Transient receptor potential melastatin 7 (TRPM7) represents melastatin TRP channel with two significant functions, cation permeability and kinase activity. TRPM7 is widely expressed among tissues and is therefore involved in a variety of cellular functions representing mainly Mg2+ homeostasis, cellular Ca2+ flickering, and the regulation of DNA transcription by a cleaved kinase domain translocated to the nucleus. TRPM7 participates in several important biological processes in the nervous and cardiovascular systems. Together with the necessary function of the TRPM7 in these tissues and its recently analyzed overall structure, this channel requires further studies leading to the development of potential therapeutic targets. Here we present the first study investigating the N-termini of TRPM7 with binding regions for important intracellular modulators calmodulin (CaM) and calcium-binding protein S1 (S100A1) using in vitro and in silico approaches. Molecular simulations of the discovered complexes reveal their potential binding interfaces with common interaction patterns and the important role of basic residues present in the N-terminal binding region of TRPM.

• Klíčová slova
• Binding region, CaM, Calcium, Fluorescence anisotropy, S100A1, TRPM7,
• Publikační typ
• časopisecké články MeSH

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

Transient receptor potential (TRPs) channels are crucial downstream targets of calcium signalling cascades. They can be modulated either by calcium itself and/or by calcium-binding proteins (CBPs). Intracellular messengers usually interact with binding domains present at the most variable TRP regions-N- and C-cytoplasmic termini. Calmodulin (CaM) is a calcium-dependent cytosolic protein serving as a modulator of most transmembrane receptors. Although CaM-binding domains are widespread within intracellular parts of TRPs, no such binding domain has been characterised at the TRP melastatin member-the transient receptor potential melastatin 6 (TRPM6) channel. Another CBP, the S100 calcium-binding protein A1 (S100A1), is also known for its modulatory activities towards receptors. S100A1 commonly shares a CaM-binding domain. Here, we present the first identified CaM and S100A1 binding sites at the N-terminal of TRPM6. We have confirmed the L520-R535 N-terminal TRPM6 domain as a shared binding site for CaM and S100A1 using biophysical and molecular modelling methods. A specific domain of basic amino acid residues (R526/R531/K532/R535) present at this TRPM6 domain has been identified as crucial to maintain non-covalent interactions with the ligands. Our data unambiguously confirm that CaM and S100A1 share the same binding domain at the TRPM6 N-terminus although the ligand-binding mechanism is different.

• Klíčová slova
• CaM and S100A1, TRPM6, binding domain, calmodulin binding motif, fluorescence anisotropy, molecular modelling,
• MeSH
• kalmodulin chemie   MeSH
• kationtové kanály TRPM chemie   MeSH
• lidé MeSH
• molekulární modely * MeSH
• proteinové domény MeSH
• proteiny S100 chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kalmodulin MeSH
• kationtové kanály TRPM MeSH
• proteiny S100 MeSH
• S100A1 protein MeSH Prohlížeč
• TRPM6 protein, human MeSH Prohlížeč