Integrative Binding Sites within Intracellular Termini of TRPV1 Receptor

<div><p>TRPV1 is a nonselective cation channel that integrates wide range of painful stimuli. It has been shown that its activity could be modulated by intracellular ligands PIP2 or calmodulin (CaM). The detailed localization and description of PIP2 interaction sites remain unclear. Here, we used synthesized peptides and purified fusion proteins of intracellular regions of TRPV1 expressed in <em>E.coli</em> in combination with fluorescence anisotropy and surface plasmon resonance measurements to characterize the PIP2 binding to TRPV1. We characterized one PIP2 binding site in TRPV1 N-terminal region, residues F189-V221, and two independent PIP2 binding sites in C–terminus: residues K688-K718 and L777-S820. Moreover we show that two regions, namely F189-V221 and L777-S820, overlap with previously localized CaM binding sites. For all the interactions the equilibrium dissociation constants were estimated. As the structural data regarding C-terminus of TRPV1 are lacking, restraint-based molecular modeling combined with ligand docking was performed providing us with structural insight to the TRPV1/PIP2 binding. Our experimental results are in excellent agreement with our <em>in silico</em> predictions.</p> </div

Equilibrium dissociation constants (K_D) and their standard deviations of synthetic peptides (pTRPV1) of three different regions on the cytoplasmic tails binding to Bodipy® FL C5, C6-PtdIns(4,5)P2 estimated by fluorescence anisotropy measurement.

<p>Equilibrium dissociation constants (K_D) and their standard deviations of synthetic peptides (pTRPV1) of three different regions on the cytoplasmic tails binding to Bodipy® FL C5, C6-PtdIns(4,5)P2 estimated by fluorescence anisotropy measurement.</p

Both PIP2 and calmodulin (CaM) shares the binding site within the C-terminal distal region of TRPV1.

<p>(<b>A</b>) SPR kinetic binding of TRPV1–CT and the complex of TRPV1–CT with calmodulin (<b>TRPV1</b>/CaM complex) to the sensor chip coated with PC/PIP2 (80∶20) liposomes. TRPV1-CT and the TRPV1-CT/CaM complex (both at 10 µM concentration) were injected in parallel over the lipid vesicles and the flow rate was maintained at 30 µl/min for both association and dissociation phase. (<b>B</b>) A typical SPR kinetic binding of TRPV1-CT to the PIP2-enriched liposomes followed by independent injection of CaM. TRPV1-CT (2 µM) was injected over the sensor chip coated with PC/PIP2 (80∶20) liposomes, left to dissociate and then calmodulin was injected onto the identical surface at 10 µM concentration. The flow rate was maintained at 30 µl/min during whole experiment. Black and white strips represent association and dissociation phase of the sensogram, respectively.</p

The sequence alignment of the C-terminus of TRPV1 A690 - K838 and the fragile histidine triad protein (FHIT) S2-D150.

<p>Identical amino acids are marked with an asterisk. Similar amino acids with the more important groups are indicated with a colon. Dots indicate similar amino acids of the less important groups that are less likely to influence the protein structure.</p

PIP2 recognizes thee independent binding sites within the TRPV1 receptor.

<p>Steady-state fluorescence anisotropy measurements of interaction between fluorescently labeled phosphatidyl inositol-4, 5-bisphosphate (PIP2-Bodipy) and synthetic peptides corresponding to cytoplasmic tails either at the N-terminal region F189-V221 of TRPV1 (pTRPV1– NT), C terminal proximal region K688-K718 of TRPV1 (pTRPV1–CTp), or C-terminal distal region L777-S820 of TRPV1 (pTRPV1–CTd), respectively. PIP2-Bodipy (10 nM) was titrated with indicated concentrations of the peptides and the bound fraction (F_B) of PIP2 Bodipy was calculated according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048437#pone.0048437.e001" target="_blank">Equation 1</a> as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048437#s4" target="_blank">Material and Methods</a>. The solid lines represent binding isotherms determined by nonlinear least-squares analysis of the isotherm using an <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048437#pone.0048437.e002" target="_blank">Equation 2</a> as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048437#s4" target="_blank">Material and Methods</a>. Values represent the mean ± SD from at least three independent experiments.</p

Analysis of the effects of charge-neutralizing mutations within the TRPV1-CT distal region (712–838) fusion protein on equilibrium dissociation constants determined by surface plasmon resonance experiments.

<p>The presented values are average ± SD from at least 3 independent measurements.</p

PIP2 binds to the C-terminal proximal region of TRPV1.

<p>Steady-state fluorescence anisotropy measurement of interaction between fluorescently labeled phosphatidyl inositol-4, 5-bisphosphate (PIP2-Bodipy) and synthetic peptide corresponding to the cytoplasmic tail at the C terminal proximal region K688-K718 of TRPV1 (pTRPV1–CTp) or its Q700A/R701A (pTRPV1–CTp-Q700A/R701A) and K694A/K698A/K710A (pTRPV1–CTp-K694A/K698A/K710A) mutant variant, respectively. PIP2-Bodipy (10 nM) was titrated with with indicated concentrations of the peptides and the bound fraction (F_B) of PIP2 Bodipy was calculated according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048437#pone.0048437.e001" target="_blank">Equation 1</a> as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048437#s4" target="_blank">Material and Methods</a>. The solid lines represent binding isotherms determined by nonlinear least-squares analysis of the isotherm using an <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048437#pone.0048437.e002" target="_blank">Equation 2</a> as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048437#s4" target="_blank">Material and Methods</a>. Values represent the mean ± SD from at least three independent experiments.</p

PIP2 binds to the C-terminal distal region of TRPV1. A.

