Biology subjects

Kohga, H.

Publications and source records attributed to Kohga, H..

7 recordsLinked to original sources

bioRxiv2026

Structural and functional insights into multiple BAM-bound conformations of BepA enabling substrate triage at the outer membrane

The outer membrane (OM) of Gram-negative bacteria acts as a selective permeability barrier against toxic compounds, and for this function, requires proper assembly of outer membrane proteins (OMPs) by the {beta}-barrel assembly machinery (BAM) complex. A periplasmic metalloprotease BepA promotes maturation of LptD, an essential OMP, while degrades LptD intermediates aberrantly stalled at BAM. However, how BepA switches between these functions remains unclear. Here, we report cryo-EM structures of BAM- BepA complexes that capture multiple BepA conformations. In vivo crosslinking and cysteine-accessibility analyses show that possible regulatory elements, 6- and 9-loops that cover the proteolytic active site, can assume open conformations in living cells. Functional analyses reveal that 6-loop opening supports substrate interaction, degradation, and membrane association, whereas 9-loop opening is required for proteolytic activation and stabilizes BAM association. These findings provide insights into how distinct loop rearrangements regulate BepA-mediated substrate triage at BAM.

molecular biology↗

bioRxiv2025

Cryo-EM structures of the BAM-P2-visible SurA complex reveal dynamic and cooperative interactions in outer membrane protein assembly

The outer membrane (OM) of Gram-negative bacteria acts as a permeability barrier against toxic compounds. Its integrity is maintained by various outer membrane proteins (OMPs), which are inserted into the OM by the {beta}-barrel assembly machinery (BAM) complex. The periplasmic chaperone SurA delivers unfolded OMPs to BAM; however, the mechanism of substrate transfer remains unclear. Here, we show that the flexible P1 and P2 domains of SurA regulate the function of its Core domain and interact with BAM components, including BamE, whose interaction with the P2 domain is crucial for efficient OMP assembly. Moreover, cryo-electron microscopy revealed four distinct Escherichia coli SurA-BAM structures, suggesting dynamic domain rearrangements of SurA. Based on these findings, we propose a dynamic model in which SurA transfers substrates to BAM through multiple conformational changes, providing a unified framework for chaperone-assisted OMP biogenesis.

molecular biology↗

bioRxiv2025

Structural basis for MurJ inhibition by phage lysis protein SglPP7 suggesting convergence

Some bacteriophages encode lysis proteins that inhibit essential bacterial processes, the elucidation of which is valuable for developing antibacterial strategies against drug-resistant pathogens. We determined the cryo-EM structure of the complex between the essential E. coli lipid II flippase MurJ and a phage lysis protein, SglPP7. MurJ was locked in an outward-facing conformation by SglPP7, similar to the MurJ/LysM complex; however, distinct interactions suggest convergent evolution among phage lysis proteins.

molecular biology↗

bioRxiv2025

Phage lysis protein LysM acts as a wedge to block MurJ conformational changes

Many antibiotics target essential cellular processes. To combat multidrug-resistant bacteria, new antibacterial strategies are needed. In the peptidoglycan biogenesis pathway in Escherichia coli, MurJ, the lipid II flippase, is an essential membrane protein. The 37-residue protein M from the Levivirus phage, known as LysM or SglM, targets MurJ and induces cell lysis; however, its molecular mechanism remains unclear. Here, we present the cryo-EM structure of the MurJ/LysM (JM) complex at 3.09 [A] resolution, revealing that LysM interacts with the crevasse between TM2 and TM7 of MurJ, locking MurJ in an outward-facing conformation, with LysM acting like a wedge. Alanine-scanning mutagenesis and pull-down assays revealed key residues responsible for LysM function, and molecular dynamics simulations showed that LysM stabilizes MurJs outward-facing state. These findings demonstrate an unprecedented phage-derived mechanism for blocking lipid II transport, providing a structural framework for designing MurJ-targeted antimicrobial agents.

molecular biology↗

bioRxiv2025

Structural basis of a phosphotransferase system xylose transporter

The bacterial phosphotransferase system (PTS) is essential for carbohydrate uptake, and IIC transporters in the system facilitate intracellular sugar transport. Here, we report the crystal and cryo-EM structures of Leminorella grimontii GatC (LgGatC), a putative xylose transporter in the IIC family, in its outward-facing state. Structural analysis indicates that the outward-facing cavity recognizes the linear form of D-xylose. A structure-based homology model of an inward-facing state supports an elevator-like sugar transport mechanism. These findings provide insights into PTS-mediated xylose transport and its potential applications in microbial engineering.

molecular biology↗

bioRxiv2025

Structural basis of lipopolysaccharide translocon assembly mediated by the small lipoprotein LptM

Gram-negative bacteria possess an outer membrane (OM) that acts as a barrier against toxic compounds. Lipopolysaccharide (LPS) in the outer leaflet of the OM is crucial for barrier function. After its synthesis in the cytoplasm and translocation to the periplasm, LPS is transported to the OM by the LPS transport system. The LPS translocon, composed of an OM protein LptD and a lipoprotein LptE, mediates LPS assembly into the OM. Recently, the small lipoprotein LptM (YifL) was identified as a novel LptD/E-interactor that facilitates LptD maturation. However, its mechanism remains unclear. Here, we investigated the detailed interaction between LptM and LptD. We found that LptM interacts with the folded LptD intermediate at the late stage of its maturation. Mutational analyses demonstrated that the N-terminal conserved region (C20GLKGPLYF28) of LptM is essential for its function. Cryo-EM structural analysis of the E. coli LptD/E/M complex, combined with biochemical analyses, revealed the molecular basis of the LptM-LptD interaction and its functional importance. Thus, we propose that LptM functions as a "barrel-rivet," stabilizing LptD for its proper assembly.

molecular biology↗

bioRxiv2023

Critical residues of the antibiotic peptide LysM that inhibits lipid II flipping

Small single-strand DNA/RNA phages that infect gram-negative bacteria encode lysis proteins that induce cell lysis without directly degrading the cell wall. One such protein, the 37-residue LysM protein derived from a lysis gene of Levivirus phage M (lysM), completely blocks the lipid II transport activity mediated by Escherichia coli MurJ, which is essential for peptidoglycan biosynthesis. LysM was proposed to be a single -helical transmembrane protein that binds to MurJ and prevents its conformational transition during lipid II transport. Although LysM possibly interacts with MurJ, the inhibition mechanism remains unclear. Here, we identified the crucial residues for LysM function via comprehensive alanine-scanning mutagenesis. These residues were located on two surfaces in an -helix model, probably providing surfaces interacting with MurJ in the membrane. This study provides fundamental information regarding the mechanism of LysM inhibition.

microbiology↗