<p>At top, the aminoacid sequences of homologous to the rat RIP2 RIPK2 proteins are aligned... more <p>At top, the aminoacid sequences of homologous to the rat RIP2 RIPK2 proteins are aligned with RIP2CARD sequence. At bottom,CARD domains of the proteins with other functions are aligned based on the 3D structure homology, according to the PDBefold server, PDB IDs are indicated. Conservative residues are highlighted with respect to their physical properties (yellow-hydrophobic, blue—positively charged, red—negatively charged, green—polar or neutral). Lines indicate the salt bridges, observed in RIP2CARD structure. Red, magenta, blue and cyan circles denote the residues which were shown important for the human RIP2-p75 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0206244#pone.0206244.ref013" target="_blank">13</a>], NOD2/RIP2, RIP2/NOD1/NOD2 and exclusively RIP2/NOD1 interactions [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0206244#pone.0206244.ref015" target="_blank">15</a>]. Brown circles denote the residues, important for the unique packing of the RIP2CARD C-terminal residues.</p
Toll-like receptors (TLRs) play an important role in the innate immune response. While a lot is k... more Toll-like receptors (TLRs) play an important role in the innate immune response. While a lot is known about the structures of their extracellular parts, many questions are still left unanswered, when the structural basis of TLR activation is analyzed for the TLR intracellular domains. Here we report the structure and dynamics of TLR1 toll-interleukin like (TIR) cytoplasmic domain in crystal and in solution. We found that the TLR1-TIR domain is capable of specific binding of Zn with nanomolar affinity. Interactions with Zn are mediated by cysteine residues 667 and 686 and C667 is essential for the Zn binding. Potential structures of the TLR1-TIR/Zn complex were predicted in silico. Using the functional assays for the heterodimeric TLR1/2 receptor, we found that both Zn addition and Zn depletion affect the activity of TLR1, and C667A mutation disrupts the receptor activity. Analysis of C667 position in the TLR1 structure and possible effects of C667A mutation, suggests that zinc-bindi...
Lynx1, membrane-bound protein co-localized with the nicotinic acetylcholine receptors (nAChRs) an... more Lynx1, membrane-bound protein co-localized with the nicotinic acetylcholine receptors (nAChRs) and regulates their function, is a three-finger protein (TFP) made of three β-structural loops, similarly to snake venom α-neurotoxin TFPs. Since the central loop II of α-neurotoxins is involved in binding to nAChRs, we have recently synthesized the fragments of Lynx1 central loop, including those with the disulfide between Cys residues introduced at N- and C-termini, some of them inhibiting muscle-type nAChR similarly to the whole-size water-soluble Lynx1 (ws-Lynx1). Literature shows that the main fragment interacting with TFPs is the C-loop of both nAChRs and acetylcholine binding proteins (AChBPs) while some ligand-binding capacity is preserved by analogs of this loop, for example, by high-affinity peptide HAP. Here we analyzed the structural organization of these peptide models of ligands and receptors and its role in binding. Thus, fragments of Lynx1 loop II, loop C from the Lymnaea s...
(2-Aminobenzylidene)-imidazolones were used as substrates for [1,5]-hydride shift triggered cycli... more (2-Aminobenzylidene)-imidazolones were used as substrates for [1,5]-hydride shift triggered cyclization under promotion by TiCl4 at room temperature.
One of the essential characteristics of any tag used in bioscience and medical applications is it... more One of the essential characteristics of any tag used in bioscience and medical applications is its size. The larger the label, the more it may affect the studied object, and the more it may distort its behavior. In this paper, using NMR spectroscopy and X-ray crystallography, we have studied the structure of fluorogen-activating protein FAST both in the apo form and in complex with the fluorogen. We shown that significant change in the protein occurs upon interaction with the ligand. While the protein is completely ordered in the complex, its apo form is characterized by higher mobility and disordering of its N-terminus. We used structural information to design the shortened FAST (which we named nanoFAST) by truncating 26 N-terminal residues. Thus, we created the shortest genetically encoded tag among all known fluorescent and fluorogen-activating proteins, which is composed of only 98 amino acids.
