Unveiling the intricate role of S100A1 in regulating RyR1 activity: A commentary on "Structural insights into the regulation of RyR1 by S100A1".

S100A1, a calcium-binding protein, plays a crucial role in regulating Ca2+ signaling pathways in skeletal and cardiac myocytes via interactions with the ryanodine receptor (RyR) to affect Ca2+ release and contractile performance. Biophysical studies strongly suggest that S100A1 interacts with RyRs but have been inconclusive about both the nature of this interaction and its competition with another important calcium-binding protein, calmodulin (CaM). Thus, high-resolution cryo-EM studies of RyRs in the presence of S100A1, with or without additional CaM, were needed. The elegant work by Weninger et al. demonstrates the interaction between S100A1 and RyR1 through various experiments and confirms that S100A1 activates RyR1 at sub-micromolar Ca2+ concentrations, increasing the open probability of RyR1 channels.

Structural insights into the regulation of RyR1 by S100A1.

S100A1, a small homodimeric EF-hand Ca2+-binding protein (~21 kDa), plays an important regulatory role in Ca2+ signaling pathways involved in various biological functions including Ca2+ cycling and contractile performance in skeletal and cardiac myocytes. One key target of the S100A1 interactome is the ryanodine receptor (RyR), a huge homotetrameric Ca2+ release channel (~2.3 MDa) of the sarcoplasmic reticulum. Here, we report cryoelectron microscopy structures of S100A1 bound to RyR1, the skeletal muscle isoform, in absence and presence of Ca2+. Ca2+-free apo-S100A1 binds beneath the bridging solenoid (BSol) and forms contacts with the junctional solenoid and the shell-core linker of RyR1. Upon Ca2+-binding, S100A1 undergoes a conformational change resulting in the exposure of the hydrophobic pocket known to serve as a major interaction site of S100A1. Through interactions of the hydrophobic pocket with RyR1, Ca2+-bound S100A1 intrudes deeper into the RyR1 structure beneath BSol than the apo-form and induces sideways motions of the C-terminal BSol region toward the adjacent RyR1 protomer resulting in tighter interprotomer contacts. Interestingly, the second hydrophobic pocket of the S100A1-dimer is largely exposed at the hydrophilic surface making it prone to interactions with the local environment, suggesting that S100A1 could be involved in forming larger heterocomplexes of RyRs with other protein partners. Since S100A1 interactions stabilizing BSol are implicated in the regulation of RyR-mediated Ca2+ release, the characterization of the S100A1 binding site conserved between RyR isoforms may provide the structural basis for the development of therapeutic strategies regarding treatments of RyR-related disorders.

S100 Proteins-Intracellular and Extracellular Function in Norm and Pathology.

The S100 proteins are small, ubiquitous, mostly homodimeric proteins containing two EF-hand structures, that is, helix-loop-helix motifs specialized in high-affinity calcium-binding (~10-6 M) [...].

Calcium mediated static and dynamic allostery in S100A12: Implications for target recognition by S100 proteins.

Structure and functions of S100 proteins are regulated by two distinct calcium binding EF hand motifs. In this work, we used solution-state NMR spectroscopy to investigate the cooperativity between the two calcium binding sites and map the allosteric changes at the target binding site. To parse the contribution of the individual calcium binding events, variants of S100A12 were designed to selectively bind calcium to either the EF-I (N63A) or EF-II (E31A) loop, respectively. Detailed analysis of the backbone chemical shifts for wildtype protein and its mutants indicates that calcium binding to the canonical EF-II loop is the principal trigger for the conformational switch between 'closed' apo to the 'open' Ca2+ -bound conformation of the protein. Elimination of binding in S100-specific EF-I loop has limited impact on the calcium binding affinity of the EF-II loop and the concomitant structural rearrangement. In contrast, deletion of binding in the EF-II loop significantly attenuates calcium affinity in the EF-I loop and the structure adopts a 'closed' apo-like conformation. Analysis of experimental amide nitrogen (15 N) relaxation rates (R1 , R2 , and 15 N-{1 H} NOE) and molecular dynamics (MD) simulations demonstrate that the calcium bound state is relatively floppy with pico-nanosecond motions induced in functionally relevant domains responsible for target recognition such as the hinge domain and the C-terminal residues. Experimental relaxation studies combined with MD simulations show that while calcium binding in the EF-I loop alone does not induce significant motions in the polypeptide chain, EF-I regulates fluctuations in the polypeptide in the presence of bound calcium in the EF-II loop. These results offer novel insights into the dynamic regulation of target recognition by calcium binding and unravels the role of cooperativity between the two calcium binding events in S100A12.

