of the full-length TRIF. The availability of full-length model of TRIF has also enabled us to examine the conformational flexibility through normal mode analyses. It is nowadays acceptable to present models of a protein, where the reliability of model is not uniformly strong – it is indeed the power of integrated modelling approaches like these. If we examine the solution of electron microscopy data of large assemblies, we frequently encounter this situation.Author response: 4. Apparently geometrical/stereochemical/structural/energetic checks were performed for the models built in this manuscript. No such data was actually presented… again, should the reader simply trust the conclusions without seeing any evidence?The basic MD validation checks and analyses  had indeed been performed on our MD simulations and now for the triplicates of long-length simulations. These are now being included in the Supplementary Materials section of the revised version. Thanks for this comment.Author response: Minor issuesPlease detail any minor comments for the authors attention  so that, once addressed, the authors may remove them from the review.Model coordinates should be made available.Model coordinates of the N-TIR complex have been provided.Author response: “Molecular dynamic simulations” should be “molecular dynamics simulations”. There are various other grammatical errors throughout the manuscript.The spelling error and other grammatical errors have been rectified.Author response: I would have liked to see some critical analysis of the results of the mutual information analysis. How much confidence can we really have in the results? This is important since all the subsequent results and models are reliant on this data.The subsequent results and models are not reliant on the mutual information analysis due to paucity of number of sequence homologues. While the co-evolution analysis is based entirely on sequence information alone, the models generated through docking are based on known experimental data, conservation of residues and the information obtained from the available structures of the N-terminal domain and the TIR domain of TRIF. The results of the co-evolution analysis complements the results of docking.Author response: Although published, at least short descriptions of the underlying methodology for the various “in-house algorithms” used in this paper should have been provided, e.g. for Dockscore, PPCheck, etc.Thanks for this comment. Additional details of the in-house algorithms used in this work are now provided in the revised version.Author response: With the new simulation data, I am mostly satisfied with the authors’ revised manuscript, and I think it is a nice body of work, with one continuing exception. I still cannot agree with the section on modelling the linker between N-terminal and TIR domain. Ab initio modelling of a 200 residue segment based on fold-recognition alone, in the absence of similar template structures, is, as I stated previously, pure fantasy, in my opinion. This is compounded by the likely intrinsically disordered nature of the segment. I note that I am not alone in having this opinion, as reviewer 2 states, “Structural modeling of the N-terminal domain-intermediate region-TIR domain does not add new insights. I suggest deleting this section.” The author response was that “we trust it is still better than *not* having any structure of the full-length TRIF.” I cannot agree with this philosophy - in fact, this is precisely the opposite of my own judgement. Publishing something that is likely to be wrong is worse than publishing nothing at all.Our response: In order to honour the referee’s continued concern on this point, we are happy to remove this part – middle domain modelling and normal mode analysis of the full-length protein out of our manuscript.Additional file 1:Appendix S1. List of organisms used for co-evolution analysis (DOCX 16 kb)Additional file 2: Figure S1.The concatenated multiple sequence alignment of the N-terminal domain and TIR domain sequences from different organisms used for predicting co-evolving pair of residues. The region of the MSA corresponding to the N-terminal domain sequences is highlighted in purple while the region corresponding to the TIR domain is highlighted in green. Different colours for different co-evolving pair of residues with one colour for each pair have also been marked on the alignment. (PDF 4 MB)Additional file 3: Table S1.Co-evolving pair of residues predicted by CMAT. The N-terminal domain corresponds to residues 1 to 153, while residues 154–349 make up the TIR domain in the concatenated multiple sequence alignment. (DOCX 16 kb)Additional file 4:Figure S2a. TIR domain of TRIF showing the position of the BB loop, B helix and the RK site. Figure S2b. Electrostatic surface potential representation of the TRIF TIR domain. (ZIP 17210 kb)Additional file 5: Figure S3.Multiple sequence alignment of the N-terminal domain of TRIF from mammals, birds and fishes. Conserved polar residues, clustering around Regions 1 and 2 are marked in light brown and cyan colours respectively. (TIF 23000 kb)Additional file 6: Table S2.Summary of interface residues identified in the different models generated by semi-guided docking using the Cluspro docking program. (DOCX 13 kb)Additional file 7: Figure S4.Our model of the N-terminal protease-resistant domain docked onto the TIR domain. The N-terminal domain is coloured in purple and the TIR domain in green. (TIF 3811 kb)Additional file 9:Figure S5a. A plot showing the variation in backbone RMSD along the trajectory, for each of the three replicates. Figure S5b. Plot of the radius of gyration for each of the three replicates. Figure S5c. The root mean square fluctuations of backbone atoms, averaged over the last 200 ns (800 ns-1000 ns) of the simulations, for each replicate. (ZIP 13857 kb)Additional file 10:Figure S6a. Profile of hydrogen bonds between Asp 21 on the N-terminal domain and Gln 443 on the TIR domain of TRIF along the MD trajectory. Figure S6b. Hydrogen bonds between Asp 21 and Leu 442 along the trajectory. Figure S6c. Hydrogen bonds between Lys 22 and Gln 471 over the MD trajectory. Figure S6d. Hydrogen bonds between Gln 20 and BB loop residue Glu 429. (ZIP 4528 kb)Additional file 11:Figure S7a. The hydrogen bond energy at the interface of the N-TIR complex calculated by PPCheck from different snapshots extracted every 1 ns from the trajectory, for all three replicates. Figure S7b. Variation in PPCheck-derived electrostatic energy at the interface during the course of the three MD simulations. Figure S7c:. van der Waals energies at the interface, calculated using PPCheck. Figure S7d. Total stabilizing energy at the interface. Figure S7e. Relative variation in the number of residues present at the interface. Figure S7f. Normalized energy per residue, as calculated using the PPCheck algorithm, at the interface. (ZIP 3290 kb)Additional file 12: Figure S8.Predicted co-evolving pairs of residues mapped onto the N-TIR docked complex. The colouring scheme follows the same scheme as in Additional file Additional file 13:Model coordinates of the N-TIR complex are provided as a Supplementary Material. (PDB 236 kb)Additional file 14: Figure S9.Our model of the mechanism of TRIF autoinhibition. (a). Upon TLR3 activation, the 522R/523K (RK) site of the TIR domain of TRIF binds to the dimerised TIR domains of the TLR3 dimer. (b). This induces a conformational change mediated by the long-range interactions between the BB loop and RK site of TRIF, exposing the BB loop. (c). This facilitates oligomerisation of TRIF leading to downstream signalling. (TIF 272014 kb)


