Iron is crucial for the metabolism and growth of most prokaryotic cells. The ferric uptake regulator (Fur) protein plays a central role in regulating iron homeostasis and metabolic processes in bacteria. It ensures the proper utilization of iron and the maintenance of cellular functions in response to environmental cues. Fur proteins are composed of an N-terminal DNA-binding domain (DBD) and a C-terminal dimerization domain (DD), typically existing as dimers in solution. Fur proteins have conserved metal-binding sites named S1, S2, and S3. Among them, site S2 serves as a regulatory site, and metal binding at S2 results in conformational changes. Additionally, as a transcriptional regulator, Fur specifically binds to a consensus DNA sequence called the Fur box. To elucidate the structural and functional properties of Fur proteins, various structures of metal- or DNA-bound Fur proteins or apo-Fur proteins have been determined. In this review, we focus on the structural properties of Fur proteins according to their ligand-bound state and the drug development strategies targeting Fur proteins. This information provides valuable insights for drug discovery.

The structure-function relationships of toxin-antitoxin (TA) systems from Mycobacterium tuberculosis have prompted the development of novel and effective antimicrobial agents that selectively target this organism. The artificial activation of toxins by peptide inhibitors can lead to the growth arrest and eventual death of bacterial cells. Optimizing candidate peptides by hydrocarbon α-helix stapling based on structural information from the VapBC TA system and in vitro systematic validation led to V26-SP-8, a VapC26 activator of M. tuberculosis. This compound exhibited highly enhanced activity and cell permeability owing to the stabilizing helical propensity of the peptide. These characteristics will increase its efficacy against multidrug-resistant tuberculosis and extensively drug-resistant tuberculosis. Similar approaches utilizing structural and biochemical information for new antibiotic targets opens a new era for developing TB therapies.

Staphylococcus aureus is a notorious and globally distributed pathogenic bacterium. New strategies to develop novel antibiotics based on intrinsic bacterial toxin–antitoxin (TA) systems have been recently reported. Because TA systems are present only in bacteria and not in humans, these distinctive systems are attractive targets for developing antibiotics with new modes of action. S. aureus PemIK is a type II TA system, comprising the toxin protein PemK and the labile antitoxin protein PemI. Here, we determined the crystal structures of both PemK and the PemIK complex, in which PemK is neutralized by PemI. Our biochemical approaches, including fluorescence quenching and polarization assays, identified Glu20, Arg25, Thr48, Thr49, and Arg84 of PemK as being important for RNase function. Our study indicates that the active site and RNA-binding residues of PemK are covered by PemI, leading to unique conformational changes in PemK accompanied by repositioning of the loop between β1 and β2. These changes can interfere with RNA binding by PemK. Overall, PemK adopts particular open and closed forms for precise neutralization by PemI. This structural and functional information on PemIK will contribute to the discovery and development of novel antibiotics in the form of peptides or small molecules inhibiting direct binding between PemI and PemK.

Bacterial toxin–antitoxin (TA) systems correlate strongly with physiological processes in bacteria, such as growth arrest, survival and apoptosis. Here, the first crystal structure of a type II TA complex structure of Klebsiella pneumoniae at 2.3 Å resolution is presented. The K. pneumoniae MazEF complex consists of two MazEs and four MazFs in a heterohexameric assembly. It was estimated that MazEF forms a dodecamer with two heterohexameric MazEF complexes in solution, and a truncated complex exists in heterohexameric form. The MazE antitoxin interacts with the MazF toxin via two binding modes, namely, hydrophobic and hydrophilic interactions. Compared with structural homologs, K. pneumoniae MazF shows distinct features in loops β1–β2, β3–β4 and β4–β5. It can be inferred that these three loops have the potential to represent the unique characteristics of MazF, especially various substrate recognition sites. In addition, K. pneumoniae MazF shows ribonuclease activity and the catalytic core of MazF lies in an RNA-binding pocket. Mutation experiments and cell-growth assays confirm Arg28 and Thr51 as critical residues for MazF ribonuclease activity. The findings shown here may contribute to the understanding of the bacterial MazEF TA system and the exploration of antimicrobial candidates to treat drug-resistant K. pneumoniae.

Peptide-based therapeutics have traditionally faced challenges, including instability in the bloodstream and limited cell membrane permeability. However, recent advancements in α-helix stapled peptide modification techniques have rekindled interest in their efficacy. Notably, these developments ensure a highly effective method for improving peptide stability and enhancing cell membrane penetration. Particularly in the realm of antimicrobial peptides (AMPs), the application of stapled peptide techniques has significantly increased peptide stability and has been successfully applied to many peptides. Furthermore, constraining the secondary structure of peptides has also been proven to enhance their biological activity. In this review, the entire process through which hydrocarbon-stapled antimicrobial peptides attain improved drug-like properties is examined. First, the essential secondary structural elements required for their activity as drugs are validated, specific residues are identified using alanine scanning, and stapling techniques are strategically incorporated at precise locations. Additionally, the mechanisms by which these structure-based stapled peptides function as AMPs are explored, providing a comprehensive and engaging discussion.

The bacterial toxin–antitoxin (TA) system regulates cell growth under various environmental stresses.Mycobacterium tuberculosis, the causative pathogen of tuberculosis (TB), has three HigBA type II TA systems with reverse gene organization, consisting of the toxin protein HigB and labile antitoxin protein HigA. Most type II TA modules are transcriptionally autoregulated by the antitoxin itself. In this report, we first present the crystal structure of theM. tuberculosisHigA3 antitoxin (MtHigA3) andMtHigA3 bound to its operator DNA complex. We also investigated the interaction betweenMtHigA3 and DNA using NMR spectroscopy. TheMtHigA3 antitoxin structure is a homodimer that contains a structurally well conserved DNA-binding domain at the N-terminus and a dimerization domain at the C-terminus. Upon comparing the HigA homologue structures, a distinct difference was found in the C-terminal region that possesses the β-lid, and diverse orientations of two helix–turn–helix (HTH) motifs from HigA homologue dimers were observed. The structure ofMtHigA3 bound to DNA reveals that the promoter DNA is bound to two HTH motifs of theMtHigA3 dimer presenting 46.5° bending, and the distance between the two HTH motifs of eachMtHigA3 monomer was increased inMtHigA3 bound to DNA. The β-lid, which is found only in the tertiary structure ofMtHigA3 among the HigA homologues, causes the formation of a tight dimerization network and leads to a unique arrangement for dimer formation that is related to the curvature of the bound DNA. This work could contribute to the understanding of the HigBA system ofM. tuberculosisat the atomic level and may contribute to the development of new antibiotics for TB treatment.