FLIM-FRET-based analysis of S100A11/annexin interactions in living cells.

Proteins achieve their biological functions in cells by cooperation in protein complexes. In this study, we employed fluorescence lifetime imaging microscopy (FLIM)-based Förster resonance energy transfer (FRET) measurements to investigate protein complexes comprising S100A11 and different members of the annexin (ANX) family, such as ANXA1, ANXA2, ANXA4, ANXA5, and AnxA6, in living cells. Using an S100A11 mutant without the capacity for Ca2+ binding, we found that Ca2+ binding of S100A11 is important for distinct S100A11/ANXA2 complex formation; however, ANXA1-containing complexes were unaffected by this mutant. An increase in the intracellular calcium concentration induced calcium ionophores, which strengthened the ANXA2/S100A11 interaction. Furthermore, we were able to show that S100A11 also interacts with ANXA4 in living cells. The FLIM-FRET approach used here can serve as a tool to analyze interactions between S100A11 and distinct annexins under physiological conditions in living cells.

Modeling the Annexin A1-S100A11 heterotetramer: a molecular dynamics investigation of structure and correlated motion.

Annexin A1 (A1) has been shown to form a tetrameric complex (A1t) with S100A11 which is implicated in calcium homeostasis and EGFR pathways. In this work, a full-length model of the A1t was generated for the first time. Multiple molecular dynamics simulations were performed on the complete A1t model for several hundred nanoseconds each to assess the structure and dynamics of A1t. These simulations yielded three structures for the A1 N-terminus (ND) which were identified via principal component analysis. The orientations and interactions of the first 11 A1-ND residues for all three structures were conserved, and their binding modes were strikingly similar to those of the Annexin A2 N-terminus in the Annexin A2-p11 tetramer. In this study, we provided detailed atomistic information for the A1t. Strong interactions were identified within the A1t between the A1-ND and both S100A11 monomers. Residues M3, V4, S5, E6, L8, K9, W12, E15, and E18 of A1 were the strongest interactions between A1 and the S100A11 dimer. The different conformations of the A1t were attributed to the interaction between W12 of the A1-ND with M63 of S100A11 which caused a kink in the A1-ND. Cross-correlation analysis revealed strong correlated motion throughout the A1t. Strong positive correlation was observed between the ND and S100A11 in all simulations regardless of conformation. This work suggests that the stable binding of the first 11 residues of A1-ND to S100A11 is potentially a theme for Annexin-S100 complexes and that the flexibility of the A1-ND allows for multiple conformations of the A1t.Communicated by Ramaswamy H. Sarma.

Ca2+ ions facilitate the organization of the Annexin A2/S100A10 heterotetramer.

Annexin A2 (A2) is a member of the Annexin family, which contains Ca2+ -regulated phospholipid-binding proteins. Annexins associate with S100 proteins to form heterotetramers. The A2/S100A10 heterotetramer (A2t) is the most extensively studied of these heterotetramers. It induces membrane microdomain formation, causes membrane budding, and facilitates proliferation of some cancers. In this work, the first molecular dynamics (MD) study on the complete A2t of 868 amino acids was performed. MD trajectories of more than 600 ns each were generated for complete A2t complexes with and without Ca2+ ions. The outward extension of membrane-binding residues A2-K279 and A2-K281 was shown to be inhibited in the absence of Ca2+ as they were captured by Ca2+ -binding residue D322. F-actin binding residue A2-D339 was observed to occupy either an exposed or buried state in the absence of Ca2+ , while it only occupied the buried state in the presence of Ca2+ . The observed motions of the A2t subunits are highly organized with a strongly correlated central region which is negatively correlated with the periphery of the complex. The central region contains the S100A10 (p11) dimer, A2-N, and A2-I, while the periphery contains A2-II, A2-III, and A2-IV. Novel interactions between A2 and p11 were identified. A2 residues outside of A2-N (K80, R77, E82, and R145) had strong interactions with p11. Residue R145 of A2 may have a significant effect on the dynamics of the system, with its interaction resulting in asymmetric motions of A2. The presented results provide novel insights to inform future experimental studies.

