Mapping of CaM, S100A1 and PIP2-Binding Epitopes in the Intracellular N- and C-Termini of TRPM4

Abstract

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.

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

“…TRPM7np (see Material and Methods) contains aromatic and hydrophobic amino acids (Y524–Y528–F533) arranged in 1-5-10 CaM-binding motif, and five interspaced basic amino acids (R525, R530, K531, R532 and R534). Due to the known shared binding epitopes of CaM and S100A1 at e.g., ryanodine (RyR) or TRP receptors [ 22 , 25 , 37 , 37 , 38 ], we also decided to investigate binding site of S100A1 at TRPM7np. The position of TRPM7np in the TRPM7 structure (PDB: 5ZXZ) proves the accessibility of the binding region to intracellular environment and the basic amino acid residues of TRPM7np are exposed out of the TRPM7 structure backbone which suggest suitable condition for complex formation with the ligands ( Figure 2 A–C).…”

“…The S100A1 and CaM binding regions often overlap, and S100A1/CaM can compete for the same binding site on the target protein [ 22 , 39 ]. Based on our previous experience of shared CaM binding regions with S100A1 [ 25 , 37 , 37 , 38 , 39 ] we also investigated the possible binding of S100A1 to the identified CaM-binding epitope to TRPM7np ( Figure 3 A). The interaction of S100A1 with TRPM7np was confirmed by steady state fluorescence anisotropy similar to that described above for CaM binding experiments.…”

“…The multiple sequence alignment analysis of TRPM binding regions revealed consensus sequences of basic amino acid residues (RxxxxR/K, where x is any amino acid) in analysed regions ( Figure 4 ) suggested the importance of R525, R530, K531, R532 and R534 in TRPM7np. This analysis of multiple TRPM sequences was performed from in vitro experimentally characterized TRPM binding regions for CaM and S100A1 [ 25 , 37 , 38 , 40 , 41 ].

Figure 4 Multiple alignment of selected CaM binding regions in TRPM channels.

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“…The multiple sequence alignment analysis of TRPM binding regions revealed consensus sequences of basic amino acid residues (RxxxxR/K, where x is any amino acid) in analysed regions (Figure 4) suggested the importance of R525, R530, K531, R532 and R534 in TRPM7np. This analysis of multiple TRPM sequences was [15,25,37,38,40,41]. The sequence alignment shows a first perfect match (indicated by an asterisk) of arginine residues and second almost accurate match (indicated by dots) of basic residues across the TRPM sequences.…”

“…The number on the right side of the sequence marks its amino acid length. performed from in vitro experimentally characterized TRPM binding regions for CaM and S100A1 [25,37,38,40,41].…”