Characterization of the S100A1 protein binding site on TRPC6 C-terminus.

The transient receptor potential (TRP) protein superfamily consists of seven major groups, among them the "canonical TRP" family. The TRPC proteins are calcium-permeable nonselective cation channels activated after the emptying of intracellular calcium stores and appear to be gated by various types of messengers. The TRPC6 channel has been shown to be expressed in various tissues and cells, where it modulates the calcium level in response to external signals. Calcium binding proteins such as Calmodulin or the family of S100A proteins are regulators of TRPC channels. Here we characterized the overlapping integrative binding site for S100A1 at the C-tail of TRPC6, which is also able to accomodate various ligands such as Calmodulin and phosphatidyl-inositol-(4,5)-bisphosphate. Several positively charged amino acid residues (Arg852, Lys856, Lys859, Arg860 and Arg864) were determined by fluorescence anisotropy measurements for their participation in the calcium-dependent binding of S100A1 to the C terminus of TRPC6. The triple mutation Arg852/Lys859/Arg860 exhibited significant disruption of the binding of S100A1 to TRPC6. This indicates a unique involvement of these three basic residues in the integrative overlapping binding site for S100A1 on the C tail of TRPC6

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

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

Summary of estimated equilibrium dissociation constants of the complex of TRPC6_(801–878)WT and its mutants with S100A1.

<p>Summary of estimated equilibrium dissociation constants of the complex of TRPC6_(801–878)WT and its mutants with S100A1.</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

Amino acid senquence of TRPC6 fusion protein.

<p>Native rat TRPC6 801–878 amino acid sequence containing integrative binding site was investigated. Predicted important basic amino acids that were replaced by alanine are in red.</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

Steady-state fluorescence anisotropy measurement of TRPC6_(801–878) WT and selected mutants to fluorescently labeled S100A1 protein.

<p>DNS- S100A1 protein (232 µM) was titrated with TRPC6 fusion protein and the Fb was calculated using equation 1 as was described in material and methods.Binding isotherms and dissociatin constants were calculated by fitting the data to the equation 2 as was described in material and methods. Values are expressed as the mean ± standard deviation (SD) measured from at least from three independent experiments. Binding isotherms of wild-type TRPC6 _(801–878) is represented as black circles, single mutant is TRPC6_(801–878) R864A as white circles and triple mutant TRPC6_(801–878) K859A/R860A/R864A as black squares.</p