S100A6 Protein-Expression and Function in Norm and Pathology.

S100A6, also known as calcyclin, is a calcium-binding protein belonging to the S100 protein family. It was first identified and purified more than 30 years ago. Initial structural studies, focused mostly on the mode and affinity of Ca2+ binding and resolution of the resultant conformational changes, were soon complemented by research on its expression, localization and identification of binding partners. With time, the use of biophysical methods helped to resolve the structure and versatility of S100A6 complexes with some of its ligands. Meanwhile, it became clear that S100A6 expression was altered in various pathological states and correlated with the stage/progression of many diseases, including cancers, indicative of its important, and possibly causative, role in some of these diseases. This, in turn, prompted researchers to look for the mechanism of S100A6 action and to identify the intermediary signaling pathways and effectors. After all these years, our knowledge on various aspects of S100A6 biology is robust but still incomplete. The list of S100A6 ligands is growing all the time, as is our understanding of the physiological importance of these interactions. The present review summarizes available data concerning S100A6 expression/localization, interaction with intracellular and extracellular targets, involvement in Ca2+-dependent cellular processes and association with various pathologies.

Binding and Functional Folding (BFF): A Physiological Framework for Studying Biomolecular Interactions and Allostery.

EF-hand Ca2+-binding proteins (CBPs), such as S100 proteins (S100s) and calmodulin (CaM), are signaling proteins that undergo conformational changes upon increasing intracellular Ca2+. Upon binding Ca2+, S100 proteins and CaM interact with protein targets and induce important biological responses. The Ca2+-binding affinity of CaM and most S100s in the absence of target is weak (CaKD > 1 µM). However, upon effector protein binding, the Ca2+ affinity of these proteins increases via heterotropic allostery (CaKD < 1 µM). Because of the high number and micromolar concentrations of EF-hand CBPs in a cell, at any given time, allostery is required physiologically, allowing for (i) proper Ca2+ homeostasis and (ii) strict maintenance of Ca2+-signaling within a narrow dynamic range of free Ca2+ ion concentrations, [Ca2+]free. In this review, mechanisms of allostery are coalesced into an empirical "binding and functional folding (BFF)" physiological framework. At the molecular level, folding (F), binding and folding (BF), and BFF events include all atoms in the biomolecular complex under study. The BFF framework is introduced with two straightforward BFF types for proteins (type 1, concerted; type 2, stepwise) and considers how homologous and nonhomologous amino acid residues of CBPs and their effector protein(s) evolved to provide allosteric tightening of Ca2+ and simultaneously determine how specific and relatively promiscuous CBP-target complexes form as both are needed for proper cellular function.

Metal sequestration by S100 proteins in chemically diverse environments.

During infection, the mammalian host initiates a metal-withholding response against invading microbial pathogens to inhibit their growth and survival, a process often termed 'nutritional immunity'. The host-defense S100 proteins calprotectin (CP) (S100A8/S100A9 oligomer), S100A12, and S100A7 play key roles in the innate immune response by sequestrating essential transition metal nutrients from microbes in the extracellular space. Accumulating evidence suggests that the antimicrobial activity of these proteins varies between infection sites and may be affected by the local chemical environment. Herein, we discuss the interplay between host metal-withholding proteins and microbial pathogens in the context of the chemical complexity of infection sites and highlight recent advances in our understanding of how chemically diverse conditions affect the properties and functions of S100 proteins.

S100A1 is Involved in Myocardial Injury Induced by Exhaustive Exercise.

