Biology subjects

Ieva, R.

Publications and source records attributed to Ieva, R..

3 recordsLinked to original sources

bioRxiv2025

Structural Basis of Lipopolysaccharide Assembly by the Outer Membrane Translocon Holo-Complex

Lipopolysaccharide (LPS) assembly at the surfaces-exposed leaflet of the bacterial outer membrane (OM) is mediated by the OM LPS translocon. An essential transmembrane {beta}-barrel protein, LptD, and a cognate lipoprotein, LptE, translocate LPS selectively into the OM external leaflet via a poorly understood mechanism. Here, we characterize two additional translocon subunits, the lipoproteins LptM and LptY (formerly YedD). We use single-particle cryo-EM analysis, functional assays and molecular dynamics simulations to visualize the roles of LptM and LptY at the translocon holo-complex LptDEMY, uncovering their impact on LptD conformational dynamics. Whereas LptY binds and stabilizes the periplasmic LptD {beta}-taco domain that functions as LPS receptor, LptM intercalates the lateral gate of the {beta}-barrel domain, promoting its opening and access by LPS. Remarkably, we demonstrate a conformational switch of the LptD {beta}-taco/{beta}-barrel interface alternating between contracted and extended states. The LptD {beta}-strand 1, which defines the mobile side of the lateral gate, binds LPS and performs a stroke movement toward the external leaflet during the contracted-to-extended state transition. Our findings establish a detailed mechanistic framework explaining the selective transport of LPS to the membrane external leaflet.

microbiology↗

bioRxiv2023

LptM promotes oxidative maturation of the lipopolysaccharide translocon by substrate binding mimicry.

Insertion of lipopolysaccharide (LPS) into the outer membrane (OM) of Gram-negative bacteria is mediated by a druggable OM translocon consisting of a {beta}-barrel membrane protein, LptD, and a lipoprotein, LptE. The {beta}-barrel assembly machinery (BAM) assembles LptD together with LptE to form a plug-and-barrel structure. In the enterobacterium Escherichia coli, formation of two native disulfide bonds in LptD controls LPS translocon activation. Here we report the discovery of LptM (formerly YifL), a conserved lipoprotein that assembles together with LptD and LptE at the BAM complex. We demonstrate that LptM stabilizes a conformation of LptD that can efficiently acquire native disulfide bonds and be released as mature LPS translocon by the BAM complex. Inactivation of LptM causes the accumulation of non-natively oxidized LptD, making disulfide bond isomerization by DsbC become essential for viability. Our structural prediction and biochemical analyses indicate that LptM binds to sites in both LptD and LptE that are proposed to coordinate LPS insertion into the OM. These results suggest that LptM facilitates oxidative maturation of LptD by mimicking LPS binding, thereby activating the LPS translocon.

microbiology↗

bioRxiv2020

Outer membrane lipoprotein DolP interacts with the BAM complex and promotes fitness during envelope stress response

In Gram-negative bacteria, coordinated remodelling of the outer membrane (OM) and the peptidoglycan is crucial for envelope integrity. Envelope stress caused by unfolded OM proteins (OMPs) activates sigmaE ({sigma}E) in Enterobacteria. {sigma}E upregulates OMP biogenesis factors, including the {beta}-barrel assembly machinery (BAM) that catalyzes OMP-folding. Elevated {sigma}E activity, however, can be detrimental for OM integrity. Here we report that DolP (YraP), a {sigma}E-upregulated OM lipoprotein important for envelope integrity, is a novel interactor of BAM and we demonstrate that OM-assembled BamA is a critical determinant of the BAM-DolP complex. Mid-cell recruitment of DolP had been previously associated to activation of septal peptidoglycan remodelling during cell division, but its role during envelope stress was unknown. We now show that DolP promotes cell fitness upon stress-induced activation of {sigma}E and opposes a detrimental effect caused by the overaccumulation of BAM in the OM. During envelope stress, DolP loses its association with the mid-cell, thus suggesting a possible link between envelope stress caused by impaired OMP biogenesis and the regulation of a late step of cell division.

microbiology↗