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

ABSTRACT: 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.

SUBMITTER: Bousova K

PROVIDER: S-EPMC8645431 | biostudies-literature |

REPOSITORIES: biostudies-literature

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TRPM6 N-Terminal CaM- and S100A1-Binding Domains.

Project description: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.

| S-EPMC6770577 | biostudies-literature

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

Project description: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.

| S-EPMC7352223 | biostudies-literature

ATXN1 N-terminal region explains the binding differences of wild-type and expanded forms.

Project description:BACKGROUND:Wild-type (wt) polyglutamine (polyQ) regions are implicated in stabilization of protein-protein interactions (PPI). Pathological polyQ expansion, such as that in human Ataxin-1 (ATXN1), that causes spinocerebellar ataxia type 1 (SCA1), results in abnormal PPI. For ATXN1 a larger number of interactors has been reported for the expanded (82Q) than the wt (29Q) protein. METHODS:To understand how the expanded polyQ affects PPI, protein structures were predicted for wt and expanded ATXN1, as well as, for 71 ATXN1 interactors. Then, the binding surfaces of wt and expanded ATXN1 with the reported interactors were inferred. RESULTS:Our data supports that the polyQ expansion alters the ATXN1 conformation and that it enhances the strength of interaction with ATXN1 partners. For both ATXN1 variants, the number of residues at the predicted binding interface are greater after the polyQ, mainly due to the AXH domain. Moreover, the difference in the interaction strength of the ATXN1 variants was due to an increase in the number of interactions at the N-terminal region, before the polyQ, for the expanded form. CONCLUSIONS:There are three regions at the AXH domain that are essential for ATXN1 PPI. The N-terminal region is responsible for the strength of the PPI with the ATXN1 variants. How the predicted motifs in this region affect PPI is discussed, in the context of ATXN1 post-transcriptional modifications.

| S-EPMC6814966 | biostudies-literature

Structures of CaV2 Ca2+/CaM-IQ domain complexes reveal binding modes that underlie calcium-dependent inactivation and facilitation.

Project description:Calcium influx drives two opposing voltage-activated calcium channel (Ca(V)) self-modulatory processes: calcium-dependent inactivation (CDI) and calcium-dependent facilitation (CDF). Specific Ca(2+)/calmodulin (Ca(2+)/CaM) lobes produce CDI and CDF through interactions with the Ca(V)alpha(1) subunit IQ domain. Curiously, Ca(2+)/CaM lobe modulation polarity appears inverted between Ca(V)1s and Ca(V)2s. Here, we present crystal structures of Ca(V)2.1, Ca(V)2.2, and Ca(V)2.3 Ca(2+)/CaM-IQ domain complexes. All display binding orientations opposite to Ca(V)1.2 with a physical reversal of the CaM lobe positions relative to the IQ alpha-helix. Titration calorimetry reveals lobe competition for a high-affinity site common to Ca(V)1 and Ca(V)2 IQ domains that is occupied by the CDI lobe in the structures. Electrophysiological experiments demonstrate that the N-terminal Ca(V)2 Ca(2+)/C-lobe anchors affect CDF. Together, the data unveil the remarkable structural plasticity at the heart of Ca(V) feedback modulation and indicate that Ca(V)1 and Ca(V)2 IQ domains bear a dedicated CDF site that exchanges Ca(2+)/CaM lobe occupants.

| S-EPMC2701236 | biostudies-literature

N-terminal region of Drosophila melanogaster Argonaute2 forms amyloid-like aggregates.

Project description:BackgroundArgonaute proteins play a central role in RNA silencing by forming protein-small RNA complexes responsible for the silencing process. While most Argonaute proteins have a short N-terminal region, Argonaute2 in Drosophila melanogaster (DmAgo2) harbors a long and unique N-terminal region. Previous in vitro biochemical studies have shown that the loss of this region does not impair the RNA silencing activity of the complex. However, an N-terminal mutant of Drosophila melanogaster has demonstrated abnormal RNA silencing activity. To explore the causes of this discrepancy between in vitro and in vivo studies, we investigated the biophysical properties of the region. The N-terminal region is highly rich in glutamine and glycine residues, which is a well-known property for prion-like domains, a subclass of amyloid-forming peptides. Therefore, the possibility of the N-terminal region functioning as an amyloid was tested.ResultsOur in silico and biochemical assays demonstrated that the N-terminal region exhibits amyloid-specific properties. The region indeed formed aggregates that were not dissociated even in the presence of sodium dodecyl sulfate. Also, the aggregates enhanced the fluorescence intensity of thioflavin-T, an amyloid detection reagent. The kinetics of the aggregation followed that of typical amyloid formation exhibiting self-propagating activity. Furthermore, we directly visualized the aggregation process of the N-terminal region under fluorescence microscopy and found that the aggregations took fractal or fibril shapes. Together, the results indicate that the N-terminal region can form amyloid-like aggregates.ConclusionsMany other amyloid-forming peptides have been reported to modulate the function of proteins through their aggregation. Therefore, our findings raise the possibility that aggregation of the N-terminal region regulates the RNA silencing activity of DmAgo2.

