Biology subjects

Josts, I.

Publications and source records attributed to Josts, I..

5 recordsLinked to original sources

bioRxiv2025

Capturing transient states of heterodimeric ABC transporter TM287/288 by Time-Resolved Small-Angle X-ray Scattering

Structures of the heterodimeric ABC transporter TM287/288 have been previously determined in several states indicating large conformational changes during its reaction cycle. However, for a complete description of the cycle, transient states (such as an occluded state) are still missing, as they are difficult to capture with static structural biology techniques. Here, we investigate the kinetics and accompanying structural changes in the course of the reaction cycle of full-length TM287/288 using time-resolved small-angle X-ray scattering, initiated by stopped-flow mixing. The use of active site mutants and state-specific sybodies/nanobodies enabled us to dissect the temporal events involved in the ATP-driven conformational cycle of TM287/288 and reveal a transient occluded state of this ABC transporter. Statement of SignificanceATP-binding cassette transporters are essential molecular machines that use the power of ATP binding and hydrolysis to move substances across cellular membranes. Here, we use time-resolved small-angle X-ray scattering combined with conformation-specific nanobodies to directly capture and assign different states to the heterodimeric ABC transporter TM287/288 in solution. Our findings advance our understanding of ABC transporter and demonstrate a broadly applicable method for dissecting the different molecular states in membrane proteins on a second-to-minute time scale. TOC figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/679171v2_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@b83fbaorg.highwire.dtl.DTLVardef@5fec81org.highwire.dtl.DTLVardef@1a022fdorg.highwire.dtl.DTLVardef@592c92_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Convergent evolution of distinct D-ribulose utilisation pathways in attaching and effacing pathogens

Attaching and effacing pathogens overcome colonisation resistance by competing with metabolically similar organisms for limited resources. Enterohaemorrhagic E. coli (EHEC) utilises the pathogenicity island-encoded Accessory -arabinose Uptake (Aau) transporter to effectively colonise the mouse gut, hypothesised to be achieved via an enhanced capacity to scavenge -arabinose. Aau is regulated exclusively in response to -arabinose, but it is unclear how this system specifically benefits EHEC in vivo. Here, we show that Aau displays a >200-fold higher affinity for the monosaccharide D-ribulose, over -arabinose. EHEC cannot grow on D-ribulose as a sole carbon source and this sugar does not trigger aau transcription. However, Aau effectively transports D-ribulose into the cell only in the presence of -arabinose, where it feeds into the pentose phosphate pathway, after phosphorylation by the -ribulokinase AraB, thus providing EHEC a significant fitness advantage. EHEC has therefore evolved a mechanism of hijacking the canonical -arabinose utilisation machinery to promote D-ribulose utilisation in vivo. Furthermore, Citrobacter rodentium encodes an analogous system that exclusively transports D-ribulose and metabolises it via a dedicated D-ribulokinase. These unique mechanisms of D-ribulose utilisation suggest that convergent evolution has driven the ability of distinct pathogenic species to exploit this nutrient during invasion of the gut niche.

microbiology↗

bioRxiv2022

A single extracellular loop of FoxA controls ligand specificity, uptake, and signaling in Pseudomonas aeruginosa

The outer membrane of Gram-negative bacteria prevents many antibiotics from reaching intracellular targets. However, some antimicrobials can take advantage of iron import transporters to cross this barrier. We showed previously that the thiopeptide antibiotic, thiocillin, exploits the nocardamine (ferrioxamine E) xenosiderophore transporter, FoxA, of the opportunistic pathogen Pseudomonas aeruginosa for uptake. Here we show that FoxA also transports the xenosiderophore bisucaberin and describe at 2.5 [A] resolution the first crystal structure of bisucaberin bound to FoxA. Bisucaberin is distinct from other siderophores because it forms a 3:2 rather than 1:1 siderophore-iron complex. Mutations in a single extracellular loop of FoxA differentially affected nocardamine, thiocillin, and bisucaberin binding, uptake, and signal transduction. These results show that in addition to modulating ligand binding, the extracellular loops of siderophore transporters are of fundamental importance for controlling ligand uptake and its regulatory consequences, which has implications for the development of siderophore-antibiotic conjugates to treat difficult infections.

microbiology↗

bioRxiv2021

Structural insights into a novel family of integral membrane siderophore reductases

Gram-negative bacteria take up the essential ion Fe3+ as ferric-siderophore complexes through their outer membrane using TonB-dependent transporters. However, the subsequent route through the inner membrane differs across many bacterial species and siderophore chemistries and is not understood in detail. Here, we report the crystal structure of the inner membrane protein FoxB (from P. aeruginosa) that is involved in Fe-siderophore uptake. The structure revealed a novel fold with two tightly-bound heme molecules. In combination with functional studies these results establish FoxB as an inner membrane reductase involved in the release of iron from ferrioxamine during Fe-siderophore uptake.

biochemistry↗

bioRxiv2019

Crystal structures of the outer membrane transporter FoxA provide novel insights into TonB-mediated siderophore uptake and signalling

Many microbes and fungi acquire the essential ion Fe3+ through the synthesis and secretion of high-affinity chelators termed siderophores. In Gram-negative bacteria, these ferric-siderophore complexes are actively taken up using highly specific TonB-dependent transporters (TBDTs) located in the outer bacterial membrane (OM). However, the detailed mechanism of how the inner-membrane protein TonB connects to the transporters in the OM as well as the interplay between siderophore- and TonB-binding to the transporter is still poorly understood. Here, we present three crystal structures of the TBDT FoxA from Pseudomonas aeruginosa (containing a signalling domain) in complex with the siderophore ferrioxamine B and TonB and combine them with a detailed analysis of binding constants. The structures show that both siderophore and TonB-binding is required to form a translocation-competent state of the FoxA transporter in a two-step TonB-binding mechanism. The complex structure also indicates how TonB-binding influences the orientation of the signalling domain.

molecular biology↗