We have isolated two orthologs expressed in the choanoflagellate Monosiga brevicollis, a unicellular relative to the metazoans. Consistent with its highly-conserved three-dimensional structure, the SH2 domain of M. brevicollis crka1 can bind to the mammalian CRK/CRKL SH2 binding consensus phospho-YxxP, and to the SRC substrate/focal adhesion protein BCAR1 (p130CAS) in the presence of activated SRC. These results demonstrate an ancient origin of the CRK/CRKL SH2-target recognition specificity. Although BCAR1 orthologs exist only in metazoans as identified by an N-terminal SH3 domain, YxxP motifs, and a C-terminal FAT-like domain, some pre-metazoan transmembrane proteins include several YxxP repeats in their cytosolic region, suggesting that they are remotely related to the BCAR1 substrate domain. Since the tyrosine kinase SRC also has a pre-metazoan origin, co-option of BCAR1-related sequences may have rewired the crka-dependent network to mediate adhesion signals in the metazoan ancestor.CRK and CRKL adapter proteins play essential roles in development and cancer through their SRC homology 2 and 3 (SH2 and SH3) domains. To gain insight into the origin of their shared functions, we have investigated their evolutionary history. We propose a term,  CRK was originally identified in CT10 avian sarcoma virus. Despite the fact that CRK does not encode a tyrosine kinase, v-CRK can increase phosphotyrosine contents in the cellChicken Tumor 10 Regulator of Kinases. In humans, the CRK gene family has two paralogous members, CRK and CRKL (CRK-Like), on chromosomes 17p13 and 22q11, respectively. CRKL is likely associated with DiGeorge syndrome2CRK has a direct involvement in congenital disorders. Overexpression of CRK or CRKL has been linked to a subset of some cancer types such as ovarian and non-small cell lung cancerCrk and Crkl are broadly expressed, and are required for normal development in mice25678The oncogene v-CRK and CRKL encode adapter proteins consisting of an N-terminal SRC homology 2 (SH2) domain followed by two SH3 domains, SH3n and SH3cCRK gene generates a full-length product  and a shorter product (isoform b or CRK-I lacking the SH3c domain similar to v-CRK). CRKL generates only a full-length form in humans. The SH2 domain binds to protein tyrosine kinase (PTK) substrates such as BCAR1 as well as receptor tyrosine kinases (RTK) such as PDGFRA and receptor-associated scaffold proteins such as GAB and DOKCrk or Crkl impairs activation of corresponding small G-proteins56111Both Monosiga brevicollis as well as the filasterea Capsaspora owczarzaki may have an elaborate network of PTK-activated signaling, evidenced by an expansive repertoire of tyrosine-specific protein kinases as well as proteins with phosphotyrosine-binding domains such as SH2 and PTB (Phospho-Tyrosine-Binding) domains141517Some components of the metazoan PTK network initially evolved in unicellular ancestors. The choanoflagellate M. brevicollisCapsaspora, but not in choanoflagellatesFAK, SRC, ABL, and their paralogous members play essential roles in integrin-induced signal transduction in mammalian cells1920CAS (CRK-associated substrate), was first discovered as a major v-CRK and SRC binding protein2425262325Among numerous PTK substrates in vertebrate cells, BCAR1 protein, widely known as p130CRK/CRKL ancestral gene evolved before the divergence of the metazoan ancestor, and that the SH2 domain of the CRK/CRKL ancestral protein already had the ability to recognize YxxP motifs, an SH2 binding consensus initially discovered with human CRKEvolution of the signaling network requires co-evolution of interactions after inventions of the primal network components. Our studies demonstrate that the M. brevicollis genome (release 1.0) predicted that two genetic loci, MONBRDRAFT_25437 and MONBRDRAFT_25438, may encode for M. brevicollis orthologs of CRK15crka1 and crka2 . One of the two transcripts identified  is a product consistent with splicing  is a product without splicing. Sequence alignments demonstrate that crka1 and crka2 differ from the mRNA models posted at NCBI, XM_001745882 and XM_001745746, respectively, in particular in the 5’ end sequences extending into the SH2 domain , to refer to invertebrate CRK/CRKL orthologs and avoid identifying them as either CRK or CRKL. The presence of crka not only in basal metazoans such as the placozoa Trichoplax adhaerens and sponge Amphimedon queenslandica but also in the pre-metazoan species Salpinogoeca rosetta, Monosiga brevicollis, and Capsaspora owczarzaki clearly indicates that the CRK gene family has a pre-metazoan origin.Despite minor differences, these trees indicate that all vertebrate species have two distinct genes,  homolog . The clain 22q11 . TherefoM. brevicollis crka1  to determine physical binding of the M. brevicollis crka1 SH2 domain with selected 21 phosphotyrosyl peptides previously reported to associate with mammalian CRK or CRKL SH2 domains253435363738M. musculus Dok7 (Y406) and Bcar1  bind the crka1 SH2 domain with KD values ranging 8–10 μM  in 293 cells, although the level of association was much lower than that of human CRK . However, similar BLAST search strategies did not identify a bcar1 ortholog in the choanozoa or C. owczarzaki genome , and the other is on chromosome 5 (XM_009860485). While these two are also annotated as crk-like at NCBI, our alignment and phylogenetic analysis produce long-branch attraction artifacts if they were included (not shown). In addition, a closely related urochordate, Ciona savignyi, has only one ortholog that corresponds to crka in the Ensembl database (ENSCSAVG00000010659). Hence, we excluded the two additional loci from the current study, and called them