Detailed Analysis of the Binding Mode of Vanilloids to Transient Receptor Potential Vanilloid Type I (TRPV1) by a Mutational and Computational Study

<div><p>Transient receptor potential vanilloid type 1 (TRPV1) is a non-selective cation channel and a multimodal sensor protein. Since the precise structure of TRPV1 was obtained by electron cryo-microscopy, the binding mode of representative agonists such as capsaicin and resiniferatoxin (RTX) has been extensively characterized; however, detailed information on the binding mode of other vanilloids remains lacking. In this study, mutational analysis of human TRPV1 was performed, and four agonists (capsaicin, RTX, [6]-shogaol and [6]-gingerol) were used to identify amino acid residues involved in ligand binding and/or modulation of proton sensitivity. The detailed binding mode of each ligand was then simulated by computational analysis. As a result, three amino acids (L518, F591 and L670) were newly identified as being involved in ligand binding and/or modulation of proton sensitivity. In addition, <i>in silico</i> docking simulation and a subsequent mutational study suggested that [6]-gingerol might bind to and activate TRPV1 in a unique manner. These results provide novel insights into the binding mode of various vanilloids to the channel and will be helpful in developing a TRPV1 modulator.</p></div

Kinetic responses of wild-type and mutant TRPV1 in the Ca²⁺ flux assay.

<p>Treatment with 5 μM capsaicin was performed at 20 s (indicated by dotted line). Because the responses of wild-type TRPV1 in each experiment were variable, the results are shown as four individual plots obtained from each experiment. Wild-type TRPV1 and all mutants except W549A responded clearly to capsaicin. The traces show representative mean data (n = 2) from 3 independent experiments.</p

EC₅₀ values of vanilloids for Y511F and S512A TRPV1 mutants.

<p>EC₅₀ values of vanilloids for Y511F and S512A TRPV1 mutants.</p

Candidate amino acid residues evaluated by mutational analysis.

<p>(A) The locations of residues thought to be involved in ligand binding are shown on the primary structure of TRPV1. C158 is thought to be a modification site of certain TRPV1 agonists [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162543#pone.0162543.ref017" target="_blank">17</a>]. Other candidate residues are located in the TM3–TM6 region of TRPV1. Candidate amino acids colored in blue are expected to contribute to the binding pocket from the adjacent monomer. (B) The locations of candidate residues are shown on the tertiary structure of the human TRPV1 model (“modified 3J5R model”; see main text). Left, whole structure of human TRPV1 viewed from the extracellular side; right, structure of the binding pocket. Each monomer is shown in a different color. The side chain of each candidate amino acid is displayed as a stick representation. Carbon atoms are depicted in the same color as the main chain. Red and gray indicate oxygen and hydrogen atoms, respectively. For clarity, non-polar hydrogen atoms are not shown. Candidate amino acids on the adjacent monomer are colored brown.</p

Influence of point mutations on proton sensitivity.

<p>(A) Kinetic response in the Ca²⁺ flux assay. Although 12 cells were evaluated in the same experiment, the results are divided into two graphs for clarity. 50 μL of D-PBS (pH 5.5) solution was added to 100μL of cells in D-PBS (pH 7.4) solution at the 20-s time point. Data are expressed as the mean from n = 3 measurements. (B) Activation of wild-type and mutant TRPV1 channels by capsaicin and proton. Maximum response in the period after stimulation was used as the representative value. W549A did not respond to either 1 μM capsaicin (grey) or proton addition (pH5.5, black). Y511A showed diminished ligand sensitivity, but maintained proton sensitivity. The F587A, F591A and L670A mutations markedly decreased proton sensitivity. Data are expressed as the mean ± SEM from n = 3 measurements.</p

Heat map of the protein–ligand interaction fingerprint.

<p>Values refer to % abundance (i.e., percentage of overall abundance in the cluster). Sur, surface contact interactions (red); Don, side chain atoms of amino acids acting as H-bond donors (blue); Acc, side chain atoms of amino acids acting as H-bond acceptors (blue). The green and brown bars on the left indicate amino acids belonging to different monomers. The residues shown in bold type were evaluated by mutational analysis in this study.</p

EC₅₀ values of four vanilloids for various TRPV1 mutants.

<p>EC₅₀ values of four vanilloids for various TRPV1 mutants.</p

Ca²⁺ flux assay in Y511F and S512 mutants for capsaicin and [6]-gingerol.

<p>(A) The potency of capsaicin against TRPV1 was decreased a little for Y511F (red) but unchanged for S512A (blue). (B) Although the S512A mutation did not affect the potency of [6]-gingerol, a rightward shift of the activation curve was observed for the Y511F mutation. The activation curves are representative mean ± SEM data (n = 3) from 3 (S512A) or 4 (Y511F) independent experiments.</p

Influence of point mutations on ligand sensitivity.

<p>The influence of point mutations on the EC₅₀ value is represented as a heat map. The ratio of each EC₅₀ value to that of wild-type TRPV1 is shown. To estimate the ratio for ligands with an EC₅₀ value of “>100,000” in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162543#pone.0162543.t001" target="_blank">Table 1</a>, 100,000 was divided by the corresponding EC₅₀ value for wild-type TRPV1. Some mutations (L518A, L547A and T550A) influenced potency in a ligand-dependent manner. Data are expressed as the mean ± SEM from n = 3–4 measurements.</p

Influence of point mutations on [³H]-RTX binding.

<p>Whole-cell fractions were prepared from CHO-K1 cells expressing wild-type or mutant TRPV1, and incubated with 1,000 or 10,000 pM [³H]-RTX. Non-specific binding was evaluated in the presence of 1 μM RTX. For Y511A, F587A, F591A and L670A, specific binding of [³H]-RTX was lost or decreased. Only wild-type TRPV1 showed specific binding in 1,000 pM [³H]-RTX. Data are expressed as the mean ± SEM (n = 3).</p