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.

2nd Faculty of Medicine Charles University 5 Uvalu 84 150 06 Prague 5 Czech Republic

Faculty of Mathematics and Physics Charles University Ke Karlovu 5 121 16 Prague 2 Czech Republic

Institute of Microbiology of the Czech Academy of Sciences Videnska 1083 142 20 Prague 4 Czech Republic

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Flemingovo namesti 2 160 00 Prague 6 Czech Republic

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Oligomerization Function of the Native Exon 5 Sequence of Ameloblastin Fused with Calmodulin

Interaction of Calmodulin with TRPM: An Initiator of Channel Modulation

TRPM7 N-terminal region forms complexes with calcium binding proteins CaM and S100A1