| S-EPMC10114355 | biostudies-literature

Targets of DNA binding, N-terminal, or C-terminal domain swapped mutant forms fo Foxa proteins in induced hepatocyte-like (iHep) cells.

Project description:We have found that three specific combinations of two transcription factors, comprising Hnf4alpha plus Foxa1, Foxa2 or Foxa3, could convert mouse embryonic fibroblasts (MEFs) into cells that closely resemble hepatocytes in vitro, and DNA binding, N-terminal, and C-terminal domains (DBD, NTD, and CTD, respectively) of Foxa proteins are swappable in the direct reprogramming of the iHep cells. Then we used ChIP-seq to explore the targets of the domain swapped mutant forms of Foxa proteins during conversion event to iHep cells.

2020-08-06 | GSE117332 | GEO

X-ray crystal structure of human calcium-bound S100A1.

Project description:S100A1 is a member of the S100 family of Ca2+-binding proteins and regulates several cellular processes, including those involved in Ca2+ signaling and cardiac and skeletal muscle function. In Alzheimer's disease, brain S100A1 is overexpressed and gives rise to disease pathologies, making it a potential therapeutic target. The 2.25 Å resolution crystal structure of Ca2+-S100A1 is solved here and is compared with the structures of other S100 proteins, most notably S100B, which is a highly homologous S100-family member that is implicated in the progression of malignant melanoma. The observed structural differences in S100A1 versus S100B provide insights regarding target protein-binding specificity and for targeting these two S100 proteins in human diseases using structure-based drug-design approaches.

| S-EPMC5379171 | biostudies-literature

The Activation of Protein Kinase A by the Calcium-Binding Protein S100A1 Is Independent of Cyclic AMP.

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Design of Calcium-Binding Proteins to Sense Calcium.

Project description:Calcium controls numerous biological processes by interacting with different classes of calcium binding proteins (CaBP's), with different affinities, metal selectivities, kinetics, and calcium dependent conformational changes. Due to the diverse coordination chemistry of calcium, and complexity associated with protein folding and binding cooperativity, the rational design of CaBP's was anticipated to present multiple challenges. In this paper we will first discuss applications of statistical analysis of calcium binding sites in proteins and subsequent development of algorithms to predict and identify calcium binding proteins. Next, we report efforts to identify key determinants for calcium binding affinity, cooperativity and calcium dependent conformational changes using grafting and protein design. Finally, we report recent advances in designing protein calcium sensors to capture calcium dynamics in various cellular environments.

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hMSH2 forms specific mispair-binding complexes with hMSH3 and hMSH6.

Project description:The genetic and biochemical properties of three human MutS homologues, hMSH2, hMSH3, and hMSH6, have been examined. The full-length hMSH6 cDNA and genomic locus were isolated and characterized, and it was demonstrated that the hMSH6 gene consisted of 10 exons and mapped to chromosome 2p15-16. The hMSH3 cDNA was in some cases found to contain a 27-bp deletion resulting in a loss of nine amino acids, depending on the individual from which the cDNA was isolated. hMSH2, hMSH3, and hMSH6 all showed similar tissue-specific expression patterns. hMSH2 protein formed a complex with both hMSH3 and hMSH6 proteins, similar to protein complexes demonstrated by studies of the Saccharomyces cerevisiae MSH2, MSH3, and MSH6. hMSH2 was also found to form a homomultimer complex, but neither hMSH3 nor hMSH6 appear to interact with themselves or each other. Analysis of the mismatched nucleotide-binding specificity of the hMSH2-hMSH3 and hMSH2-hMSH6 protein complexes showed that they have overlapping but not identical binding specificity. These results help to explain the distribution of mutations in different mismatch-repair genes seen in hereditary nonpolyposis colon cancer.

| S-EPMC19374 | biostudies-literature

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