crkb and crkc, respectively. It is possible that the crkb and crkc loci accumulated extensive sequence degeneration after they were duplicated in the C. intestinalis lineage. It is noteworthy that C. intestinalis chromosome 12 contains several genes that are orthologous to human chromosomes 17 or 22 and to Xenopus tropicalis genomic scaffolds to which crk and crkl are localized  were present in the pre-metazoan species29M. brevicollis (XP_001742769) and C. owczarzaki (XP_004345254) nor in two basal metazoans, Amphimedon queenslandica and Trichoplax adhaerens . The conserved YxxP motifs exist in zebrafish paxillin (XP_005172507), but not in Hydra vulgaris, S. kowalevskii, or Ciona intestinalis . On a related note, paralogous members of the human paxillin family, leupaxin (LPXN) and HIC5 (TGFB1I1), do not have YxxP motifs that correspond to paxillin Y31 and Y118. Therefore, it is likely that Bcar1 has been a major player in mediating metazoan PTK signals until co-option has recruited paxillin to participate in networking with CRK and CRKL at focal adhesions in vertebrates.Our current study has offered evidence that the focal adhesion protein M. brevicollis crka1 SH2 to the conserved binding consensus phospho-YxxP, although it does not exclude the possibility that pre-metazoan crka SH2 domains may have distinct binding preferences. We speculate that a recruitment of crka to bcar1 may have become an integral mechanism to mediate cell community-based signaling in the metazoan ancestor likely through co-option of bcar1 as a multi domain signaling center . In these sequences, we identified a significant difference between the Xenopus tropicalis crkl homolog sequences available at NCBI and Ensembl. We chose the sequence from Ensembl as it was more in line with the criteria described below. In the species in which genes are not fully annotated, tblastn was used to identify candidate nucleotide sequences . When available, genomic information was also used to confirm RNA sequences. In species in which database information provided no or partial information, we sequenced EST plasmids to obtain full sequence information, or isolated cDNA using RT-PCR from RNA samples obtained from the organism.CRK and/or CRKL homologs have been annotated in RefSeq databases for several species, although some sequences are still listed as “provisional” at NCBI  by 147 bases corresponding to approximately half the SH2 domain  were identified from a single gene, we chose the longest transcript for phylogenic analysis.Total RNA samples were obtained from Nicole King for the choanoflagellate P. marinus or M. brevicollis per manufacturer’s protocol. Crk/Crkl homologs were then amplified by high-fidelity PCR with PrimeSTAR (TAKARA) using specific-primers designed for the Gateway cloning system (Invitrogen). Isolated cDNA fragments as well as full-length EST plasmids were sequenced for both upper and lower strands at the University of Chicago Sequencing Core Facility.Reverse transcriptase  was used with oligo dT primers to generate cDNA pools from total RNA isolated from www.uniprot.org . Geneious program suite (Biomatters) was used to visualize trees and alignments.GUIDANCE2 server was used with MAFFT alignment options (\-\-genafpair \-\-maxiterate 1000) to generate iterative nucleotide and protein alignments and to provide a confidence score for each aligned column4951M. brevicollis crka1 were generated initially as polyhistidine-tagged proteins as describedThe SH2 domains of human CRK, CRKL, and M. brevicollis crka1 was synthesized as above, then labeled with 13C and 15N using the dialysis mode of the Escherichia coli cell-free protein synthesis system2H11 Tris–HCl buffer, pH 7.0, 100 mM NaCl, 0.02% (w/v) NaN3, and 1 mM 2H10 dithiothreitol) was prepared with Vivaspin 2 Concentrator MWCO 5000 PES (Sartorius). All NMR experiments were performed at 298 K on 600, 700, or 800 MHz Avance spectrometers (Bruker) equipped with CryoProbes. Acquired NMR spectra for the chemical shift assignments and the distance constraints were 1H-15N HSQC, 1H-13C HSQC, HNCO, HN(CA)CO, HNCA, HN(CO)CA, HNCACB, CBCA(CO)NH, HBHA(CO)NH, C(CCO)NH, HC(C)H-TOCSY, (H)CCH-TOCSY, HC(C)H-COSY, (HB)CB(CGCD)HD, 15N-edited NOESY-HSQC (80 ms mixing time), and 13C-edited NOESY-HSQC (80 ms mixing time). The NMR spectra were processed and analyzed as previously described15N labeled proteinsFor structure determinations, the SH2 domain of Synthesized phosphotyrosyl peptides (95% or greater purity by HPLC) were purchased from Toray Research Center. Based on a pilot experiment with different peptide lengths, we found that 8-mer was the minimal length to obtain reliable ITC and NMR titration results (not shown). Isothermal titration calorimetry (ITC) was performed in MicroCal Auto-iTC200  at 25 °C as described in the manufacturer’s instruction manual. The SH2 domain and peptide were dissolved in 25 mM HEPES, pH7, 100 mM NaCl at a final concentration of 40 μM or 400 μM, respectively. Titration was carried out by injecting the peptide solution into the reaction cell holding the SH2 domain containing solution every 150 seconds. The results were analyzed by the software ORIGIN 7 supplied by the manufacturer.M. brevicollis crka1 was subcloned into a 3′ in-frame cloning site of a CMV-driven humanized GFP vector to generate GFP-Mb crka1. GFP-Hs CRK plasmid was constructed by subcloning the cDNA into pEGFP C1 plasmid (Clontech/TAKARA). HA-Mm Bcar1 is a mouse Bcar1 tagged with a hemagglutinin (HA)-epitope at the N-terminus and subcloned into a CMV expression plasmid. A constitutively active mouse Src in which the negative regulatory tyrosine Y529 was replaced with a phenylalanine residue, Src(Y529F), was subcloned into a Pol2 promoter-driven expression plasmidHH . HA-tag, GFP, Src, and Rapgef1 (C3G) were probed in Western blots using anti-HA , anti-GFP , anti-Src , and anti-C3G , respectively.The human Embryonic Kidney 293 cell line  was used to construct a heterologous expression system by plasmid transfection. Monosiga brevicollis crka1 and crka2, Saccoglossus kowalevskii crka, Branchiostoma floridae crka1 and crka2, and Petromyzon marinus crkl have been assigned GenBank accession numbers . In addition to these sequences, Ciona intestinalis crka (cima833d10) transcript sequence and the GENSCAN-predicted coding sequence for Trichoplax adhaerens crka. The NMR structure data for Monosiga brevicollis crka1 SH2 domain have been assigned Protein Data Bank (PDB) ID number 2RVF.The mRNA (cDNA) sequences of How to cite this article: Shigeno-Nakazawa, Y. et al. A pre-metazoan origin of the CRK gene family and co-opted signaling network. Sci. Rep.6, 34349; doi: 10.1038/srep34349 (2016).


