Biology subjects

Isom, G. L.

Publications and source records attributed to Isom, G. L..

9 recordsLinked to original sources

bioRxiv2026

Structural modelling and functional analysis support lipid binding by the dimeric E. coli DedA protein YqjA

Maintenance of membrane homeostasis is essential for bacterial viability, yet the molecular functions of many membrane proteins involved in this process remain poorly understood. The widely distributed DedA superfamily of integral membrane proteins has been implicated in membrane homeostasis, with deletion of DedA genes resulting in sensitivities to temperature, pH and a range of antimicrobial compounds. Several bacterial DedA proteins have been linked to lipid transport, providing a potential connection between function and phenotype. However, a lack of direct functional and structural data means that the precise role of DedA proteins in bacterial membrane homeostasis remains unclear. Here, using analytical ultracentrifugation (AUC), we show that the Escherichia coli DedA protein YqjA exists predominantly as a dimer and, by combining structural modelling with site-specific cysteine crosslinking, we have identified the most likely dimer interface. Modelling of dimeric YqjA in the presence of lipids predicts an interfacial lipid-binding site located in close proximity to several conserved, functionally important residues. Consistent with lipid binding, addition of lipid substantially increased the thermal stability of YqjA. Furthermore, mutation of residues associated with the predicted binding site impaired YqjA function. Together, these findings provide new structural and functional insights into the DedA family and support a role for lipid binding in the activity of YqjA.

biochemistry↗

bioRxiv2026

Homologues of the inner-membrane LPS transport proteins are required for sphingolipid transport in Caulobacter crescentus

Recent elucidation of the bacterial sphingolipid synthesis pathway has revealed that these lipids are produced by a range of taxonomically diverse species. In contrast to the biosynthetic pathways, the mechanism by which sphingolipids are transported from the inner membrane to the cell surface in Gram-negative bacteria remains a mystery. Here, we identify and characterize paralogs of the well-characterized lipopolysaccharide (LPS) inner membrane ABC transporter proteins encoded within the sphingolipid locus. Using Caulobacter crescentus as a model system, we analyzed three putative inner membrane proteins with homology to LptF, LptG, and LptC. Deletion of these genes was lethal, likely due to the accumulation of anionic sphingolipids in the inner membrane. We further show that the LptF and LptG homologues form a complex like their LPS counterparts and discover that they interact with the LPS ATPase LptB. Together, our data suggest that ceramide transport to the outer membrane is facilitated by an ABC transporter consisting of a sphingolipid-specific LptFG homolog coupled to the LPS LptB, supporting a model in which sphingolipid transport partially converges with the LPS transport system. Together, these findings reveal an unexpected evolutionary relationship between sphingolipid and lipopolysaccharide transport.

microbiology↗

bioRxiv2025

Live-Cell Chemoproteomic Profiling Identifies the Uncharacterised Protein YbaA as a Direct Target of Ciprofloxacin in Escherichia coli

Fluoroquinolone antibiotics, such as ciprofloxacin, are important broad-spectrum agents for a range of bacterial infections; however, fluoroquinolone usage is increasingly challenged by the emergence of resistance. Ciprofloxacin resistance mechanisms include mutations in the antibiotic target DNA gyrase, downregulation of porins required for bacterial cell penetration, and upregulation of efflux pumps to expel the antibiotic. However, new pathways driving bacterial tolerance to fluoroquinolones are still being discovered, suggesting that additional ciprofloxacin-binding proteins may exist in bacteria. In this study, we report the use of affinity-based protein profiling (AfBPP) with photo-crosslinking chemical probes to identify protein binding partners of ciprofloxacin in live E. coli cells. AfBPP identified novel ciprofloxacin binding proteins including YjdN and YbaA, whose molecular functions are as yet unannotated. Target engagement was validated using genetic knockout and biophysical binding assays, and key interactions identified in the ciprofloxacin binding site of YbaA. Collectively, this study demonstrates that additional and previously unreported biological interactions can exist for well-established antibiotics, and provides methodology to identify and interrogate these interactions in detail.

