Molecular determinants of the binding of various endogenous modulators to transient receptor potential (TRP) channels are crucial for the understanding of necessary cellular pathways, as well as new paths for rational drug designs. The aim of this study was to characterise interactions between the TRP cation channel subfamily melastatin member 4 (TRPM4) and endogenous intracellular modulators-calcium-binding proteins (calmodulin (CaM) and S100A1) and phosphatidylinositol 4, 5-bisphosphate (PIP2). We have found binding epitopes at the N- and C-termini of TRPM4 shared by CaM, S100A1 and PIP2. The binding affinities of short peptides representing the binding epitopes of N- and C-termini were measured by means of fluorescence anisotropy (FA). The importance of representative basic amino acids and their combinations from both peptides for the binding of endogenous TRPM4 modulators was proved using point alanine-scanning mutagenesis. In silico protein-protein docking of both peptides to CaM and S100A1 and extensive molecular dynamics (MD) simulations enabled the description of key stabilising interactions at the atomic level. Recently solved cryo-Electron Microscopy (EM) structures made it possible to put our findings into the context of the entire TRPM4 channel and to deduce how the binding of these endogenous modulators could allosterically affect the gating of TRPM4. Moreover, both identified binding epitopes seem to be ideally positioned to mediate the involvement of TRPM4 in higher-order hetero-multimeric complexes with important physiological functions.

• Klíčová slova
• CaM, PIP2, S100A1, TRPM4 channel, binding epitope, docking, fluorescence anisotropy, molecular dynamics simulations,
• MeSH
• akvaporiny chemie   metabolismus   MeSH
• interakční proteinové domény a motivy * MeSH
• kalmodulin chemie   metabolismus   MeSH
• kationtové kanály TRPM chemie   metabolismus   MeSH
• kinetika MeSH
• konformace proteinů MeSH
• lidé MeSH
• molekulární modely MeSH
• multiproteinové komplexy chemie   metabolismus   MeSH
• peptidové fragmenty MeSH
• proteiny S100 chemie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• vazba proteinů MeSH
• vazebná místa * MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• akvaporiny MeSH
• kalmodulin MeSH
• kationtové kanály TRPM MeSH
• multiproteinové komplexy MeSH
• peptidové fragmenty MeSH
• proteiny S100 MeSH
• S100A1 protein MeSH Prohlížeč
• TRPM4 protein, human MeSH Prohlížeč