Immunoprecipitated full length TRPA1 was shown to be phosphorylated by Cdk5 and this interaction was blocked by TFP5, an inhibitor that prevents activation of Cdk5. In vitro peptide-based kinase assay revealed that four of six TRPA1 Cdk5 consensus sites acted as substrates for Cdk5, and modeling of the ankyrin repeats disclosed that phosphorylation would occur at characteristic pockets within the (T/S)PLH motifs. Calcium imaging of trigeminal ganglion neurons from genetically engineered mice overexpressing or lacking the Cdk5 activator p35 displayed increased or decreased responsiveness, respectively, to stimulation with the TRPA1 agonist allylisothiocyanate (AITC). AITC-induced chemo-nociceptive behavior was also heightened in vivo in mice overexpressing p35 while being reduced in p35 knockout mice. Our findings demonstrate that TRPA1 is a substrate of Cdk5 and that Cdk5 activity is also able to modulate TRPA1 agonist-induced calcium influx and chemo-nociceptive behavioral responses.Cyclin-dependent kinase 5 (Cdk5) is a key neuronal kinase that is upregulated during inflammation, and can subsequently modulate sensitivity to nociceptive stimuli. We conducted an  Cdk5 is unlike other cyclin-dependent kinases because it is mostly active in post-mitotic neurons. Additionally, Cdk5 is not activated by a cyclin, but, instead, binds to two regulatory subunits, either p35 or p39, that are primarily restricted to neurons. Cdk5 activity is upregulated following peripheral administration of inflammatory agents such as carrageenan and Complete Freund’s Adjuvant (CFA)3. Increased Cdk5 activity can, in turn, promote both mechanical and heat hyperalgesia2. Cdk5 activity can also affect morphine tolerance, where reduced Cdk5 activity correlates with delayed tolerance4. Therapeutically, intrathecal injection of the Cdk5 inhibitor roscovitine appears to attenuate CFA-induced heat hyperalgesia but not mechanical allodynia3 while also reducing formalin-induced nociceptive behavior5. Cdk5 substrates include P/Q-type voltage-dependent calcium channels, which control calcium influx during nociception, and N-methyl-D-aspartate (NMDA) receptors, which relay pain signaling via the excitatory neurotransmitter glutamate4. Cdk5, however, is most directly linked to pain signaling by phosphorylating the pain transducing ion channel Transient Receptor Potential Vanilloid 1 (TRPV1). Cdk5 phosphorylates TRPV1 at T407, which, in turn, reduces channel desensitization7. Because of its role in modifying pain sensitivity, we wanted to screen for potential Cdk5 phosphorylation targets specifically enriched within pain-transmitting TRPV1+ nociceptors11. Our in silico search identified multiple potential macromolecular substrates, but the chemo-nociceptive channel Transient Receptor Potential Ankyrin 1 (TRPA1) was of particular interest for having 6 potential Cdk5 phosphorylation sites  located within its intracellular N-terminal ankyrin repeat domain. TRPA1 is also of interest for being associated with inflammatory and neuropathic pain12 and for its genetic link to familial episodic pain syndrome13.Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase that can modulate pain signaling14 where it is primarily regarded as a “chemo-sensor”15. TRPA1 is activated by not only natural pungent plant products  but also by pollutants such as acrolein and formalin16. Endogenous activators of TRPA1 include the 4-hydroxynonenal aldehyde17 and the prostaglandin 15d-PGJ2 (15-deoxy-Δ-prostaglandin J2). These noxious chemicals are capable of binding to and activating TRPA1, which leads to acute pain. In addition, TRPA1 contributes to pathological pain and mediates mechanical and cold hyperalgesia22 following inflammation or nerve injury. Such pathological pain mediated by TRPA1 can be induced by multiple mechanisms. A variety of pro-inflammatory mediators25 or endogenous ligands18 can sensitize or activate TRPA1. Inflammation can cause upregulation of TRPA1 expression or sensitization of TRPA1 through phosphorylation via induction of downstream protein kinases26. Protein kinase A and Protein kinase C, for example, enhance activation of TRPA1 in vitro, which can then be attenuated by mutation of the prospective phosphorylated residues28. However, the mechanisms of TRPA1 sensitization through phosphorylation under pathophysiological conditions are largely unclear.TRPA1 is expressed in peripheral C-fiber nociceptors29 in TRPA1 essentially overlap with a T/SxxH motif that is found in about 41% of ankyrin repeats30. Ankyrin repeats mainly contain a stereotypic motif composed of a pair of α-helices, a finger loop, and a β-hairpin. The T/SxxH tetrapeptide located in the ankyrin repeats aids in establishing the helix–turn–helix conformation in the ankyrin repeat while also promoting intra-molecular hydrogen bonds to confer conformational stability. Ankyrin repeats are thought to be involved in protein-protein interactions, but have also been suspected to display elastic qualities31. A unique feature that distinguishes TRPA1 from other TRP channels is the large number of cytoplasmic ankyrin repeats. The N-terminal domain of TRPA1 contains a total of 17 ankyrin repeats that are thought to confer sensitivity to chemical irritants32 and are believed to be essential for plasma membrane localization33. Another feature within the N-terminal cytoplasmic domain is a number of reactive cysteines 34 that are thought to covalently bond to electrophilic compounds, thereby allowing TRPA1 to respond to multiple chemical irritants.Within TRPA1, all six potential Cdk5 phosphorylation sites are situated in the N-terminal cytoplasmic domain. All of