The neurotrophin receptor p75NTR plays crucial roles in neuron development and regulates importan... more The neurotrophin receptor p75NTR plays crucial roles in neuron development and regulates important neuronal processes like degeneration, apoptosis and cell survival. At the same time the detailed mechanism of signal transduction is unclear. One of the main hypotheses known as the snail-tong mechanism assumes that in the inactive state, the death domains interact with each other and in response to ligand binding there is a conformational change leading to their exposure. Here, we show that neither rat nor human p75NTR death domains homodimerize in solution. Moreover, there is no interaction between the death domains in a more native context: the dimerization of transmembrane domains in liposomes and the presence of activating mutation in extracellular juxtamembrane region do not lead to intracellular domain interaction. These findings suggest that the activation mechanism of p75NTR should be revised. Thus, we propose a novel model of p75NTR functioning based on interaction with “help...
Cyclopentane fragment in the hydrophobic tail expands operational range of the surfactant into th... more Cyclopentane fragment in the hydrophobic tail expands operational range of the surfactant into the low-temperature region.
The molecular mechanism of TrkA activation have been poorly studied in comparison with those of o... more The molecular mechanism of TrkA activation have been poorly studied in comparison with those of other RTK family members. TrkA, like most RTKs, is in a monomer-dimer equilibrium in the absence of their ligand, and an unsolved issue is how ligand-binding can activate a pre-formed inactive dimer. Here we show that the transmembrane domain (TMD) of TrkA can form dimers using two different interfaces. We characterized the structural determinants of TrkA-TMD dimerization and identified the dimer interface of the active receptor. We validated this interface using site-directed mutagenesis together with functional and cell differentiation studies with the full-length TrkA receptor expressed in mammalian cells. Using in vivo crosslinking we identified a reordering of the extracellular juxtamembrane region after ligand binding. This conformational change could be mimicked by replacement of some residues with cysteine and the spontaneous formation of ligand-independent active dimers. In addit...
Lipid-protein nanodiscs (LPNs) are nanoscaled fragments of a lipid bilayer stabilized in solution... more Lipid-protein nanodiscs (LPNs) are nanoscaled fragments of a lipid bilayer stabilized in solution by the apolipoprotein or a special membrane scaffold protein (MSP). In this work, the applicability of LPN-based membrane mimetics in the investigation of water-soluble membrane-active peptides was studied. It was shown that a pore-forming antimicrobial peptide arenicin-2 from marine lugworm (charge of +6) disintegrates LPNs containing both zwitterionic phosphatidylcholine (PC) and anionic phosphatidylglycerol (PG) lipids. In contrast, the spider toxin VSTx1 (charge of +3), a modifier of Kv channel gating, effectively binds to the LPNs containing anionic lipids (POPC/DOPG, 3 : 1) and does not cause their disruption. VSTx1 has a lower affinity to LPNs containing zwitterionic lipids (POPC), and it weakly interacts with the protein component of nanodiscs, MSP (charge of -6). The neurotoxin II (NTII, charge of +4) from cobra venom, an inhibitor of the nicotinic acetylcholine receptor, shows...
Soil fungi are known to contain a rich variety of defense metabolites that allow them to compete ... more Soil fungi are known to contain a rich variety of defense metabolites that allow them to compete with other organisms (fungi, bacteria, nematodes, and insects) and help them occupy more preferential areas at the expense of effective antagonism. These compounds possess antibiotic activity towards a wide range of other microbes, particularly fungi that belong to different taxonomical units. These compounds include peptaibols, which are non-ribosomal synthesized polypeptides containing non-standard amino acid residues (alpha-aminoisobutyric acid mandatory) and some posttranslational modifications. We isolated a novel antibiotic peptide from the culture medium of Emericellopsis alkalina, an alkalophilic strain. This peptide, called emericellipsin A, exhibited a strong antifungal effect against the yeast Candida albicans, the mold fungus Aspergillus niger, and human pathogen clinical isolates. It also exhibited antimicrobial activity against some Gram-positive and Gram-negative bacteria....
