The YhcH/YjgK/YiaL (DUF386) family, widely conserved across bacterial species, is involved in essential cellular processes yet remains poorly characterized. YjgK from Salmonella enterica serovar Typhimurium has drawn attention because of its potential role in biofilm formation associated with metal homeostasis, which may be critical for bacterial survival. In this study, we report the crystal structure of YjgK at 1.76 Å resolution, revealing a dimeric arrangement where each monomer consists of a jelly roll-type β-sandwich fold. This fold forms a funnel-shaped cavity, suggesting potential ligand binding. YjgK contains two zinc ions per dimer, which were identified through structural analysis and confirmed by inductively coupled plasma mass spectrometry (ICP-MS). The zinc ions are coordinated by conserved residues (Glu62, His64, Asp69, and His128) to form a tetrahedral geometry. Structural comparisons with homologous proteins revealed significant similarities in their overall fold but distinct differences in their metal ion specificity, with YhcH binding copper and HP1029 binding zinc. Salmonella lacking YjgK increased biofilm formation, while YjgK overexpression hardly influenced biofilm formation. Our findings suggest that the zinc-binding capability of YjgK may play a key role in metal ion homeostasis, contributing to the ability of Salmonella to form biofilm in response to metal-limited environments, such as those encountered during infection. The conservation of DUF386 fold across species, along with variations in metal ion coordination, indicates functional diversification within this family.

Methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) infection has rapidly spread through various routes. A genomic analysis of clinical MRSA samples revealed an unknown protein, Sav2152, predicted to be a haloacid dehalogenase (HAD)-like hydrolase, making it a potential candidate for a novel drug target. In this study, we determined the crystal structure of Sav2152, which consists of a C2-type cap domain and a core domain. The core domain contains four motifs involved in phosphatase activity that depend on the presence of Mg²⁺ ions. Specifically, residues D10, D12, and D233, which closely correspond to key residues in structurally homolog proteins, are responsible for binding to the metal ion and are known to play critical roles in phosphatase activity. Our findings indicate that the Mg²⁺ ion known to stabilize local regions surrounding it, however, paradoxically, destabilizes the local region. Through mutant screening, we identified D10 and D12 as crucial residues for metal binding and maintaining structural stability via various uncharacterized intra-protein interactions, respectively. Substituting D10 with Ala effectively prevents the interaction with Mg²⁺ ions. The mutation of D12 disrupts important structural associations mediated by D12, leading to a decrease in the stability of Sav2152 and an enhancement in binding affinity to Mg²⁺ ions. Additionally, our study revealed that D237 can replace D12 and retain phosphatase activity. In summary, our work uncovers the novel role of metal ions in HAD-like phosphatase activity.

The YhcH/YjgK/YiaL (DUF386) family, widely conserved across bacterial species, is involved in essential cellular processes yet remains poorly characterized. YjgK from Salmonella enterica serovar Typhimurium has drawn attention because of its potential role in biofilm formation associated with metal homeostasis, which may be critical for bacterial survival. In this study, we report the crystal structure of YjgK at 1.76 Å resolution, revealing a dimeric arrangement where each monomer consists of a jelly roll-type β-sandwich fold. This fold forms a funnel-shaped cavity, suggesting potential ligand binding. YjgK contains two zinc ions per dimer, which were identified through structural analysis and confirmed by inductively coupled plasma mass spectrometry (ICP-MS). The zinc ions are coordinated by conserved residues (Glu62, His64, Asp69, and His128) to form a tetrahedral geometry. Structural comparisons with homologous proteins revealed significant similarities in their overall fold but distinct differences in their metal ion specificity, with YhcH binding copper and HP1029 binding zinc. Salmonella lacking YjgK increased biofilm formation, while YjgK overexpression hardly influenced biofilm formation. Our findings suggest that the zinc-binding capability of YjgK may play a key role in metal ion homeostasis, contributing to the ability of Salmonella to form biofilm in response to metal-limited environments, such as those encountered during infection. The conservation of DUF386 fold across species, along with variations in metal ion coordination, indicates functional diversification within this family.

Methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) infection has rapidly spread through various routes. A genomic analysis of clinical MRSA samples revealed an unknown protein, Sav2152, predicted to be a haloacid dehalogenase (HAD)-like hydrolase, making it a potential candidate for a novel drug target. In this study, we determined the crystal structure of Sav2152, which consists of a C2-type cap domain and a core domain. The core domain contains four motifs involved in phosphatase activity that depend on the presence of Mg²⁺ ions. Specifically, residues D10, D12, and D233, which closely correspond to key residues in structurally homolog proteins, are responsible for binding to the metal ion and are known to play critical roles in phosphatase activity. Our findings indicate that the Mg²⁺ ion known to stabilize local regions surrounding it, however, paradoxically, destabilizes the local region. Through mutant screening, we identified D10 and D12 as crucial residues for metal binding and maintaining structural stability via various uncharacterized intra-protein interactions, respectively. Substituting D10 with Ala effectively prevents the interaction with Mg²⁺ ions. The mutation of D12 disrupts important structural associations mediated by D12, leading to a decrease in the stability of Sav2152 and an enhancement in binding affinity to Mg²⁺ ions. Additionally, our study revealed that D237 can replace D12 and retain phosphatase activity. In summary, our work uncovers the novel role of metal ions in HAD-like phosphatase activity.

Endolysins produced by bacteriophages play essential roles in the release of phage progeny by degrading the peptidoglycan layers of the bacterial cell wall. Bacteriophage-encoded endolysins have emerged as a new class of antibacterial agents to combat surging antibiotic resistance. The crystal structure of mtEC340M, an engineered endolysin EC340 from the PBEC131 phage that infects Escherichia coli, was determined. The crystal structure of mtEC340M at 2.4 Å resolution consists of eight α-helices and two loops. The three active residues of mtEC340M were predicted by structural comparison with peptidoglycan-degrading lysozyme.

Protein phosphorylation is one of the most widely observed and important post-translational modification (PTM) processes. Protein phosphorylation is regulated by protein kinases, each of which covalently attaches a phosphate group to an amino acid side chain on a serine (Ser), threonine (Thr), or tyrosine (Tyr) residue of a protein, and by protein phosphatases, each of which, conversely, removes a phosphate group from a phosphoprotein. These reversible enzyme activities provide a regulatory mechanism by activating or deactivating many diverse functions of proteins in various cellular processes. In this review, their structures and substrate recognition are described and summarized, focusing on Ser/Thr protein kinases and protein Ser/Thr phosphatases, and the regulation of protein structures by phosphorylation. The studies reviewed here and the resulting information could contribute to further structural, biochemical, and combined studies on the mechanisms of protein phosphorylation and to drug discovery approaches targeting protein kinases or protein phosphatases.

Interactions involving Epstein-Barr virus (EBV) LMP2A and Nedd4 family E3 ubiquitin-protein ligases promote the ubiquitination of LMP2A-associated proteins, which results in the perturbation of normal B-cell signaling. Here, we solved the solution structure of the WW2 domain of hAIP4 and investigated the binding mode involving the N-terminal domain of LMP2A and the WW2 domain. The WW2 domain presented a conserved WW domain scaffold with a three-stranded anti-parallel β-sheet and bound two PY motifs via different binding mechanisms. Our NMR titration and ITC data demonstrated that the PY motifs of LMP2A can recognize and interact weakly with the XP groove of the WW2 domain (residues located around the third β-strand), and then residues between two PY motifs optimize the binding by interacting with the loop 1 region of the WW2 domain. In particular, the residue Val15 in the hairpin loop region between β1 and β2 of the WW2 domain exhibited unique changes depending on the terminal residues of the PY motif. This result suggested that the hairpin loop is responsible for additional interactions outside the XP groove, and this hypothesis was confirmed in a deuterium exchange experiment. These weak but wide interactions can stabilize the complex formed between the PY and WW domains.

We report the crystal structure and bioinformatic analysis of SF173, a functionally uncharacterized protein from the human enteropathogenic bacteria Shigella flexneri. The structure shows a tightly interlinked dimer formed by adimeric core comprising α2 and α3 helices from both subunits and swapping the N-terminal α1 helix of each monomer. Structural inspection and genomic analysis results suggest that the SF173 might play its putative function by binding to SF172, the partially overlapped upstream product in the operon. As YaeO (an SF172 orthologue) has been identified to be an inhibitor of the bacterial transcription terminator Rho protein, SF173 is suggested to be involved in the regulation of Rho-dependent transcription termination, by inhibiting the Rho protein binding to SF172/YaeO.