the typical consensus Cdk5 sites of (S/T)PX(K/H/R)in vitro kinase assay. The immunopreciptiated TRPA1 was phosphorylated by Cdk5 and this modification could be blocked using an inhibitory peptide that disrupts Cdk5/p35 interactions35. To identify which of the 6 candidate sites were most likely phosphorylated by Cdk5, we screened 10 residue amino acid peptides containing the Cdk5 consensus sequence in an in vitro kinase assay and determined that 4 of the 6 potential Cdk5 phosphorylation motifs were highly phosphorylated. Calcium imaging studies in cultured trigeminal ganglion (TG) neurons from mice with genetically altered Cdk5 activity showed that Cdk5 enzymatic activity modulated the number of responsive neurons to the TRPA1 agonist allylisothiocyanate (AITC). Lastly, mice with increased Cdk5 activity had increased aversion to consumption of AITC, while the converse was true for mice with decreased Cdk5 activity. These data are consistent with the idea that Cdk5 can modulate noci-responsive ion channel activity of primary afferent neurons through post-translational phosphorylation with subsequent change in the sensitivity of the nociceptive neurons to multiple pain modalities.In order to determine if TRPA1 was a substrate of Cdk5, we immunoprecipitated a FLAG-tagged version TRPA1 for testing in an 37. While Cdk5 is highly expressed in all neuronal tissue, most identified candidate substrates have not been shown to be directly applicable to nociception, even though Cdk5 is a major regulator of pain signaling1. Within the dorsal root ganglia (DRG), Cdk5 is uniformly expressed throughout all types of sensory neurons10. The Cdk5 activator p35 is also expressed regularly throughout all DRG neurons, suggesting that further restriction of targets to the nociceptive subpopulation9 would be more informative. To specifically identify pain-related Cdk5 substrates, we narrowed our search of candidates to a nociceptive subset of neurons. This subset can be broadly identified by genetic labeling of the TRPV1-lineage since TRPV1 expression precedes full developmental differentiation of the various nociceptive subpopulations. We cross-referenced a RNA-Seq database of genes highly expressed in the TRPV1-lineage (TRPV1L) for proteins that contain the typical (S/T)PX(K/H/R) phosphorylation motif that is found in 49% of Cdk5 substrates29 . All of the potential Cdk5 sites in TRPA1 essentially consist of a T/SxxH tetrapeptide motif that is common to 41% of ankyrin repeats30. The structure of TRPA1 has recently been modeled using single-particle cryo-electron microscopy (CryoEM), and this three-dimensional structural data was used to determine if these T/SxxH containing ankyrin repeats within TRPA1 were structurally permissive as phosphorylation sites for Cdk540. Because the TRPA1 CryoEM structure lacks information on ankyrin repeat domains 1 to 11, we constructed a model of these ankyrin repeats 41, noxious chemical accomodating sites, reactive cysteines, and the structurally conserved consensus ankyrin repeat sequence.TRP channels have been implicated in sensory physiology to detect environmental cues such as temperature, light, and chemicals, and several members of the TRP family are known to have ankyrin repeats (AR) in their N-terminal domainsats Fig.  linked t485 phosphorylation site in further detail 34.After determining that TRPA1 was a substrate of Cdk5, the next objective was to determine which of the six potential Cdk5 sites in TRPA1 were most amenable to phosphorylation. We, therefore, conducted an due Fig. . We had und Fig. . In conttro Fig. . Both th242 Fig. , which i2+ influx in cultured trigeminal ganglia (TG) neurons. We first verified the co-expression of TRPA1 with Cdk5 and p35 in cultured neurons using immunofluorescence. In Fig. 47. Like Cdk5, p35 is co-expressed with TRPA1 in dissociated TG sensory neurons as well  or Tgp35 mice (a transgenic mouse line overexpressing p35 that has a >50% increase in Cdk5 activity)2. To determine the extent of TRPA1 activation in dissociated neurons, we used allyl isothiocyanate (AITC), a pungent plant compound found in mustard oil. AITC is an electrophilic agonist of TRPA1 that covalently binds to the reactive cysteines Cys415, Cys422, and Cys622 in the mouse TRPA134. In particular, Cys415 (Cys414 in humans) is thought to be important for channel activation with both cysteine and non-cysteine reactive TRPA1 agonists48 and this electrophilic site is intriguingly situated adjacent to the potential Cdk5 phosphorylation site T416. Cultured trigeminal neurons from p35KO and Tgp35 mice were loaded with Fura 2-AM, tested with a low concentration of AITC, washed, bathed in a high concentration of AITC, washed, then treated with 30 mM KCl to determine the total excitable neuronal component of the culture  but the difference was not statistically significant  in the drinking water. Mice were first habituated to the lickometer with water only prior to testing with AITC. During the training sessions, we observed no change in the number of licks between the different genotypes of mice and their corresponding controls  to conditionally knockout Cdk5 (Cdk5CoKo)6. We further investigated the extent to which Cdk5 phosphorylation could be a multifactorial regulator of nociceptive inputs by trying to identify new Cdk5 substrates that are highly expressed in nociceptive afferents, particularly the TRPV1+ set of ganglionic neurons which are critical for the detection of many painful modalities, especially heat50. Within the general population of nociceptive neurons, we identified multiple candidate proteins that contain putative Cdk5 phosphorylation sites, which