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

Ca2+ /CaM-dependent protein kinase kinases 1 and 2 (CaMKK1 and CaMKK2) phosphorylate and enhance the catalytic activity of downstream kinases CaMKI, CaMKIV, and protein kinase B. Accordingly, CaMKK1 and CaMKK2 regulate key physiological and pathological processes, such as tumorigenesis, neuronal morphogenesis, synaptic plasticity, transcription factor activation, and cellular energy homeostasis, and promote cell survival. Both CaMKKs are partly inhibited by phosphorylation, which in turn triggers adaptor and scaffolding protein 14-3-3 binding. However, 14-3-3 binding only significantly affects CaMKK1 function. CaMKK2 activity remains almost unchanged after complex formation for reasons still unclear. Here, we aim at structurally characterizing CaMKK1:14-3-3 and CaMKK2:14-3-3 complexes by SAXS, H/D exchange coupled to MS, and fluorescence spectroscopy. The results revealed that complex formation suppresses the interaction of both phosphorylated CaMKKs with Ca2+ /CaM and affects the structure of their kinase domains and autoinhibitory segments. But these effects are much stronger on CaMKK1 than on CaMKK2 because the CaMKK1:14-3-3γ complex has a more compact and rigid structure in which the active site of the kinase domain directly interacts with the last two C-terminal helices of the 14-3-3γ protein, thereby inhibiting CaMKK1. In contrast, the CaMKK2:14-3-3 complex has a looser and more flexible structure, so 14-3-3 binding only negligibly affects the catalytic activity of CaMKK2. Therefore, Ca2+ /CaM binding suppression and the interaction of the kinase active site of CaMKK1 with the last two C-terminal helices of 14-3-3γ protein provide the structural basis for 14-3-3-mediated CaMKK1 inhibition.

• Keywords
• 14-3-3 proteins, CaMKK, SAXS, calcium/calmodulin-dependent protein kinase, fluorescence spectroscopy, hydrogen/deuterium exchange coupled to MS, protein-protein interaction,
• MeSH
• X-Ray Diffraction MeSH
• Phosphorylation MeSH
• Catalytic Domain MeSH
• Calcium-Calmodulin-Dependent Protein Kinase Kinase * chemistry   metabolism   MeSH
• Scattering, Small Angle MeSH
• 14-3-3 Proteins * metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Calcium-Calmodulin-Dependent Protein Kinase Kinase * MeSH
• 14-3-3 Proteins * MeSH

Death-associated protein kinase 2 (DAPK2) is a CaM-regulated Ser/Thr protein kinase, involved in apoptosis, autophagy, granulocyte differentiation and motility regulation, whose activity is controlled by autoinhibition, autophosphorylation, dimerization and interaction with scaffolding proteins 14-3-3. However, the structural basis of 14-3-3-mediated DAPK2 regulation remains unclear. Here, we structurally and biochemically characterize the full-length human DAPK2:14-3-3 complex by combining several biophysical techniques. The results from our X-ray crystallographic analysis revealed that Thr369 phosphorylation at the DAPK2 C terminus creates a high-affinity canonical mode III 14-3-3-binding motif, further enhanced by the diterpene glycoside Fusicoccin A. Moreover, concentration-dependent DAPK2 dimerization is disrupted by Ca2+/CaM binding and stabilized by 14-3-3 binding in solution, thereby protecting the DAPK2 inhibitory autophosphorylation site Ser318 against dephosphorylation and preventing Ca2+/CaM binding. Overall, our findings provide mechanistic insights into 14-3-3-mediated DAPK2 inhibition and highlight the potential of the DAPK2:14-3-3 complex as a target for anti-inflammatory therapies.

• MeSH
• Dimerization MeSH
• Phosphorylation MeSH
• Humans MeSH
• Death-Associated Protein Kinases genetics   metabolism   MeSH
• 14-3-3 Proteins genetics   metabolism   MeSH
• Gene Expression Regulation MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DAPK2 protein, human MeSH Browser
• Death-Associated Protein Kinases MeSH
• 14-3-3 Proteins MeSH
• YWHAG protein, human MeSH Browser

