The evolution of proteins is primarily driven by the combinatorial assembly of a limited set of pre-existing modules known as protein domains. This modular architecture not only supports the diversity of natural proteins but also provides a robust strategy for protein engineering, enabling the design of artificial proteins with enhanced or novel functions for various industrial applications. Among these functions, oligomerization plays a crucial role in enhancing protein activity, such as by increasing the binding capacity of antibodies. To investigate the potential of engineering oligomerization, we examined the transferability of the sequence domain encoded by exon 5 (Ex5), which was originally responsible for the oligomerization of ameloblastin (AMBN). We designed a two-domain protein composed of Ex5 in combination with a monomeric, globular, and highly stable protein, specifically calmodulin (CaM). CaM represents the opposite protein character to AMBN, which is highly disordered and has a dynamic character. This engineered protein, termed eCaM, successfully acquired an oligomeric function, inducing self-assembly under specific conditions. Biochemical and biophysical analyses revealed that the oligomerization of eCaM is both concentration- and time-dependent, with the process being reversible upon dilution. Furthermore, mutating a key oligomerization residue within Ex5 abolished the self-assembly of eCaM, confirming the essential role of the Ex5 motif in driving oligomerization. Our findings demonstrate that the oligomerization properties encoded by Ex5 can be effectively transferred to a new protein context, though the positioning of Ex5 within the protein structure is critical. This work highlights the potential of enhancing monomeric proteins with oligomeric functions, paving the way for industrial applications and the development of proteins with tailored properties.

• Publication type
• Journal Article MeSH

Transient receptor potential melastatin (TRPM) channels, a subfamily of the TRP superfamily, constitute a diverse group of ion channels involved in mediating crucial cellular processes like calcium homeostasis. These channels exhibit complex regulation, and one of the key regulatory mechanisms involves their interaction with calmodulin (CaM), a cytosol ubiquitous calcium-binding protein. The association between TRPM channels and CaM relies on the presence of specific CaM-binding domains in the channel structure. Upon CaM binding, the channel undergoes direct and/or allosteric structural changes and triggers down- or up-stream signaling pathways. According to current knowledge, ion channel members TRPM2, TRPM3, TRPM4, and TRPM6 are directly modulated by CaM, resulting in their activation or inhibition. This review specifically focuses on the interplay between TRPM channels and CaM and summarizes the current known effects of CaM interactions and modulations on TRPM channels in cellular physiology.

• Keywords
• TRPM channels, calcium homeostasis, calmodulin, calmodulin binding site, regulation,
• MeSH
• Calmodulin * metabolism   MeSH
• TRPM Cation Channels * metabolism   MeSH
• Calcium-Binding Proteins metabolism   MeSH
• Calcium metabolism   MeSH
• Calcium Signaling MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Calmodulin * MeSH
• TRPM Cation Channels * MeSH
• Calcium-Binding Proteins MeSH
• Calcium MeSH

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.

• Keywords
• Binding region, CaM, Calcium, Fluorescence anisotropy, S100A1, TRPM7,
• Publication type
• Journal Article 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.

• Keywords
• CaM, PIP2, S100A1, TRPM4 channel, binding epitope, docking, fluorescence anisotropy, molecular dynamics simulations,
• MeSH
• Aquaporins chemistry   metabolism   MeSH
• Protein Interaction Domains and Motifs * MeSH
• Calmodulin chemistry   metabolism   MeSH
• TRPM Cation Channels chemistry   metabolism   MeSH
• Kinetics MeSH
• Protein Conformation MeSH
• Humans MeSH
• Models, Molecular MeSH
• Multiprotein Complexes chemistry   metabolism   MeSH
• Peptide Fragments MeSH
• S100 Proteins chemistry   metabolism   MeSH
• Amino Acid Sequence MeSH
• Protein Binding MeSH
• Binding Sites * MeSH
• Structure-Activity Relationship MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Aquaporins MeSH
• Calmodulin MeSH
• TRPM Cation Channels MeSH
• Multiprotein Complexes MeSH
• Peptide Fragments MeSH
• S100 Proteins MeSH
• S100A1 protein MeSH Browser
• TRPM4 protein, human MeSH Browser

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.

• Keywords
• CaM and S100A1, TRPM6, binding domain, calmodulin binding motif, fluorescence anisotropy, molecular modelling,
• MeSH
• Calmodulin chemistry   MeSH
• TRPM Cation Channels chemistry   MeSH
• Humans MeSH
• Models, Molecular * MeSH
• Protein Domains MeSH
• S100 Proteins chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Calmodulin MeSH
• TRPM Cation Channels MeSH
• S100 Proteins MeSH
• S100A1 protein MeSH Browser
• TRPM6 protein, human MeSH Browser

The transient receptor potential (TRP) protein superfamily consists of seven major groups, among them the "canonical TRP" family. The TRPC proteins are calcium-permeable nonselective cation channels activated after the emptying of intracellular calcium stores and appear to be gated by various types of messengers. The TRPC6 channel has been shown to be expressed in various tissues and cells, where it modulates the calcium level in response to external signals. Calcium binding proteins such as Calmodulin or the family of S100A proteins are regulators of TRPC channels. Here we characterized the overlapping integrative binding site for S100A1 at the C-tail of TRPC6, which is also able to accomodate various ligands such as Calmodulin and phosphatidyl-inositol-(4,5)-bisphosphate. Several positively charged amino acid residues (Arg852, Lys856, Lys859, Arg860 and Arg864) were determined by fluorescence anisotropy measurements for their participation in the calcium-dependent binding of S100A1 to the C terminus of TRPC6. The triple mutation Arg852/Lys859/Arg860 exhibited significant disruption of the binding of S100A1 to TRPC6. This indicates a unique involvement of these three basic residues in the integrative overlapping binding site for S100A1 on the C tail of TRPC6.

• MeSH
• Anisotropy MeSH
• Circular Dichroism MeSH
• Protein Interaction Domains and Motifs MeSH
• TRPC Cation Channels chemistry   genetics   MeSH
• TRPC6 Cation Channel MeSH
• Humans MeSH
• Molecular Sequence Data MeSH
• Mutagenesis, Site-Directed MeSH
• S100 Proteins chemistry   MeSH
• Protein Structure, Secondary MeSH
• Amino Acid Sequence MeSH
• Amino Acid Substitution MeSH
• Calcium chemistry   MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• TRPC Cation Channels MeSH
• TRPC6 Cation Channel MeSH
• S100 Proteins MeSH
• S100A1 protein MeSH Browser
• TRPC6 protein, human MeSH Browser
• Calcium MeSH