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.
- MeSH
- akvaporiny chemie metabolismus MeSH
- interakční proteinové domény a motivy * MeSH
- kalmodulin chemie metabolismus MeSH
- kationtové kanály TRPM chemie metabolismus MeSH
- kinetika MeSH
- konformace proteinů MeSH
- lidé MeSH
- molekulární modely MeSH
- multiproteinové komplexy chemie metabolismus MeSH
- peptidové fragmenty MeSH
- proteiny S100 chemie metabolismus MeSH
- sekvence aminokyselin MeSH
- vazba proteinů MeSH
- vazebná místa * MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
- Publikační typ
- tisková chyba MeSH
Working at the border between innate and adaptive immunity, natural killer (NK) cells play a key role in the immune system by protecting healthy cells and by eliminating malignantly transformed, stressed or virally infected cells. NK cell recognition of a target cell is mediated by a receptor "zipper" consisting of various activating and inhibitory receptors, including C-type lectin-like receptors. Among this major group of receptors, two of the largest rodent receptor families are the NKR-P1 and the Clr receptor families. Although these families have been shown to encode receptor-ligand pairs involved in MHC-independent self-nonself discrimination and are a target for immune evasion by tumour cells and viruses, structural mechanisms of their mutual recognition remain less well characterized. Therefore, we developed a non-viral eukaryotic expression system based on transient transfection of suspension-adapted human embryonic kidney 293 cells to produce soluble native disulphide dimers of NK cell C-type lectin-like receptor ectodomains. The expression system was optimized using green fluorescent protein and secreted alkaline phosphatase, easily quantifiable markers of recombinant protein production. We describe an application of this approach to the recombinant protein production and characterization of native rat NKR-P1B and Clr-11 proteins suitable for further structural and functional studies.
- MeSH
- HEK293 buňky MeSH
- krysa rodu rattus MeSH
- lektinové receptory NK-buněk - podrodina B chemie genetika metabolismus MeSH
- lidé MeSH
- multimerizace proteinu MeSH
- protein podobný kalcitoninovému receptoru chemie genetika metabolismus MeSH
- proteinové domény MeSH
- proteinové inženýrství metody MeSH
- rekombinantní proteiny chemie genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- kalmodulin chemie MeSH
- kationtové kanály TRPM chemie MeSH
- lidé MeSH
- molekulární modely * MeSH
- proteinové domény MeSH
- proteiny S100 chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
We have investigated structural changes of peptides related to antimicrobial peptide Halictine-1 (HAL-1) induced by interaction with various membrane-mimicking models with the aim to identify a mechanism of the peptide mode of action and to find a correlation between changes of primary/secondary structure and biological activity. Modifications in the HAL-1 amino acid sequence at particular positions, causing an increase of amphipathicity (Arg/Lys exchange), restricted mobility (insertion of Pro) and consequent changes in antimicrobial and hemolytic activity, led to different behavior towards model membranes. Secondary structure changes induced by peptide-membrane interaction were studied by circular dichroism, infrared spectroscopy, and fluorescence spectroscopy. The experimental results were complemented by molecular dynamics calculations. An α-helical structure has been found to be necessary but not completely sufficient for the HAL-1 peptides antimicrobial action. The role of alternative conformations (such as β-sheet, PPII or 310-helix) also seems to be important. A mechanism of the peptide mode of action probably involves formation of peptide assemblies (possibly membrane pores), which disrupt bacterial membrane and, consequently, allow membrane penetration.