Many studies have confirmed that exhaustive exercise has adverse effects on the heart by generating reactive oxygen species (ROS). S100A1 calcium-binding protein A1 (S100A1) is a regulator of myocardial contractility and a protector against myocardial injury. However, few studies have investigated the role of S100A1 in the regulation of myocardial injury induced by exhaustive exercise. In the present study, we suggested that exhaustive exercise led to increased ROS, downregulation of S100a1, and myocardial injury. Downregulation of S100a1 promoted exhaustive exercise-induced myocardial injury and overexpression of S100A1 reversed oxidative stress-induced cardiomyocyte injury, indicating S100A1 is a protective factor against myocardial injury caused by exhaustive exercise. We also found that downregulation of S100A1 promoted damage to critical proteins of the mitochondria by inhibiting the expression of Ant1, Pgc1a, and Tfam under exhaustive exercise. Our study indicated S100A1 as a potential prognostic biomarker or therapeutic target to improve the myocardial damage induced by exhaustive exercise and provided new insights into the molecular mechanisms underlying the myocardial injury effect of exhaustive exercise.

Mechanism of Zn2+ and Ca2+ Binding to Human S100A1.

S100A1 is a member of the S100 family of small ubiquitous Ca2+-binding proteins, which participates in the regulation of cell differentiation, motility, and survival. It exists as homo- or heterodimers. S100A1 has also been shown to bind Zn2+, but the molecular mechanisms of this binding are not yet known. In this work, using ESI-MS and ITC, we demonstrate that S100A1 can coordinate 4 zinc ions per monomer, with two high affinity (KD~4 and 770 nm) and two low affinity sites. Using competitive binding experiments between Ca2+ and Zn2+ and QM/MM molecular modeling we conclude that Zn2+ high affinity sites are located in the EF-hand motifs of S100A1. In addition, two lower affinity sites can bind Zn2+ even when the EF-hands are saturated by Ca2+, resulting in a 2Ca2+:S100A1:2Zn2+ conformer. Finally, we show that, in contrast to calcium, an excess of Zn2+ produces a destabilizing effect on S100A1 structure and leads to its aggregation. We also determined a higher affinity to Ca2+ (KD~0.16 and 24 µm) than was previously reported for S100A1, which would allow this protein to function as a Ca2+/Zn2+-sensor both inside and outside cells, participating in diverse signaling pathways under normal and pathological conditions.

Regulation of the tubulin polymerization-promoting protein by Ca2+/S100 proteins.

To elucidate S100 protein-mediated signaling pathways, we attempted to identify novel binding partners for S100A2 by screening protein arrays carrying 19,676 recombinant glutathione S-transferase (GST)-fused human proteins with biotinylated S100A2. Among newly discovered putative S100A2 interactants, including TMLHE, TRH, RPL36, MRPS34, CDR2L, OIP5, and MED29, we identified and characterized the tubulin polymerization-promoting protein (TPPP) as a novel S100A2-binding protein. We confirmed the interaction of TPPP with Ca2+/S100A2 by multiple independent methods, including the protein array method, S100A2 overlay, and pulldown assay in vitro and in transfected COS-7 cells. Based on the results from the S100A2 overlay assay using various GST-TPPP mutants, the S100A2-binding region was identified in the C-terminal (residues 111-160) of the central core domain of a monomeric form of TPPP that is involved in TPPP dimerization. Chemical cross-linking experiments indicated that S100A2 suppresses dimer formation of His-tagged TPPP in a dose-dependent and a Ca2+-dependent manner. In addition to S100A2, TPPP dimerization is disrupted by other multiple S100 proteins, including S100A6 and S100B, in a Ca2+-dependent manner but not by S100A4. This is consistent with the fact that S100A6 and S100B, but not S100A4, are capable of interacting with GST-TPPP in the presence of Ca2+. Considering these results together, TPPP was identified as a novel target for S100A2, and it is a potential binding target for other multiple S100 proteins, including S100A6 and S100B. Direct binding of the S100 proteins with TPPP may cause disassembly of TPPP dimer formation in response to the increasing concentration of intracellular Ca2+, thus resulting in the regulation of the physiological function of TPPP, such as microtubule organization.

S100P Interacts with p53 while Pentamidine Inhibits This Interaction.

