Conformational Plasticity of TRPV1 Ankyrin Repeat Domain in Complex with Cysteine Reactive Agonist Allicin

Abstract

Chemosensation is a signal transduction process in which exogenous compounds interact with receptors and give a taste, odor or other sensation. The TRPV1 ion channel is one of the most important chemoreceptors associated with pain. This channel is activated by pungent compounds like capsaicin (Chili peppers), low pH, high temperature, allicin (Garlic) and others. Allicin (diallylthiosulfinate) is non polar compound that reacts with cysteines forming an allylcystine.The rat TRPV1 is activated by allicin reacting with the cisteine at position 157 generating calcium entry into cells. C157 is located in the N-terminal Ankyrin Repeat Domain (ARD). The ARD crystallographic structure showed a hidden C157, but reactivity of this supposes an alternative conformation were C157 is more accessible. The conformational changes of the TRPV1 structure after the modification with allicin are not known. In this study we used the wild type TRPV1 ARD to investigate the reaction of allicin with the ARD using circular dichroism, fluorescence spectroscopy and molecular modeling to assess the reaction rate and the degree of perturbation to the ARD structure. Our findings show that pure allicin in complex with ARD gives a reaction that showed changes into the far-UV circular dichroism spectra. Mainly the peak at 190 nm shows an increment, presumably due to an increment in alpha helical content after covalent modification of C157. Fluorescence spectra showed a shifted signal from tryptophane 272 from 332 nm to 339 nm, suggesting a conformational change that exposed W272. Accessible surface area analysis of C157 in crystallographic ARD showed a hidden cysteine into the second motif of the ARD. We used molecular dynamics of the ARD in solution to look for a conformation with an accessible path for allicin.