- MeSH
- antibakteriální látky chemie metabolismus MeSH
- fosfatidylcholiny chemie MeSH
- fosfatidylglyceroly chemie MeSH
- hydrofobní a hydrofilní interakce MeSH
- kationické antimikrobiální peptidy chemie metabolismus MeSH
- kinetika MeSH
- konformace proteinů, alfa-helix MeSH
- konformace proteinů, beta-řetězec MeSH
- lipidové dvojvrstvy chemie MeSH
- permeabilita MeSH
- sekvence aminokyselin MeSH
- simulace molekulární dynamiky MeSH
- Publikační typ
- časopisecké články MeSH
β-N-acetylhexosaminidase from the fungus Aspergillus oryzae is a secreted extracellular enzyme that cleaves chitobiose into constituent monosaccharides. It belongs to the GH 20 glycoside hydrolase family and consists of two N-glycosylated catalytic cores noncovalently associated with two 10-kDa O-glycosylated propeptides. We used X-ray diffraction and mass spectrometry to determine the structure of A. oryzae β-N-acetylhexosaminidase isolated from its natural source. The three-dimensional structure determined and refined to a resolution of 2.3 Å revealed that this enzyme is active as a uniquely tight dimeric assembly further stabilized by N- and O-glycosylation. The propeptide from one subunit forms extensive noncovalent interactions with the catalytic core of the second subunit in the dimer, and this chain swap suggests the distinctive structural mechanism of the enzyme's activation. Unique structural features of β-N-acetylhexosaminidase from A. oryzae define a very stable and robust framework suitable for biotechnological applications. The crystal structure reported here provides structural insights into the enzyme architecture as well as the detailed configuration of the active site. These insights can be applied to rational enzyme engineering. DATABASE: Structural data are available in the PDB database under the accession number 5OAR. ENZYME: β-N-acetylhexosaminidase (EC 3.2.1.52).
- MeSH
- Aspergillus oryzae enzymologie MeSH
- beta-N-acetylhexosaminidasy chemie metabolismus MeSH
- dimerizace MeSH
- fungální proteiny chemie metabolismus MeSH
- G(M2) aktivátorový protein chemie metabolismus MeSH
- G(M2) gangliosid chemie metabolismus MeSH
- glykosylace MeSH
- interakční proteinové domény a motivy MeSH
- katalytická doména MeSH
- konzervovaná sekvence MeSH
- krystalografie rentgenová MeSH
- ligandy MeSH
- molekulární modely * MeSH
- posttranslační úpravy proteinů MeSH
- prekurzory enzymů chemie metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- stabilita proteinů MeSH
- strukturní homologie proteinů MeSH
- substrátová specifita MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The protein sequences found in nature represent a tiny fraction of the potential sequences that could be constructed from the 20-amino-acid alphabet. To help define the properties that shaped proteins to stand out from the space of possible alternatives, we conducted a systematic computational and experimental exploration of random (unevolved) sequences in comparison with biological proteins. In our study, combinations of secondary structure, disorder, and aggregation predictions are accompanied by experimental characterization of selected proteins. We found that the overall secondary structure and physicochemical properties of random and biological sequences are very similar. Moreover, random sequences can be well-tolerated by living cells. Contrary to early hypotheses about the toxicity of random and disordered proteins, we found that random sequences with high disorder have low aggregation propensity (unlike random sequences with high structural content) and were particularly well-tolerated. This direct structure content/aggregation propensity dependence differentiates random and biological proteins. Our study indicates that while random sequences can be both structured and disordered, the properties of the latter make them better suited as progenitors (in both in vivo and in vitro settings) for further evolution of complex, soluble, three-dimensional scaffolds that can perform specific biochemical tasks.
- MeSH
- cirkulární dichroismus MeSH
- databáze proteinů MeSH
- datové soubory jako téma MeSH
- molekulární modely * MeSH
- nukleární magnetická rezonance biomolekulární MeSH
- peptidová knihovna * MeSH
- proteinové agregáty MeSH
- rekombinantní proteiny chemie izolace a purifikace toxicita MeSH
- rozpustnost MeSH
- sbalování proteinů MeSH
- sekundární struktura proteinů * MeSH
- sekvence aminokyselin MeSH
- výpočetní biologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Ligand binding of neutral progesterone, basic propranolol, and acidic warfarin to human α1-acid glycoprotein (AGP) was investigated by Raman spectroscopy. The binding itself is characterized by a uniform conformational shift in which a tryptophan residue is involved. Slight differences corresponding to different contacts of the individual ligands inside the β-barrel are described. Results are compared with in silico ligand docking into the available crystal structure of deglycosylated AGP using quantum/molecular mechanics. Calculated binding energies are -18.2, -14.5, and -11.5 kcal/mol for warfarin, propranolol, and progesterone, respectively. These calculations are consistent with Raman difference spectroscopy; nevertheless, minor discrepancies in the precise positions of the ligands point to structural differences between deglycosylated and native AGP. Thermal dynamics of AGP with/without bounded warfarin was followed by Raman spectroscopy in a temperature range of 10-95 °C and analyzed by principal component analysis. With increasing temperature, a slight decrease of α-helical content is observed that coincides with an increase in β-sheet content. Above 45 °C, also β-strands tend to unfold, and the observed decrease in β-sheet coincides with an increase of β-turns accompanied by a conformational shift of the nearby disulfide bridge from high-energy trans-gauche-trans to more relaxed gauche-gauche-trans. This major rearrangement in the vicinity of the bridge is not only characterized by unfolding of the β-sheet but also by subsequent ligand release. Hereby, ligand binding alters the protein dynamics, and the more rigid protein-ligand complex shows an improved thermal stability, a finding that contributes to the reported chaperone-like function of AGP.
