Integrative binding sites within intracellular termini of TRPV1 receptor

Abstract

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 E.coli 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 in silico predictions.

Figure 1. PIP2 binds to the C-terminal distal region of TRPV1. A.

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 Equation 1 as described in Material and Methods. Binding isotherm and the equilibrium dissociation constant KD (3.48+/−0.93 µM) was determined by fitting the data to the Equation 2 as described in Material and Methods. 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. C. 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.

Figure 2. Surface plasmon resonance (SPR) analysis of interactions between TRPV1-CT and PIP2-enriched liposomes.

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.

Figure 3. SPR kinetic binding of TRPV1-CT to PIP2-enriched liposomes (A) and to liposomes made from phosphatidyl choline (PC) (B).

Both PIP2-enriched (PIP2/PC 80∶20) and PC liposomes were immobilized to the sensor chip at the same density (∼1000 RU), and the TRPV1-CT protein at indicated concentrations was injected in parallel over the lipid vesicles at flow rate of 30 µl/min.

Figure 4. PIP2 recognizes thee independent binding sites within the TRPV1 receptor.

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 Equation 1 as described in Material and Methods. The solid lines represent binding isotherms determined by nonlinear least-squares analysis of the isotherm using an Equation 2 as described in Material and Methods. Values represent the mean ± SD from at least three independent experiments.

Figure 5. Both PIP2 and calmodulin (CaM) shares the binding site within the C-terminal distal region of TRPV1.

(A) SPR kinetic binding of TRPV1–CT and the complex of TRPV1–CT with calmodulin (TRPV1/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) 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.

Figure 6. PIP2 binds to the C-terminal proximal region of TRPV1.

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 Equation 1 as described in Material and Methods. The solid lines represent binding isotherms determined by nonlinear least-squares analysis of the isotherm using an Equation 2 as described in Material and Methods. Values represent the mean ± SD from at least three independent experiments.

Figure 7. Model of interactions of TPRV1 – CT (V746 - K838) with PIP2. A.

Model of TRPV1-CT V746 - K838 generated by homology modeling using Modeller 9v9 software. This model is based on known structure of fragile histidine triad (FHIT) protein from Serin 2 to Aspartate 150 (pdb. accession number: 1FIT). The primary structure of this protein shows a high degree of similarity (44%). B. Conformation of the TRPV1 – CT (V746 - K838)/PIP2 complexes after the initial docking using Autodock4 software. Searching was done along the whole surface of the TRPV1 molecule. All suggested TRPV1-CT V746 - K838/PIP2 molecule interaction states are visualized. C. Detailed view of the region of interest of TRPV1 – CT (V746 - K838) determined in the initial docking step. Residues hydrogen bonded to PIP2 are highlighted namely R778, R781, R785. All side chains are shown as sticks. The colors representation is following: the backbone of the protein (grey), carbons (yellow), phosphorus (orange) and oxygen (red).

Figure 8. The sequence alignment of the C-terminus of TRPV1 A690 - K838 and the fragile histidine triad protein (FHIT) S2-D150.

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.