could then affect the excitability of these neurons, and thereby modulate the nociceptive tone. Out of this group, we focused on TRPA1 because further examination revealed six potential sites that were in the same general location within the N-terminal ankyrin repeat-containing domains. TRPV1 is also regulated by Cdk56 and our analyses of the single cell DRG data sets show approximately 50% coexpression, further supporting a role for Cdk5 phosphorylation in regulating these two key transducers of noxious stimuli in multiple subpopulations of afferent nerve terminals.Upregulated Cdk5 activity within primary sensory neurons modulates pain sensitivity by directly adjusting the responses of these neurons to painful stimuli51. TRP channels, in general, are known to be modulated by phospholipase C (PLC), as the sensitization of TRPA1 following stimulation of either PAR2 or the bradykinin receptor 2 (B2R) is mediated by PLC activity25. TRPA1 channel activity induced by bradykinin is inhibited by protein kinase A inhibitors, and activation of either PKA or PLC leads to membrane trafficking of TRPA152. Recently, TRPA1 was shown to be phosphorylated by PKA at the amino acid residues S86, S317, S428, and S972, which, in turn, has a sensitizing effect on TRPA127. Brackley et al. concurrently demonstrated PKA-mediated phosphorylation of TRPA1, with the mouse S87 residue being key to sensitizing mustard oil responses. In addition, Brackley et al. showed phosphorylation of TRPA1 by protein kinase C (PKC) as well, where the S119, T281, and T529 PKC sites are essential for sensitization28. During both PKA and PKC-mediated phosphorylation of TRPA1, the scaffolding protien AKAP (A-Kinase Anchoring Protein 79/150) plays an essential role in anchoring these kinases to TRPA1.TRPA1 is a pain-transducing ion channel that is primarily known as a chemo-sensor in nociceptive neurons, but is also implicated in cold and mechanical hypersensitivity31. Structural modeling revealed a stereotypical pocket within these ankyrin repeats that can accept addition of the phosphate group. Using in vitro kinase assays, we demonstrated that recombinant full-length TRPA1 is a substrate for Cdk5, and identified which putative potential Cdk5 sites could act as preferred substrates for phosphorylation. These sites are highly conserved in both mammals and lower organisms such as mosquitos and fruit flies where TRPA1 serves as a thermosensor54.During our bioinformatics search for Cdk5 substrates associated with pain signaling, we observed that TRPA1 has many putative Cdk5 sites within its ankyrin repeats, which could potentially modify channel opening or conductance and, ultimately, modulate the efficacy of signal transduction through this channel. The six potential Cdk5 phosphorylation sites in TRPA1 are all located in the ankyrin repeats that contain a canonical pattern of [G-(X)-TPLH-(X)-A-(X3)-G-(X7)-LL-(X2)-GA-(X5)]2+ imaging on dissociated cultured AITC-responsive nociceptors from our mouse models with genetically altered levels of p35, but with endogenous expression of TRPA1 and Cdk5. We observed that primary neurons from p35 knockout mice, which lack the required cofactor for Cdk5 activity, exhibited a reduced percentage of responders to AITC PLH consensus motif within the ankyrin repeats of TRPA1 were predicted to be Cdk5 phosphorylation sites at a high stringency level. The amino acid analogous to the human TRPA1 T673, however, is an alanine (A675) in the mouse TRPA1 PLH motif common to many ankyrin repeats can be highly phosphorylated by Cdk5. Our in vitro kinase data suggests that these tetrapeptide motifs are still the most likely Cdk5 phosphorylation sites, but further analysis needs to be conducted to determine which ankyrin repeat phosphorylation site is most critical for sensitizing TRPA1.During the course of our study, Hynkova + neurons in the DRG suggest a range between 3.6% to 32.4% in rodents20. TRPA1 expression, however, can also be increased following tissue injury20. Taking advantage of the two available single cell transcriptomic murine data sets58 a comparative analysis suggests that 43% of DRG neurons express TRPA1 in the mouse. Lennertz et al.51 suggest that there may be a nociceptor population of C-Mechano Cold (CMC) fibers involved in mechanical hyperalgesia that express TPRA1 only, while inflammatory heat hypersensitivity occurs via a separate population of neurons expressing TRPV1. Other studies, however, show that TRPA1+ neurons predominantly co-express TRPV159. Our transcriptome analysis shows that TRPA1 is highly enriched in TRPV1+ neurons10. Co-expression of both of these TRP channels in nociceptive neurons is additionally supported by examples of cross-desensitization between TRPV1 and TRPA1 agonists, as capsaicin pretreatment in rats reduces subsequent AITC responses and vice versa60. Bautista et al.61 also proposed that TRPV1-mediated calcium influx activates TRPA1 downstream of bradykinin signaling, which is known to promote TRPA1 sensitization. Weng et al.62 showed that Tmem100 links TRPV1 and TRPA1 in nociceptive neurons, which then promotes TRPA1 Ca2+ influx by weakening the association between these two channels to promote TRPA1 activity. Conditional deletion of Tmem100 was, therefore, able to reduce inflammatory-induced mechanical hypersensitivity but did not alter heat responses. In contrast to Tmem100-related trafficking of these two TRP channels, TNF-α signaling was also shown to promote plasma membrane localization of both TRPA1 and TRPV1, mediated by Munc18-1/syntaxin1/Snap-25 complexes63. Interestingly, TNF-α signaling is known to induce p35 expression, which, in