- MeSH
- lidé MeSH
- molekulární modely MeSH
- orosomukoid chemie metabolismus MeSH
- progesteron chemie metabolismus MeSH
- propranolol chemie metabolismus MeSH
- Ramanova spektroskopie MeSH
- sekundární struktura proteinů MeSH
- simulace molekulového dockingu MeSH
- stabilita proteinů MeSH
- termodynamika MeSH
- tryptofan metabolismus MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- warfarin chemie metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
BACKGROUND: The handling of cerebrospinal fluid (CSF) affects the biomarker quantification used to diagnose Alzheimer's disease (AD). Only specialized centers can test for AD markers. The precise timing and freezing is required to correctly measure these biomarkers. Therefore, the effects of CSF storage temperature and repeated freeze/thaw cycles on CSF stability were investigated. METHODS: Drop coating deposition Raman spectroscopy in combination with principal component analysis was used to analyze CSF and its dialyzed form (ELISA confirmed the removal of up to 80% of the AD markers). The advantage of this approach is that no prior knowledge of the biomarkers is necessary and that both the concentration and the protein structure of intact CSF are analyzed. RESULTS: Dialyzed CSF was stable for up to 5 h after its collection, while native CSF started to denature nearly immediately. Most of the unstable proteins were denatured within 24 h. The dialyzed CSF was not affected by freeze/thaw cycles, but the native CSF exhibited significant progressive changes, even after the first freezing. The mechanism as well as the resulting structures of the freeze-denatured proteins differed from those of the temporally denatured proteins, although both protein sets began with the same initial proteins. CONCLUSIONS: CSF must be processed immediately, within 5 h of collection. Flash cooling is recommended for freezing CSF, but any freeze/thaw cycle will affect the protein component of CSF.
Raman microscopy permits structural analysis of protein crystals in situ in hanging drops, allowing for comparison with Raman measurements in solution. Nevertheless, the two methods sometimes reveal subtle differences in structure that are often ascribed to the water layer surrounding the protein. The novel method of drop-coating deposition Raman spectropscopy (DCDR) exploits an intermediate phase that, although nominally "dry," has been shown to preserve protein structural features present in solution. The potential of this new approach to bridge the structural gap between proteins in solution and in crystals is explored here with extrinsic protein PsbP of photosystem II from Spinacia oleracea. In the high-resolution (1.98 Å) x-ray crystal structure of PsbP reported here, several segments of the protein chain are present but unresolved. Analysis of the three kinds of Raman spectra of PsbP suggests that most of the subtle differences can indeed be attributed to the water envelope, which is shown here to have a similar Raman intensity in glassy and crystal states. Using molecular dynamics simulations cross-validated by Raman solution data, two unresolved segments of the PsbP crystal structure were modeled as loops, and the amino terminus was inferred to contain an additional beta segment. The complete PsbP structure was compared with that of the PsbP-like protein CyanoP, which plays a more peripheral role in photosystem II function. The comparison suggests possible interaction surfaces of PsbP with higher-plant photosystem II. This work provides the first complete structural picture of this key protein, and it represents the first systematic comparison of Raman data from solution, glassy, and crystalline states of a protein.
- MeSH
- aminokyselinové motivy MeSH
- fotosystém II - proteinový komplex chemie MeSH
- krystalografie rentgenová MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- Ramanova spektroskopie MeSH
- rostlinné proteiny chemie MeSH
- sekundární struktura proteinů MeSH
- sekvence aminokyselin MeSH
- Spinacia oleracea chemie MeSH
- terciární struktura proteinů MeSH
- vodíková vazba MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