<p>At top, the aminoacid sequences of homologous to the rat RIP2 RIPK2 proteins are aligned... more <p>At top, the aminoacid sequences of homologous to the rat RIP2 RIPK2 proteins are aligned with RIP2CARD sequence. At bottom,CARD domains of the proteins with other functions are aligned based on the 3D structure homology, according to the PDBefold server, PDB IDs are indicated. Conservative residues are highlighted with respect to their physical properties (yellow-hydrophobic, blue—positively charged, red—negatively charged, green—polar or neutral). Lines indicate the salt bridges, observed in RIP2CARD structure. Red, magenta, blue and cyan circles denote the residues which were shown important for the human RIP2-p75 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0206244#pone.0206244.ref013" target="_blank">13</a>], NOD2/RIP2, RIP2/NOD1/NOD2 and exclusively RIP2/NOD1 interactions [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0206244#pone.0206244.ref015" target="_blank">15</a>]. Brown circles denote the residues, important for the unique packing of the RIP2CARD C-terminal residues.</p
Toll-like receptors (TLRs) play an important role in the innate immune response. While a lot is k... more Toll-like receptors (TLRs) play an important role in the innate immune response. While a lot is known about the structures of their extracellular parts, many questions are still left unanswered, when the structural basis of TLR activation is analyzed for the TLR intracellular domains. Here we report the structure and dynamics of TLR1 toll-interleukin like (TIR) cytoplasmic domain in crystal and in solution. We found that the TLR1-TIR domain is capable of specific binding of Zn with nanomolar affinity. Interactions with Zn are mediated by cysteine residues 667 and 686 and C667 is essential for the Zn binding. Potential structures of the TLR1-TIR/Zn complex were predicted in silico. Using the functional assays for the heterodimeric TLR1/2 receptor, we found that both Zn addition and Zn depletion affect the activity of TLR1, and C667A mutation disrupts the receptor activity. Analysis of C667 position in the TLR1 structure and possible effects of C667A mutation, suggests that zinc-bindi...
Lynx1, membrane-bound protein co-localized with the nicotinic acetylcholine receptors (nAChRs) an... more Lynx1, membrane-bound protein co-localized with the nicotinic acetylcholine receptors (nAChRs) and regulates their function, is a three-finger protein (TFP) made of three β-structural loops, similarly to snake venom α-neurotoxin TFPs. Since the central loop II of α-neurotoxins is involved in binding to nAChRs, we have recently synthesized the fragments of Lynx1 central loop, including those with the disulfide between Cys residues introduced at N- and C-termini, some of them inhibiting muscle-type nAChR similarly to the whole-size water-soluble Lynx1 (ws-Lynx1). Literature shows that the main fragment interacting with TFPs is the C-loop of both nAChRs and acetylcholine binding proteins (AChBPs) while some ligand-binding capacity is preserved by analogs of this loop, for example, by high-affinity peptide HAP. Here we analyzed the structural organization of these peptide models of ligands and receptors and its role in binding. Thus, fragments of Lynx1 loop II, loop C from the Lymnaea s...
(2-Aminobenzylidene)-imidazolones were used as substrates for [1,5]-hydride shift triggered cycli... more (2-Aminobenzylidene)-imidazolones were used as substrates for [1,5]-hydride shift triggered cyclization under promotion by TiCl4 at room temperature.
One of the essential characteristics of any tag used in bioscience and medical applications is it... more One of the essential characteristics of any tag used in bioscience and medical applications is its size. The larger the label, the more it may affect the studied object, and the more it may distort its behavior. In this paper, using NMR spectroscopy and X-ray crystallography, we have studied the structure of fluorogen-activating protein FAST both in the apo form and in complex with the fluorogen. We shown that significant change in the protein occurs upon interaction with the ligand. While the protein is completely ordered in the complex, its apo form is characterized by higher mobility and disordering of its N-terminus. We used structural information to design the shortened FAST (which we named nanoFAST) by truncating 26 N-terminal residues. Thus, we created the shortest genetically encoded tag among all known fluorescent and fluorogen-activating proteins, which is composed of only 98 amino acids.