turn, increases Cdk5 activity47. The resulting active Cdk5/p35 complex could then, in turn, phosphorylate both Munc1864 to regulate exocytosis, and the ion channels TRPV16 and TRPA1, which subsequently encourages Ca2+ influx and depolarization. With TRPA1 primarily localized within many TRPV1+ neurons14 and both of these pain-transducing ion channels acting as Cdk5 substrates, we decided to develop a gene co-expression map of putative Cdk5 substrates using single cell RNA-sequencing data from mouse dorsal root ganglia  TRPV1-expressing lineage as determined by previously published RNA-Seq datasets of this tissue11 . The theoretical model of TRPA1 was obtained from The School of Theoretical Modeling where it was generated using methods described previously73. The homology with ankyrin repeats of TRPV1 and TRPV2 was used as an aid in the contrstruction of our model of AR1 to 11 because of similarities to the repeats in TRPA1  and in a predicted model of the ankyrin repeat domain .Atomic coordinates of proteins determined using structural data from the Brookhaven Protein Databank (PDB)2/5 mM EDTA/50 μM NaF/5 μM Na2VO3/5 mM DTT) containing either 10 μg of histone H1 (Sigma-Aldrich), immunoprecipitated TRPA1, or the following mouse TRPA1 peptides : TRPA11 YGNTPLHCAA, TRPA12 NMMSPLHIAV, TRPA13 KKASPLHLAV, TRPA14 DGCTPLHYAC, TRPA15 DKKSPLHFAA, TRPA16 HGMTPLHLAA. Kinase assays were carried out at 30 °C for 60 min by adding 5 μCi of [γ-32P] ATP (0.5 mM). The peptide assay was stopped by adding 10% trichloroacetic acid to precipitate the proteins. 20-μl aliquots of trichloroacetic acid supernatant were transferred onto P81 phosphocellulose squares (spotted in duplicates), air-dried, and washed five times for 15 min each in 75 mM phosphoric acid and once in 95% ethanol. After air drying, squares were transferred to vials containing Bio-Safe II scintillation fluid  for counting in a Beckman Coulter  scintillation counter (model no. SL 3801).Recombinant full-length human Cdk5 and p35  were incubated in 45 μl of kinase assay buffer (100 mM Tris·HCl [pH 7.4]/50 mM MgCl20. TRPA1 expression was confirmed using the following antibodies: FLAG M2 (Sigma-Aldrich), TRPA1 , p35 , Cdk5 , and Actin . Immunoprecipitation of FLAG tagged TRPA1 was performed using ANTI-FLAG M2 Magnetic Beads (Sigma-Aldrich), according to the manufacturer’s protocol.FLAG tagged and untagged mouse TRPA1 expression vectors were purchased from Origene . The mouse neuroblastoma cell line Neuro-2a was cultured in DMEM and 10% fetal bovine serum and the cells were transfected with the TRPA1 constructs using Neuro-2a Cell Avalanche Transfection Reagent . Western blot was performed as previously describedTrigeminal ganglia (TG) neurons were plated onto Lab-Tek 8 chamber cover-slides  coated with laminin/poly-D-lysine (Sigma-Aldrich) with F12 medium (Thermo Fisher Scientific) containing 10% FBS  and 1% penicillin/streptomycin (Thermo Fisher Scientific). The following day, cultured cells were fixed in 4% paraformaldehyde  and permeabilized for 10 minutes with 0.1% Triton X-100 (Sigma-Aldrich). Cells were probed with the following primary antibodies: TRPA1 , p35 (C-19) and Cdk5 (C-8) (both Santa Cruz Biotechnology). After several washes with PBS (Thermo Fisher Scientific), cultured neurons were incubated with secondary antibodies conjugated with either Alexa 488 or Alexa 647  and finally labeled with rhodamine-phalloidin  for 20 minutes. All the images were acquired using a Zeiss LSM 700 microscope.6 where an SNS (NaV1.8) promoter-driven Cre is used to conditionally delete Cdk5 in small-diameter nociceptive neurons. All animals were housed in standard cages in climate- and light-controlled rooms with free access to food and water. All experimental procedures were approved by the Animal Care and Use Committee of the National Institute of Dental and Craniofacial Research, National Institutes of Health and we adhered to the guidelines of the IASP Committee for Research and Ethical Issue74.p35 knockout (p35KO) and Cdk5 conditional knockout (Cdk5CoKo) mice were maintained in a C57BL/6:129/SvJ background. Transgenic p35 (Tgp35) mice were conserved in an FVB/N background. Age-matched wild-type mice served as controls. C-fiber neuron-specific Cdk5CoKo mice were generated as previously described2+ imaging was performed as previously described75. Essentially, the trigeminal ganglia (TG) were extracted from p35KO and Tgp35 mice and their respective wild type (WT) littermate controls. Minced TG were incubated in Ca2+-free, Mg2+-free HBSS with 1 mg/ml collagenase type IV  at 37 °C for 30 min with agitation. EDTA, DNAse, and 2.5% trypsin were then added and the tissues were incubated for an additional 15 min at 37 °C. Following enzymatic digestion, TG neurons were centrifuged, resuspended in culture medium, and mechanically dissociated using a descending diameter firepolished glass micropipettes. Dissociated neurons were plated onto glass coverslips (8-mm) coated with poly-ornithine and laminin with DMEM/F12 medium containing 10% FBS and 1% penicillin/streptomycin. The following day, cultured neurons were loaded with Fura2-AM and pluronic acid for 45 minutes at 37 °C in calcium imaging buffer . Ratiometric Ca2+ imaging was performed using an inverted fluorescence microscope , an excitation filter changer , and a CCD camera (Nikon Instrument). Paired images  were collected every 2 s with NIS Element (Nikon Instrument). The Fura response was defined as the ratio of emissions measured during excitation at 340 and 380 nm, and the relative Fura response was defined as the ratio normalized to the five ratio values prior to the application of