Structural Classification Insights Into the Plant Defensive Peptides.

This study presents a structural phylogenetic analysis of plant defensive peptides, revealing their evolutionary relationships, structural diversification, and functional adaptations. Utilizing a robust dataset comprising both experimental and predicted structures sourced from the RCSB Protein Data Bank and AlphaFold DB, we constructed a detailed phylogenetic tree to elucidate the distinct evolutionary paths of plant defensive peptide families. Our findings showcase the evolutionary intricacies of defensive peptides, highlighting their diversity and the conservation of key structural motifs critical to their antimicrobial or defensive functions. The results also underscore the adaptive significance of defensive peptides in plant evolution, highlighting their roles in responding to ecological pressures and pathogen interactions.

Identification of inhibitors targeting the energy-coupling factor (ECF) transporters.

The energy-coupling factor (ECF) transporters are a family of transmembrane proteins involved in the uptake of vitamins in a wide range of bacteria. Inhibition of the activity of these proteins could reduce the viability of pathogens that depend on vitamin uptake. The central role of vitamin transport in the metabolism of bacteria and absence from humans make the ECF transporters an attractive target for inhibition with selective chemical probes. Here, we report on the identification of a promising class of inhibitors of the ECF transporters. We used coarse-grained molecular dynamics simulations on Lactobacillus delbrueckii ECF-FolT2 and ECF-PanT to profile the binding mode and mechanism of inhibition of this novel chemotype. The results corroborate the postulated mechanism of transport and pave the way for further drug-discovery efforts.

Probing antibacterial drugs for Fusobacterium nucleatum subsp. nucleatum ATCC 25586 targeting UDP-N-acetylglucosamine 1-carboxyltransferase.

Fusobacterium nucleatum is a Gram-negative anaerobic bacteria that is commonly found in oral cavities and is associated with connective tissue destruction in periodontitis. UDP-N-acetylglucosamine 1-carboxyltransferase with enzyme commission number 2.5.1.7 is a transferases enzyme that plays a role in bacterial pathogenesis. Inhibiting binding sites of UDP-N-acetylglucosamine 1-carboxyltransferase is needed to find potential antibiotic candidates for periodontitis treatment. Hence, the research aimed to present potential UDP-N-acetylglucosamine 1-carboxyltransferase inhibiting compounds through molecular docking simulation by in silico analysis. DrugBank database was used to obtain the antibacterial candidates, which were further screened computationally using the AutoDock Vina program on Google Colab Pro. The top nine compounds yielded binding affinity ranging from -12.1 to -12.8 kcal/mol, with conivaptan as one of the three compounds having the highest binding affinity. Molecular dynamic study revealed that the ligand-protein complex for conivaptan had root-mean-square deviation values of 0.05-1.1 nm, indicating likeliness for stable interaction. Our findings suggest that conivaptan is the potent UDP-N-acetylglucosamine 1-carboxyltransferase inhibitor, hence its efficacy against periodontitis-causing bacteria.

In vitro reconstitution of dynamically interacting integral membrane subunits of energy-coupling factor transporters.

Energy-coupling factor (ECF) transporters mediate import of micronutrients in prokaryotes. They consist of an integral membrane S-component (that binds substrate) and ECF module (that powers transport by ATP hydrolysis). It has been proposed that different S-components compete for docking onto the same ECF module, but a minimal liposome-reconstituted system, required to substantiate this idea, is lacking. Here, we co-reconstituted ECF transporters for folate (ECF-FolT2) and pantothenate (ECF-PanT) into proteoliposomes, and assayed for crosstalk during active transport. The kinetics of transport showed that exchange of S-components is part of the transport mechanism. Competition experiments suggest much slower substrate association with FolT2 than with PanT. Comparison of a crystal structure of ECF-PanT with previously determined structures of ECF-FolT2 revealed larger conformational changes upon binding of folate than pantothenate, which could explain the kinetic differences. Our work shows that a minimal in vitro system with two reconstituted transporters recapitulates intricate kinetics behaviour observed in vivo.

An ECF-type transporter scavenges heme to overcome iron-limitation in Staphylococcus lugdunensis.

Energy-coupling factor type transporters (ECF) represent trace nutrient acquisition systems. Substrate binding components of ECF-transporters are membrane proteins with extraordinary affinity, allowing them to scavenge trace amounts of ligand. A number of molecules have been described as substrates of ECF-transporters, but an involvement in iron-acquisition is unknown. Host-induced iron limitation during infection represents an effective mechanism to limit bacterial proliferation. We identified the iron-regulated ECF-transporter Lha in the opportunistic bacterial pathogen Staphylococcus lugdunensis and show that the transporter is specific for heme. The recombinant substrate-specific subunit LhaS accepted heme from diverse host-derived hemoproteins. Using isogenic mutants and recombinant expression of Lha, we demonstrate that its function is independent of the canonical heme acquisition system Isd and allows proliferation on human cells as sources of nutrient iron. Our findings reveal a unique strategy of nutritional heme acquisition and provide the first example of an ECF-transporter involved in overcoming host-induced nutritional limitation.

Dynamic Combinatorial Chemistry to Identify Binders of ThiT, an S-Component of the Energy-Coupling Factor Transporter for Thiamine.

We applied dynamic combinatorial chemistry (DCC) to identify ligands of ThiT, the S-component of the energy-coupling factor (ECF) transporter for thiamine in Lactococcus lactis. We used a pre-equilibrated dynamic combinatorial library (DCL) and saturation-transfer difference (STD) NMR spectroscopy to identify ligands of ThiT. This is the first report in which DCC is used for fragment growing to an ill-defined pocket, and one of the first reports for its application with an integral membrane protein as target.