The neurotrophin receptor p75NTR plays crucial roles in neuron development and regulates importan... more The neurotrophin receptor p75NTR plays crucial roles in neuron development and regulates important neuronal processes like degeneration, apoptosis and cell survival. At the same time the detailed mechanism of signal transduction is unclear. One of the main hypotheses known as the snail-tong mechanism assumes that in the inactive state, the death domains interact with each other and in response to ligand binding there is a conformational change leading to their exposure. Here, we show that neither rat nor human p75NTR death domains homodimerize in solution. Moreover, there is no interaction between the death domains in a more native context: the dimerization of transmembrane domains in liposomes and the presence of activating mutation in extracellular juxtamembrane region do not lead to intracellular domain interaction. These findings suggest that the activation mechanism of p75NTR should be revised. Thus, we propose a novel model of p75NTR functioning based on interaction with “help...
Cyclopentane fragment in the hydrophobic tail expands operational range of the surfactant into th... more Cyclopentane fragment in the hydrophobic tail expands operational range of the surfactant into the low-temperature region.
The molecular mechanism of TrkA activation have been poorly studied in comparison with those of o... more The molecular mechanism of TrkA activation have been poorly studied in comparison with those of other RTK family members. TrkA, like most RTKs, is in a monomer-dimer equilibrium in the absence of their ligand, and an unsolved issue is how ligand-binding can activate a pre-formed inactive dimer. Here we show that the transmembrane domain (TMD) of TrkA can form dimers using two different interfaces. We characterized the structural determinants of TrkA-TMD dimerization and identified the dimer interface of the active receptor. We validated this interface using site-directed mutagenesis together with functional and cell differentiation studies with the full-length TrkA receptor expressed in mammalian cells. Using in vivo crosslinking we identified a reordering of the extracellular juxtamembrane region after ligand binding. This conformational change could be mimicked by replacement of some residues with cysteine and the spontaneous formation of ligand-independent active dimers. In addit...
Lipid-protein nanodiscs (LPNs) are nanoscaled fragments of a lipid bilayer stabilized in solution... more Lipid-protein nanodiscs (LPNs) are nanoscaled fragments of a lipid bilayer stabilized in solution by the apolipoprotein or a special membrane scaffold protein (MSP). In this work, the applicability of LPN-based membrane mimetics in the investigation of water-soluble membrane-active peptides was studied. It was shown that a pore-forming antimicrobial peptide arenicin-2 from marine lugworm (charge of +6) disintegrates LPNs containing both zwitterionic phosphatidylcholine (PC) and anionic phosphatidylglycerol (PG) lipids. In contrast, the spider toxin VSTx1 (charge of +3), a modifier of Kv channel gating, effectively binds to the LPNs containing anionic lipids (POPC/DOPG, 3 : 1) and does not cause their disruption. VSTx1 has a lower affinity to LPNs containing zwitterionic lipids (POPC), and it weakly interacts with the protein component of nanodiscs, MSP (charge of -6). The neurotoxin II (NTII, charge of +4) from cobra venom, an inhibitor of the nicotinic acetylcholine receptor, shows...
Soil fungi are known to contain a rich variety of defense metabolites that allow them to compete ... more Soil fungi are known to contain a rich variety of defense metabolites that allow them to compete with other organisms (fungi, bacteria, nematodes, and insects) and help them occupy more preferential areas at the expense of effective antagonism. These compounds possess antibiotic activity towards a wide range of other microbes, particularly fungi that belong to different taxonomical units. These compounds include peptaibols, which are non-ribosomal synthesized polypeptides containing non-standard amino acid residues (alpha-aminoisobutyric acid mandatory) and some posttranslational modifications. We isolated a novel antibiotic peptide from the culture medium of Emericellopsis alkalina, an alkalophilic strain. This peptide, called emericellipsin A, exhibited a strong antifungal effect against the yeast Candida albicans, the mold fungus Aspergillus niger, and human pathogen clinical isolates. It also exhibited antimicrobial activity against some Gram-positive and Gram-negative bacteria....
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