Biology subjects

North, R. A.

Publications and source records attributed to North, R. A..

4 recordsLinked to original sources

bioRxiv2026

Mechanism of phospholipid transport to the bacterial outer membrane by TAM.

Gram-negative bacteria transport phospholipids from the inner membrane (IM) to the outer membrane (OM) via poorly understood processes. These processes are essential for cell growth and the establishment of an antibiotic-resistant barrier. Here, we conducted single-particle cryo-electron microscopy, in vivo functional assays, and lipidomics to investigate the role of the "translocation and assembly module" (TAM) in lipid transport. We found that the OM-embedded subunit TamA anchors the IM-embedded bridge-like subunit TamB to the OM by forming a functional stable hybrid-barrel structure with the highly conserved C-terminal domain of unknown function 490 (DUF490). Using in vivo disulfide-tethering experiments we found that a highly conserved amphipathic helix within TamB DUF490 is important for TAM to function in OM maintenance. We also found that TamB DUF490 forms a {beta}-taco channel containing lipid-like densities and that the lipophilic property of the channel is important for TAM to maintain the levels of cardiolipin in the OM. Not only do our data support a novel model in which TAM acts to direct specific lipid classes into the OM, but it also supports the notion that TamB is a bacterial evolutionary prototype of a structurally homologous superfamily of eukaryotic bridge-like lipid transfer proteins.

biochemistry↗

bioRxiv2024

On the function of TRAP substrate-binding proteins: conformational variation of the sialic acid binding protein SiaP

Tripartite ATP-independent periplasmic (TRAP) transporters are analogous to ABC transporters in that they use a substrate-binding proteins to scavenge metabolites (e.g., N-acetylneuraminate) and deliver them to the membrane components for import. TRAP substrate-binding proteins are thought to bind the substrate using a two-state (open and closed) induced-fit mechanism. We solved the structure of the TRAP N-acetylneuraminate substrate-binding protein from Aggregatibacter actinomycetemcomitans (AaSiaP) in both the open ligand-free and closed liganded conformations. Surprisingly, we also observed an intermediate conformation, where AaSiaP is mostly closed and is bound to a non-cognate ligand, acetate, which hints at how N-acetylneuraminate binding stabilises a fully closed state. AaSiaP preferentially binds N-acetylneuraminate (KD = 0.4 {micro}M) compared to N-glycolylneuraminate (KD = 4.4 {micro}M), which is explained by the closed-N-acetylneuraminate bound structure. Small-angle X-ray scattering data alongside molecular dynamics simulations suggest the AaSiaP adopts a more open state in solution than in crystal. However, the open unliganded conformation can also sample closed conformations. Molecular dynamics simulations also demonstrate the importance of water molecules for stabilising the closed conformation. Although our data is consistent with an induced fit model of binding, it is likely that the open unliganded conformation encompasses multiple states capable of binding substrate. The mechanism by which the ligand is released for import remains to be determined.

biochemistry↗

bioRxiv2022

Structure and mechanism of the tripartite ATP-independent periplasmic (TRAP) transporter

In bacteria and archaea, tripartite ATP-independent periplasmic (TRAP) transporters uptake essential carboxylate- and sulfonate-containing nutrients into the cytoplasm. Unlike other secondary active transporters, TRAP transporters cannot receive their substrates directly, but do so indirectly via a secreted soluble substrate-binding protein. How a sodium-driven secondary active transporter is strictly coupled to a passenger-carrying substrate-binding domain is poorly understood. Here, we report the cryo-EM structure of the sialic acid TRAP transporter SiaQM from Photobacterium profundum at 2.97 [A] resolution. SiaM has 12-TMs that come together to form a "transport" domain and a "scaffold" domain, with the transport domain consisting of helical hairpins as seen in the sodium-coupled elevator transporter VcINDY. Interestingly, the SiaQ protein forms intimate contacts with SiaM to extend the size of the scaffold domain, indicating TRAP transporters may operate as monomers, rather than the typically observed oligomers. We have identified the Na+ and sialic acid binding sites in SiaM and confirmed a strict dependence on the substrate-binding protein SiaP for uptake. We have determined the SiaP crystal structure that, together with co-evolution driven docking studies, provides a molecular basis for how sialic acid is delivered to the SiaQM transporter complex. We conclude that TRAP proteins are conceptually a marriage between an ABC importer and a secondary active transporter, which we describe herein as an elevator-with-an-operator mechanism.

molecular biology↗

bioRxiv2020

Mechanism of NanR gene repression and allosteric induction of bacterial sialic acid metabolism

Bacteria respond to environmental changes by inducing transcription of some genes and repressing others. Sialic acids, which coat human cell surfaces, are a nutrient source for pathogenic and commensal bacteria. The Escherichia coli GntR-type transcriptional repressor, NanR, regulates sialic acid metabolism, but the mechanism is unclear. Here, we demonstrate that three NanR dimers bind a (GGTATA)3-repeat operator cooperatively and with high affinity. Truncation of an N-terminal extension abolishes cooperative binding. The effector, N-acetylneuraminate, binds NanR and attenuates DNA binding. Crystal structure data show that N-acetylneuraminate binding to NanR causes a domain rearrangement that locks the protein in a conformation that prevents DNA binding. Single-particle cryo-electron microscopy structures of NanR bound to DNA reveal the DNA binding domain is reorganized to engage DNA, while the three dimers assemble in close proximity across the (GGTATA)3-repeat operator allowing protein-protein interactions to form via the N-terminal extensions. Our data provides a molecular basis for the regulation of bacterial sialic acid metabolism.

molecular biology↗