S100P, a small calcium-binding protein, associates with the p53 protein with micromolar affinity. It has been hypothesized that the oncogenic function of S100P may involve binding-induced inactivation of p53. We used 1H-15N HSQC experiments and molecular modeling to study the molecular interactions between S100P and p53 in the presence and absence of pentamidine. Our experimental analysis indicates that the S100P-53 complex formation is successfully disrupted by pentamidine, since S100P shares the same binding site for p53 and pentamidine. In addition, we showed that pentamidine treatment of ZR-75-1 breast cancer cells resulted in reduced proliferation and increased p53 and p21 protein levels, indicating that pentamidine is an effective antagonist that interferes with the S100P-p53 interaction, leading to re-activation of the p53-21 pathway and inhibition of cancer cell proliferation. Collectively, our findings suggest that blocking the association between S100P and p53 by pentamidine will prevent cancer progression and, therefore, provide a new avenue for cancer therapy by targeting the S100P-p53 interaction.

Specificity of Molecular Fragments Binding to S100B versus S100A1 as Identified by NMR and Site Identification by Ligand Competitive Saturation (SILCS).

S100B, a biomarker of malignant melanoma, interacts with the p53 protein and diminishes its tumor suppressor function, which makes this S100 family member a promising therapeutic target for treating malignant melanoma. However, it is a challenge to design inhibitors that are specific for S100B in melanoma versus other S100-family members that are important for normal cellular activities. For example, S100A1 is most similar in sequence and structure to S100B, and this S100 protein is important for normal skeletal and cardiac muscle function. Therefore, a combination of NMR and computer aided drug design (CADD) was used to initiate the design of specific S100B inhibitors. Fragment-based screening by NMR, also termed "SAR by NMR," is a well-established method, and was used to examine spectral perturbations in 2D [1H, 15N]-HSQC spectra of Ca2+-bound S100B and Ca2+-bound S100A1, side-by-side, and under identical conditions for comparison. Of the 1000 compounds screened, two were found to be specific for binding Ca2+-bound S100A1 and four were found to be specific for Ca2+-bound S100B, respectively. The NMR spectral perturbations observed in these six data sets were then used to model how each of these small molecule fragments showed specificity for one S100 versus the other using a CADD approach termed Site Identification by Ligand Competitive Saturation (SILCS). In summary, the combination of NMR and computational approaches provided insight into how S100A1 versus S100B bind small molecules specifically, which will enable improved drug design efforts to inhibit elevated S100B in melanoma. Such a fragment-based approach can be used generally to initiate the design of specific inhibitors for other highly homologous drug targets.

Structural characterization of a dimeric complex between the short cytoplasmic domain of CEACAM1 and the pseudo tetramer of S100A10-Annexin A2 using NMR and molecular dynamics.

AIIt, a heterotetramer of S100A10 (P11) and Annexin A2, plays a key role in calcium dependent, membrane associations with a variety of proteins. We previously showed that AIIt interacts with the short cytoplasmic domain (12 amino acids) of CEACAM1 (CEACAM1-SF). Since the cytoplasmic domains of CEACAM1 help regulate the formation of cis- or trans-dimers at the cell membrane, we investigated the possible role of their association with AIIt in this process. Using NMR and molecular dynamics, we show that AIIt and its pseudoheterodimer interacts with two molecules of short cytoplasmic domain isoform peptides, and that interaction depends on the binding motif 454-Phe-Gly-Lys-Thr-457 where Phe-454 binds in a hydrophobic pocket of AIIt, the null mutation Phe454Ala reduces binding by 2.5 fold, and the pseudophosphorylation mutant Thr457Glu reduces binding by three fold. Since these two residues in CEACAM1-SF were also found to play a role in the binding of calmodulin and G-actin at the membrane, we hypothesize a sequential set of three interactions are responsible for regulation of cis- to trans-dimerization of CEACAM1. The hydrophobic binding pocket in AIIt corresponds to a previously identified binding pocket for a peptide found in SMARCA3 and AHNAK, suggesting a conserved functional motif in AIIt allowing multiple proteins to reversibly interact with integral membrane proteins in a calcium dependent manner.