microbiology↗

bioRxiv2021

Role of Ring6 in the function of the E. coli MCE protein LetB

LetB is a tunnel-forming protein found in the cell envelope of some double-membraned bacteria, and is thought to be important for the transport of lipids between the inner and outer membranes. In Escherichia coli the LetB tunnel is formed from a stack of seven rings (Ring1 - Ring7), in which each ring is composed of a homo-hexameric assembly of MCE domains. The primary sequence of each MCE domain of the LetB protein is substantially divergent from the others, making each MCE ring unique in nature. The role of each MCE domain and how it contributes to the function of LetB is not well understood. Here we probed the importance of each MCE ring for the function of LetB, using a combination of bacterial growth assays and cryo-EM. Surprisingly, we find that {Delta}Ring3 and {Delta}Ring6 mutants, in which Ring3 and Ring6 have been deleted, confer increased resistance to membrane perturbing agents. Specific mutations in the pore-lining loops of Ring6 similarly confer increased resistance. A cryo-EM structure of the {Delta}Ring6 mutant shows that despite the absence of Ring6, which leads to a shorter assembly, the overall architecture is maintained, highlighting the modular nature of MCE proteins. Previous work has shown that Ring6 is dynamic and in its closed state, may restrict the passage of substrate through the tunnel. Our work suggests that removal of Ring6 may relieve this restriction. The deletion of Ring6 combined with mutations in the pore-lining loops leads to a model for the tunnel gating mechanism of LetB. Together, these results provide insight into the functional roles of individual MCE domains and pore-lining loops in the LetB protein. HighlightsO_LIDeleting MCE domains 3 or 6 from LetB confers increased resistance to membrane-perturbing agents C_LIO_LICryo-EM structure of {Delta}Ring6 LetB mutant highlights the modular nature of MCE domains C_LIO_LIMutations along LetB pore-lining loops modulate detergent resistance C_LI

biochemistry↗

bioRxiv2021

YhcB coordinates peptidoglycan and LPS biogenesis with phospholipid synthesis during Escherichia coli cell growth.

The cell envelope is essential for viability in all kingdoms of life. It retains enzymes and substrates within a confined space while providing a protective barrier to the external environment. Destabilising the envelope of bacterial pathogens is a common strategy employed by antimicrobial treatment. However, even in one of the most well studied organisms, Escherichia coli, there remain gaps in our understanding of how the synthesis of the successive layers of the cell envelope are coordinated during growth and cell division. Here, we used a whole genome phenotypic screen to identify mutants with a defective cell envelope. We report that loss of yhcB, a conserved gene of unknown function, results in loss of envelope stability, increased cell permeability and dysregulated control of cell size. Using whole genome transposon mutagenesis strategies we report the complete genetic interaction network of yhcB, revealing all genes with a synthetic negative and a synthetic positive relationship. These genes include those previously reported to have a role in cell envelope biogenesis. Surprisingly, we identified genes previously annotated as essential that became non-essential in a {Delta}yhcB background. Subsequent analyses suggest that YhcB sits at the junction of several envelope biosynthetic pathways coordinating the spatiotemporal growth of the cell, highlighting YhcB as an as yet unexplored antimicrobial target.

microbiology↗

bioRxiv2020

Structure of MlaFEDB lipid transporter reveals an ABC exporter fold and two bound phospholipids

In double-membraned bacteria, phospholipids must be transported across the cell envelope to maintain the outer membrane barrier, which plays a key role in antibiotic resistance and pathogen virulence. The Mla system has been implicated in phospholipid trafficking and outer membrane integrity, and includes an ABC transporter complex, MlaFEDB. The transmembrane subunit, MlaE, has minimal sequence similarity to other ABC transporters, and the structure of the entire inner membrane MlaFEDB complex remains unknown. Here we report the cryo-EM structure of the MlaFEDB complex at 3.05 [A] resolution. Our structure reveals that while MlaE has many distinct features, it is distantly related to the LPS and MacAB transporters, as well as the eukaryotic ABCA/ABCG families. MlaE adopts an outward-open conformation, resulting in a continuous pathway for phospholipid transport from the MlaE substrate-binding site to the pore formed by the ring of MlaD. Unexpectedly, two phospholipids are bound in the substrate-binding pocket of MlaFEDB, raising the possibility that multiple lipid substrates may be translocated each transport cycle. Site-specific crosslinking confirms that lipids bind in this pocket in vivo. Our structure provides mechanistic insight into substrate recognition and transport by the MlaFEDB complex.