drug. Conversion of Fura ratios to absolute [Ca2+]i was performed following system calibration76 using a calcium calibration kit . The amplitude of response was calculated by fold increase from baseline level. The response level was defined as the level 3 standard deviations above the mean of the nonresponsive peak, which was 1.15 times (15% increase) greater than their average baseline response prior to the application of AITC. The percentage of responders was calculated by normalizing the number of responders to the number of KCl responders in entire neurons derived from one mouse. AITC-evoked Ca2+ responses were various in different experiments. To control for the effects of different experimental conditions, we tested a pair of transgenic mouse and a littermate WT in the same day. The averaged response amplitudes and percentage of responders were obtained from each experiment and data from 6 to 7 mice were compared. In the p35 KO mice, a total of 217 neurons from 7 mice (31 ± 7.0 neurons per mouse) were analyzed. In the corresponding C57BL/6 littermate WT controls, a total 238 neurons from 7 mice (34 ± 9.7 neurons per mouse) were analyzed. In Tgp35 mice, a total 231 neurons from 6 mice (34 ± 8.4 neurons per mouse) were analyzed. In the corresponding littermate FVB/N WT mice, a total 204 neurons from 6 mice (38.5 ± 6.1 neurons per mouse) were analyzed.CaAn operant lickometer test was used to test nociceptive responses to noxious oral stimuli. Nociceptive sensitization was induced by different concentrations of the TRPA1 agonist AITC . Water-deprived (15 h) animals were placed in the lickometer cages  where a computer-operated system monitored their licking behavior for a period of 1 hour. Initially, the animals were tested with water (N = 5 sessions). Afterwards, consumption/aversion to water with different concentrations of AITC was monitored (N = 5 sessions per single AITC concentration). All mice were tested at the same time each day and then retested under the same conditions on non-consecutive days.All data are expressed as mean ± SEM. The statistical evaluation was done with GraphPad Prism 7 software . Statistical differences between the groups were assessed using either ANOVA or an unpaired t-test with significance level was set at p < 0.05.58 . The threshold for positive gene expression was 5 FPKM (fragments per kilobase of transcript per 106 mapped reads). Network visualization was constructed with R software using package ‘igraph’77. RNA-sequencing data from whole tissue rat DRG2  was used to quantitate DRG expression levels for putative Cdk5 substrates.The gene coexpression network was built using publicly available RNA-sequencing data of mouse dorsal root ganglionSupplementary Information


Dear Editor,Deleted in colorectal cancer (DCC) is a single pass transmembrane glycoprotein that was originally identified in humans as a candidate tumor suppressor  in complex with netrin-1. The structure revealed two distinct binding sites located between DCCFN56 and netrin-1  . The two structures are complementary to each other, encompassing FN4, FN5, and FN6 domains of DCC, interacting with the netrin-1 N-terminal LN domain and all three of the netrin-1 EGF domains  was predominantly expressed, whereas between D10.5 and D11.5 of embryogenesis, the full-length DCClong isoform (with 28-residue linker) mRNA was upregulated  repeats (herein referred to as SGG-22) between the FN4 and FN5 domains. We have crystallized and analyzed the structure of this DCCFN456 construct. From the structure, we discuss how a consolidated FN5 can serve as the key netrin-1 binding domain, and how a closed configuration, where the FN4 domain “lies” on the rod-like FN5-FN6 domains, can be proposed as a resting state of the DCC receptor for stability.Located between the functional DCCFN456 with different linkers of different length between FN4 and FN5. The cell-binding assay has been previously utilized to describe interactions between DCC and netrin-1 in the binding site 1 and binding site 2 . This construct shows significantly reduced binding to human netrin-1 7. The P21 crystal structure was determined at 3.0 Å resolution with an Rfree/R = 0.223/0.278 via molecular replacement. The search models used were the DCCFN56 domain (PDB 4URT)   domains, they form modular structures as components of cell surface receptors that function in the immune system as well as the nervous system. Unlike Ig-like domains though, FN domains do not have disulfide bonds between the two opposing β sheets. Nevertheless, the FN domains have an invariant tryptophan at the center of the β strand B in place of the cysteine in the Ig-like domain that assists to form a hydrophobic core to stabilize the FN domain. Still, compared to the Ig-like domains, the FN domain is less robust and may be subject to mechanical deformation  of DCC, which has ten domains in tandem located on the cell surface.A high proportion of cell-surface receptors are modular. Usually, there will be just a couple of residues in between domains to provide some flexibility to the receptors. However, there are also cases where a long enough linker between domains exists, like the one between the FN4 and FN5 domains of DCC. Apparently, this kind of long linker is prone to be cleaved in the extra-cellular environment. If such a long linker does specifically serve a function, there might likely be a way to protect it from cleavage in the unliganded state. One previously reported example is found in the human fibronectin, where there is a 21-residue linker between its first and second type III FN domains Below is the link to the electronic supplementary material.