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

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

TRPV1 is a nonselective cation channel that integrates wide range of painful stimuli. It has been shown that its activity could be modulated by intracellular ligands PIP2 or calmodulin (CaM). The detailed localization and description of PIP2 interaction sites remain unclear. Here, we used synthesized peptides and purified fusion proteins of intracellular regions of TRPV1 expressed in E.coli in combination with fluorescence anisotropy and surface plasmon resonance measurements to characterize the PIP2 binding to TRPV1. We characterized one PIP2 binding site in TRPV1 N-terminal region, residues F189-V221, and two independent PIP2 binding sites in C-terminus: residues K688-K718 and L777-S820. Moreover we show that two regions, namely F189-V221 and L777-S820, overlap with previously localized CaM binding sites. For all the interactions the equilibrium dissociation constants were estimated. As the structural data regarding C-terminus of TRPV1 are lacking, restraint-based molecular modeling combined with ligand docking was performed providing us with structural insight to the TRPV1/PIP2 binding. Our experimental results are in excellent agreement with our in silico predictions.

• MeSH
• Ankyrins chemistry   MeSH
• Phosphatidylinositol Phosphates metabolism   MeSH
• Protein Interaction Domains and Motifs MeSH
• Calmodulin chemistry   metabolism   MeSH
• TRPV Cation Channels chemistry   genetics   metabolism   MeSH
• Protein Conformation MeSH
• Rats MeSH
• Ligands MeSH
• Liposomes metabolism   MeSH
• Mutation MeSH
• Recombinant Fusion Proteins chemistry   genetics   metabolism   MeSH
• Molecular Docking Simulation MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Ankyrins MeSH
• Phosphatidylinositol Phosphates MeSH
• Calmodulin MeSH
• TRPV Cation Channels MeSH
• Ligands MeSH
• Liposomes MeSH
• Recombinant Fusion Proteins MeSH
• TRPV1 receptor MeSH Browser

TRPM3 has been reported to play an important role in Ca(2+) homeostasis, but its gating mechanisms and regulation via Ca(2+) are unknown. Ca(2+) binding proteins such as calmodulin (CaM) could be probable modulators of this ion channel. We have shown that this protein binds to two independent domains, A35-K124 and H291-G382 on the TRPM3 N-terminus, which contain conserved hydrophobic as well as positively charged residues in specific positions, and that these residues have a crucial impact on its binding. We also showed that the other Ca(2+) binding protein, S100A1, is able to bind to these regions and that CaM and S100A1 compete for these binding sites on the TRPM3 N-terminus. Moreover, our results suggest that another very important TRP channel activity modulator, PtdIns(4,5)P(2), interacts with the CaM/S100A1 binding sites on the TRPM3 N-terminus with high affinity.

• MeSH
• Fluorescence Polarization MeSH
• Phosphatidylinositol 4,5-Diphosphate metabolism   MeSH
• Calmodulin metabolism   MeSH
• TRPM Cation Channels chemistry   metabolism   MeSH
• Liposomes metabolism   MeSH
• Models, Molecular MeSH
• Surface Plasmon Resonance MeSH
• S100 Proteins metabolism   MeSH
• Protein Structure, Tertiary MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Phosphatidylinositol 4,5-Diphosphate MeSH
• Calmodulin MeSH
• TRPM Cation Channels MeSH
• Liposomes MeSH
• S100 Proteins MeSH
• S100A1 protein MeSH Browser

Transient receptor potential melastatin 3 ion channel (TRPM3) belongs to the TRP family of cation-permeable ion channels involved in many important biological functions such as pain transduction, thermosensation, and mechanoregulation. The channel was reported to play an important role in Ca(2+) homeostasis, but its gating mechanisms, functions, and regulation are still under research. Utilizing biophysical and biochemical methods, we characterized two independent domains, Ala-35-Lys-124 and His-291-Gly-382, on the TRPM3 N terminus, responsible for interactions with the Ca(2+)-binding proteins calmodulin (CaM) and S100A1. We identified several positively charged residues within these domains as having a crucial impact on CaM/S100A1 binding. The data also suggest that the interaction is calcium-dependent. We also performed competition assays, which suggested that CaM and S100A1 are able to compete for the same binding sites within the TRPM3 N terminus. This is the first time that such an interaction has been shown for TRP family members.

• MeSH
• Calmodulin chemistry   genetics   metabolism   MeSH
• TRPM Cation Channels chemistry   genetics   metabolism   MeSH
• Humans MeSH
• Mutation, Missense MeSH
• S100 Proteins chemistry   genetics   metabolism   MeSH
• Amino Acid Substitution 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
• Calmodulin MeSH
• TRPM Cation Channels MeSH
• S100 Proteins MeSH
• S100A1 protein MeSH Browser
• TRPM3 protein, human MeSH Browser