<p>Fluorescence anisotropy measurements of interaction between fluorescently labeled phosphatidyl inositol-4, 5-bisphosphate (PIP2-Bodipy) and the distal region of TRPV1 (amino acids 712–838) fusion protein. PIP2-Bodipy (10 nM) was titrated with TRPV1-CT fusion protein WT and the bound fraction (FB) was calculated according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048437#pone.0048437.e001" target="_blank">Equation 1</a> as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048437#s4" target="_blank">Material and Methods</a>. Binding isotherm and the equilibrium dissociation constant KD (3.48+/−0.93 µM) was determined by fitting the data to the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048437#pone.0048437.e002" target="_blank">Equation 2</a> as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048437#s4" target="_blank">Material and Methods</a>. <b>B.</b> Fluorescence anisotropy measurements of interaction between PIP2-Bodipy and thioredoxin. PIP2-Bodipy (10 nM) was titrated with thioredoxin and the bound fraction (FB) of PIP2 Bodipy was calculated as above. <b>C.</b> Steady-state fluorescence anisotropy measurement of interaction between fluorescently labeled phosphatidyl choline (NBD–PC) and TRPV1-CT. NBD-PC (10 nM) was titrated with indicated concentrations of TRPV1-CT fluorescence anisotropy was recorded. Values are expressed as the mean ± standard deviation (SD) measured from at least from six independent experiments.</p

Surface plasmon resonance (SPR) analysis of interactions between TRPV1-CT and PIP2-enriched liposomes.

<p>Kinetic binding measurements of TRPV1-CT (A) and the TRPV1-CT-K770A/R778A/R785A triple mutant (B) to the sensor chip coated with PC/PIP2 (80∶20) liposomes. The proteins at indicated concentrations were injected in parallel over the lipid vesicles and the flow rate was maintained at 30 µl/min for both association and dissociation phases of the sensograms. (C) SPR equilibrium binding of the TRPV1-CT, TRPV1-CT-K770A/R778A/R785A, and TRPV1-CT-R778A proteins to the sensor chip coated with PC/PIP2 (80∶20) liposomes. The proteins were injected at 25 µl/min at different concentrations and washed over the lipid surface and Req values were deduced from steady state (equilibrium) SPR response. The solid lines represent binding isotherms determined by nonlinear least-squares analysis of the isotherm using an equation Req = Rmax/1+Kd/P0), where Req stands for SPR response value near -equilibrium, Rmax is the maximum response and P0 is the protein concentration. Values represent the mean ± S.D from four independent experiments.</p

dCyaA-KP toxoid unable to promote calcium influx and relocate into lipid rafts is rapidly taken up by J774A.1 cells.

<p>(A) J774A.1 cells were loaded with the calcium probe Fura-2/AM at a 3 µM final concentration at 25°C for 30 min. After washing in HBSS medium, toxoid variants (3 µg/ml) or buffer were added at time zero (indicated by arrow) and time course of calcium entry into cytosol of cells (elevation of [Ca²⁺]_i) was followed by spectrofluorimetry <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002580#ppat.1002580-Fiser1" target="_blank">[14]</a>. (B) J774A.1 cells were incubated for 10 minutes with 500 ng/ml of dCyaA or dCyaA-KP and detergent-resistant membrane microdomains (DRMs) were extracted with cold Triton X-100, separated by flotation in sucrose density gradient and probed in Western blots with the 9D4 antibody. The DRM fractions were defined as fractions enriched in the lipid raft marker NTAL. The transferrin receptor CD71 was used as a non-raft marker that remained in the bottom fractions of the gradient. (C) J774A.1 cells grown on glass bottom microwell dishes were incubated for 20 minutes at 37°C with 5 µg/ml of Dyomics 647-labeled dCyaA or Alexa Fluor 488-labeled dCyaA-KP proteins in the presence or absence of the anti-CD11b MAb M1/70 (20 µg/ml). Endocytic uptake of toxoids was analyzed at indicated time points by live cell imaging using an Olympus Cell^R IX 81 microscope with a 60×/1.35 oil objective (UPlanSApo). (D) The numbers of endosomes localized in cytoplasm of cells and loaded with dCyaA or dCyaA-KP were counted as described in detail in the legend to Supplementary <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002580#ppat.1002580.s002" target="_blank">figure S2</a>, using a script based on WCIF ImageJ software (<a href="http://rsb.info.nih.gov/ij" target="_blank">http://rsb.info.nih.gov/ij</a>, <a href="http://www.uhnresearch.ca/facilities/wcif/imagej" target="_blank">http://www.uhnresearch.ca/facilities/wcif/imagej</a>). The plot shows mean values plus standard deviations, as calculated on 20 to 40 cells per time point for one representative experiment out of three performed (n = 3). (E) J774A.1 cells (3×10⁵) were incubated for 30 min on ice at three different toxoid concentrations within the linear range of the dose-response curve (0.5, 1 and 5 µg/ml) and in the presence or absence of the anti-CD11b monoclonal antibody M1/70 (20 µg/ml, 15 min of preincubation of cells). Binding to cells was assessed as cell-associated toxoid fluorescence by FACS analysis. The % of toxoid binding at each concentration was calculated, taking the value for dCyaA toxoid in the absence of M1/70 as 100%. The average ± standard deviations are shown.</p