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Toll-like receptors (TLRs) are key players in the innate immune system. Despite the great efforts in TLR structural biology, today we know the spatial structures of only four human TLR intracellular TIR domains. All of them belong to one of five subfamilies of receptors. One of the main bottlenecks is the high-level production of correctly folded proteins in soluble form. Here we used a rational approach to find the optimal parameters to produce TIR domains of all ten human TLR family members in soluble form in E . coli cells. We showed that dozens of milligrams of soluble His-tagged TLR2/3/6/7 TIR and MBP-tagged TLR3/5/7/8 TIR can be produced. We also developed the purification protocols and demonstrated by CD and NMR spectroscopy that purified TLR2/3/7 TIR demonstrate a structural organization inherent to TIR domains. This illustrates the correct folding of produced proteins and their suitability for further structural and functional investigations.

Natural toxins are highly effective at targeting ion channels with high selectivity and potency. To date, all identified spider venom peptide toxins that modulate voltage-gated potassium (K V ) channels inhibit Shab (K V 2) or Shal-related isoforms (K V 4) by interacting with their voltage-sensing domains. In this study, we report novel spider-derived pore-blocking toxins that selectively target Shaker-type (K V 1) channels with nanomolar potency. We isolated murinotoxins MnTx-1 and MnTx-2 from the orange baboon tarantula Pterinochilus murinus and sequenced them using a combination of Edman degradation, mass spectrometry, and venom gland nanopore transcriptomics. MnTx-1 was produced recombinantly, and its NMR solution structure was determined. Although MnTx-1 shares sequence motifs common to spider toxins, it displays a distinctly different three-dimensional structure, featuring an alternative disulfide linkage, which we have termed the Disulfide-Reined Hairpin (DRH). We attribute the unique pharmacology of MnTx-1 to its unusual spatial structure. The DRH motif represents a promising new miniature scaffold for future bioengineering applications.

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. Additionally, emericellipsin A showed a significant cytotoxic effect and was highly active against Hep G2 and HeLa tumor cell lines. We used NMR spectroscopy to reveal that this peptaibol is nine amino acid residues long and contains non-standard amino acids. The mode of molecular action of emericellipsin A is most likely associated with its effects on the membranes of cells. Emericellipsin A is rather short peptaibol and could be useful for the development of antifungal, antibacterial, or anti-tumor remedies.

Nicotinic acetylcholine receptors (nAChRs) play an important role in the functioning of the central and peripheral nervous systems, and other organs of living creatures. There are several subtypes of nAChRs, and almost all of them are considered as pharmacological targets in different pathological states. The crude venom of the sea anemone Metridium senile showed the ability to interact with nAChRs. Four novel peptides (Ms11a-1–Ms11a-4) with nAChR binding activity were isolated. These peptides stabilized by three disulfide bridges have no noticeable homology with any known peptides. Ms11a-1–Ms11a-4 showed different binding activity towards the muscle-type nAChR from the Torpedo californica ray. The study of functional activity and selectivity for the most potent peptide (Ms11a-3) revealed the highest antagonism towards the heterologous rat α9α10 nAChR compared to the muscle and α7 receptors. Structural NMR analysis of two toxins (Ms11a-2 and Ms11a-3) showed that they belong to a new variant of the inhibitor cystine knot (ICK) fold but have a prolonged loop between the fifth and sixth cysteine residues. Peptides Ms11a-1–Ms11a-4 could represent new pharmacological tools since they have structures different from other known nAChRs inhibitors.

A new series of AT-specific minor groove DNA ligands (DB 3 P( n ); n  = 1,2,3,4) was synthesized and their spectral, biological and virological properties were investigated. With a methylene spacers of different lengths blocks of three AT pairs located at different distances from each other could be recognized. The compounds synthesized suppressed the activity of HIV-1 integrase at submicromolar concentrations (0.25–0.50 µМ). Also, DB 3 P( n ) were found to be effective inhibitors of simplex virus type I and DNA topoisomerase I. The synthesized DB 3 P( n ) demonstrated good solubility in water, could penetrate through cell and nuclear membranes, and stain DNA in live cells. Graphical Abstract

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-binding ability of TLR1-TIR domain is critical for the receptor activation.Lushpa et al report the structure and dynamics of the TLR1 toll-interleukin like (TIR) cytoplasmic domain in both crystal and solution. They demonstrate that the TLR1 TIR domain is capable of specific binding of Zn with nanomolar affinity, which appears to be critical for receptor activation, and provide potential structures TLR1-TIR/Zn complex based on in silico data.