biochemistry↗

bioRxiv2020

Structure of MlaFB uncovers novel mechanisms of ABC transporter regulation

ABC transporters facilitate the movement of a diverse array of molecules across cellular membranes, using power from ATP hydrolysis. While the overall mechanism of the transport cycle has been characterized in detail for several important members of this transporter family, it is less well understood how the activity of ABC transporters is regulated in the cell post-translationally. Here we report the X-ray crystal structure of MlaFB from E. coli, an ABC nucleotide binding domain (MlaF) in complex with its putative regulatory subunit (MlaB). MlaFB constitutes the cytoplasmic portion of the larger MlaFEDB ABC transporter complex, which drives phospholipid transport across the bacterial envelope and is important for maintaining the integrity of the outer membrane barrier. Our data show that the regulatory subunit MlaB, a STAS domain protein, binds to the nucleotide binding domain and is required for its stability. Our structure also implicates a unique C-terminal tail of the ABC subunit, MlaF, in self-dimerization. Both the C-terminal tail of MlaF and the interaction with MlaB are required for the proper assembly of the MlaFEDB complex and its function in cells. This work leads to a new model for how the activity of an important bacterial lipid transporter may be regulated by small binding proteins, and raises the possibility that similar regulatory mechanisms may exist more broadly across the ABC transporter family, from bacteria to humans.

microbiology↗

bioRxiv2019

Structure of LetB reveals a tunnel for lipid transport across the bacterial envelope

Gram-negative bacteria are surrounded by an outer membrane composed of phospholipids and lipopolysaccharide (LPS), which acts as a barrier to the environment and contributes to antibiotic resistance. While mechanisms of LPS transport have been well characterised, systems that translocate phospholipids across the periplasm, such as MCE (Mammalian Cell Entry) transport systems, are less well understood. Here we show that E. coli MCE protein LetB (formerly YebT), forms a [~]0.6 megadalton complex in the periplasm. Our cryo-EM structure reveals that LetB consists of a stack of seven modular rings, creating a long hydrophobic tunnel through the centre of the complex. LetB is sufficiently large to span the gap between the inner and outer membranes, and mutations that shorten the tunnel abolish function. Lipids bind inside the tunnel, suggesting that it functions as a pathway for lipid transport. Cryo-EM structures in the open and closed states reveal a dynamic tunnel lining, with implications for gating or substrate translocation. Together, our results support a model in which LetB establishes a physical link between the bacterial inner and outer membranes, and creates a hydrophobic pathway for the translocation of lipids across the periplasm, to maintain the integrity of the outer membrane permeability barrier.

biochemistry↗

bioRxiv2018

Evidence for phospholipid export from the gram-negative inner membrane: time to rethink the Mla pathway?

The Mla pathway is believed to be involved in maintaining the asymmetrical Gram-negative outer membrane via retrograde phospholipid transport. The pathway is composed of 3 components: the outer membrane MlaA-OmpC/F complex, a soluble periplasmic protein, MlaC, and the inner membrane ATPase, MlaFEDB complex. Here we solve the crystal structure of MlaC in its phospholipid free closed apo conformation, revealing a novel pivoting {beta}-sheet mechanism which functions to open and close the phospholipid-binding pocket. Using the apo form of MlaC we provide evidence that the Mla pathway functions in an anterograde rather than a retrograde direction by showing the inner membrane MlaFEDB machinery exports phospholipids and transfers them to MlaC in the periplasm. We confirm that the lipid export process occurs through the MlaD component of the MlaFEDB complex. This lipid export process is shown to be independent of ATP. Our data provides, for the first time, evidence of an apparatus for lipid export to the outer membrane.

biophysics↗