Interferon Beta Activity Is Modulated via Binding of Specific S100 Proteins.

Interferon-ß (IFN-ß) is a pleiotropic cytokine used for therapy of multiple sclerosis, which is also effective in suppression of viral and bacterial infections and cancer. Recently, we reported a highly specific interaction between IFN-ß and S100P lowering IFN-ß cytotoxicity to cancer cells (Int J Biol Macromol. 2020; 143: 633-639). S100P is a member of large family of multifunctional Ca2+-binding proteins with cytokine-like activities. To probe selectivity of IFN-ß-S100 interaction with respect to S100 proteins, we used surface plasmon resonance spectroscopy, chemical crosslinking, and crystal violet assay. Among the thirteen S100 proteins studied S100A1, S100A4, and S100A6 proteins exhibit strictly Ca2+-dependent binding to IFN-ß with equilibrium dissociation constants, Kd, of 0.04-1.5 µM for their Ca2+-bound homodimeric forms. Calcium depletion abolishes the S100-IFN-ß interactions. Monomerization of S100A1/A4/A6 decreases Kd values down to 0.11-1.0 nM. Interferon-α is unable of binding to the S100 proteins studied. S100A1/A4 proteins inhibit IFN-ß-induced suppression of MCF-7 cells viability. The revealed direct influence of specific S100 proteins on IFN-ß activity uncovers a novel regulatory role of particular S100 proteins, and opens up novel approaches to enhancement of therapeutic efficacy of IFN-ß.

Learning peptide recognition rules for a low-specificity protein.

Many proteins interact with short linear regions of target proteins. For some proteins, however, it is difficult to identify a well-defined sequence motif that defines its target peptides. To overcome this difficulty, we used supervised machine learning to train a model that treats each peptide as a collection of easily-calculated biochemical features rather than as an amino acid sequence. As a test case, we dissected the peptide-recognition rules for human S100A5 (hA5), a low-specificity calcium binding protein. We trained a Random Forest model against a recently released, high-throughput phage display dataset collected for hA5. The model identifies hydrophobicity and shape complementarity, rather than polar contacts, as the primary determinants of peptide binding specificity in hA5. We tested this hypothesis by solving a crystal structure of hA5 and through computational docking studies of diverse peptides onto hA5. These structural studies revealed that peptides exhibit multiple binding modes at the hA5 peptide interface-all of which have few polar contacts with hA5. Finally, we used our trained model to predict new, plausible binding targets in the human proteome. This revealed a fragment of the protein α-1-syntrophin that binds to hA5. Our work helps better understand the biochemistry and biology of hA5, as well as demonstrating how high-throughput experiments coupled with machine learning of biochemical features can reveal the determinants of binding specificity in low-specificity proteins.

Zooming into the Dark Side of Human Annexin-S100 Complexes: Dynamic Alliance of Flexible Partners.

Annexins and S100 proteins form two large families of Ca2+-binding proteins. They are quite different both structurally and functionally, with S100 proteins being small (10-12 kDa) acidic regulatory proteins from the EF-hand superfamily of Ca2+-binding proteins, and with annexins being at least three-fold larger (329 ± 12 versus 98 ± 7 residues) and using non-EF-hand-based mechanism for calcium binding. Members of both families have multiple biological roles, being able to bind to a large cohort of partners and possessing a multitude of functions. Furthermore, annexins and S100 proteins can interact with each other in either a Ca2+-dependent or Ca2+-independent manner, forming functional annexin-S100 complexes. Such functional polymorphism and binding indiscrimination are rather unexpected, since structural information is available for many annexins and S100 proteins, which therefore are considered as ordered proteins that should follow the classical "one protein-one structure-one function" model. On the other hand, the ability to be engaged in a wide range of interactions with multiple, often unrelated, binding partners and possess multiple functions represent characteristic features of intrinsically disordered proteins (IDPs) and intrinsically disordered protein regions (IDPRs); i.e., functional proteins or protein regions lacking unique tertiary structures. The aim of this paper is to provide an overview of the functional roles of human annexins and S100 proteins, and to use the protein intrinsic disorder perspective to explain their exceptional multifunctionality and binding promiscuity.

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

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.