Peptaibols are linear fungal peptides featuring α,α-dialkylated amino acids (e.g., α-aminoisobutyric acid (Aib), isovaline (Iva)) and characteristic C-terminal alcohol groups. Despite their promising antibacterial and antiplasmodial activities, detailed biosynthetic studies remain limited. A genome-guided study of the fungus Trichodema sp. SK1-7, isolated from decaying wood, revealed the production of previously described trichorozin IV (1), along with novel SF4-type peptaibol 2 (trichorozin V). The structures of these compounds were elucidated through MS analysis, NMR study and advanced Marfey’s method. The genome of Trichoderma sp. SK1-7 harbors two PKS-NRPS hybrid gene clusters containing 14 and 18 adenylation domains. Analysis of the modular architecture suggested that trichorozins are synthesized by a 14-module protein via a module skipping mechanism. Genome mining revealed several types of short peptaibol synthase architectures (10–14 adenylation domains) across various Trichoderma species, accompanied by similar long peptaibol synthases. Furthermore, putative Aib/Iva biosynthesis machinery in Trichoderma was identified, showing specific architectures potentially involved in regulating peptaibol biosynthesis. Feeding experiments demonstrated that peptaibol production depends on the ratio of Iva/Aib. The isolated compounds exhibited moderate antibacterial and cytotoxic activities along with a synergistic effect when combined with membrane-targeting antibiotics. Our findings suggest that genome-guided approaches hold promise for further development of peptabiotics with a wide range of applications, including antibiotic adjuvants.

NanoFAST is a fluorogen-activating protein and can be considered one of the smallest encodable fluorescent tags. Being a shortened variant of another fluorescent tag, FAST, nanoFAST works nicely only with one out of all known FAST ligands. This substantially limits the applicability of this protein. To find the reason for such a behavior, we investigated the spatial structure and dynamics of nanoFAST, both in the apo state and in the complex with its fluorogen molecule, using the solution NMR spectroscopy. We showed that the truncation of FAST did not affect the structure of the remaining part of the protein. Our data suggest that the deleted N-terminus of FAST destabilizes the C-terminal domain in the apo state. While it does not contact the fluorogen directly, it serves as a free energy reservoir that enhances the ligand binding propensity of the protein. The structure of nanoFAST/HBR-DOM2 complex reveals the atomistic details of nanoFAST interactions with the rhodanine-based ligands and explains the ligand specificity. NanoFAST selects ligands with the lowest dissociation constants, 2,5-disubstituted 4-hydroxybenzyldienerhodainines, which allow the non-canonical intermolecular CH–N hydrogen bonding and provide the optimal packing of the ligand within the hydrophobic cavity of the protein.

The transition to organic farming is one of the most desirable achievements of our time. Rational use of organic farming approaches not only enables a reduction in costs and increased yields but also limits the risks associated with the use of pesticides and chemicals. Despite the widest practical application of numerous biocontrol agents based on Bacillus strains, their metabolome, including the main active substances, often remains unknown. In order to understand the basic principles of the functioning of the Bacillus velezensis K-3618 strain, widely used in organic farming, we studied its spectrum of antimicrobial metabolites in detail. It was shown that the main antimicrobial agents of B. velezensis K-3618 are representatives of the macrolactin family. The identified macrolactin A (MLN A) and its acylated analogs 7-O-malonyl macrolactin A (mal-MLN A) and 7-O-succinyl macrolactin A (suc-MLN A) are active against Gram-positive bacterial pathogens, including multidrug-resistant strains. Among them, suc-MLN A is the most potent antimicrobial, highly active (MIC = 0.1 μg/mL) against the common human pathogen methicillin-resistant Staphylococcus aureus (MRSA). It was revealed that the primary mechanism of action of MLN A-based macrolactins is protein translation inhibition. Acylated macrolactins outperform MLN A in the prokaryotic cell-free system, displaying high efficiency in low micromolar concentrations. We observed that acylated MLN A analogs undergo pathogen-mediated biotransformation into MLN F analogs, having their antimicrobial activity reduced by two orders of magnitude. Hence, both acylation of MLNs and stabilization of the MLN A core are essential for the creation of new synthetic MLNs with improved antimicrobial activity and stability. However, we speculate that these degradability modes are of prime importance for bacterial ecology, and they are highly conserved in Bacillus species from various ecological niches.

Biochemistry of bioluminescence of the marine parchment tubeworm Chaetopterus has been in research focus for over a century; however, the results obtained by various groups contradict each other. Here, we report the isolation and structural elucidation of three compounds from Chaetomorpha linum algae, which demonstrate bioluminescence activity with Chaetopterus luciferase in the presence of Fe2+ ions. These compounds are derivatives of polyunsaturated fatty acid peroxides. We have also obtained their structural analogues and demonstrated their activity in the bioluminescence reaction, thus confirming the broad substrate specificity of the luciferase.