Biology subjects

Bartels, K.

Publications and source records attributed to Bartels, K..

7 recordsLinked to original sources

bioRxiv2026

Structural basis of substrate recognition and transport in bacterial ACS transporters

Membrane transporters of the major facilitator superfamily (MFS) mediate uptake of diverse metabolites, yet the molecular basis of substrate recognition within many bacterial families remains unclear. The anion:cation symporter (ACS) family is conserved from bacteria to humans and includes medically relevant solute carrier transporters, but only few bacterial members have been functionally characterized. Here, we combine genetics, biochemistry, transport assays, and structural biology to define the substrate specificity of five ACS transporters from Escherichia coli. Using systematic growth complementation assays in deletion strains, we assign physiological substrates to each transporter, identifying DgoT, LgoT, and ExuT as specific uptake systems for D-galactonate, L-galactonate, and galacturonate/glucuronate, respectively, while revealing overlapping roles for GarP and GudP in C6 sugar acid uptake. NanoDSF ligand binding assays show highly selective recognition of sugar acids but do not predict transport activity. Proton-coupled uptake was directly demonstrated using reconstituted proteoliposomes, defining strict stereoselectivity of LgoT and DgoT. We determined the 2.2 [A] X-ray structure of LgoT in an inward-open conformation, revealing a canonical MFS fold with a conserved but differentially tuned substrate-binding cavity. Comparative structural and sequence analyses across ACS members identify a conserved core for coordination of sugar acid carboxylate and hydroxyl groups, while localized substitutions modulate steric and electrostatic properties to enable discrimination of substrate size and stereochemistry. These results provide a framework for substrate recognition in bacterial ACS transporters and establish LgoT as a structural model for stereo-selective proton-coupled organic anion transport.

biochemistry↗

bioRxiv2025

Cefdinir binding to a class-A β-lactamase revealed by serial cryo-crystallography

One of the most common resistance mechanisms against antibiotics employed by Gram-negative bacteria involves the production of {beta}-lactamases, resulting in rapid hydrolysis of the antibiotic. Extensive use of the early generation cephalosporins led to the rise of extended-spectrum {beta}-lactamases (ESBLs) like CTX-Ms. Cefdinir is an extended-spectrum third-generation cephalosporin administered since the late 90s; despite this, there is no reported 3D-structure of the antibiotic bound to any {beta}-lactamase or Penicillin-Binding-Protein (PBP) in the PDB. Here we report the X-ray crystallographic structure of Cefdinir-bound CTX-M-14 E166A mutant obtained via serial cryo-crystallography (cryo-SSX). SynopsisSerial cryo-crystallography reveals the structure of the extended spectrum {beta}-lactamase CTX-M-14, in complex with the third-generation cephalosporin antibiotic Cefdinir.

biophysics↗

bioRxiv2025

Binding mode of isoxazolyl penicillins to a Class-A beta-lactamase at ambient conditions

The predominant resistance mechanism observed in Gram-negative bacteria involves the production of {beta}-lactamases, which catalyse the hydrolysis of {beta}-lactam antibiotics, thereby rendering them ineffective. Although isoxazolyl penicillins are available since the 1970s, there are currently no structures in complex with class-A {beta}-lactamases available. In order to support the rational development of new {beta}-lactamase inhibitors, we have analysed the structure of the clinically relevant {beta}-lactamase CTX-M-14 from Klebsiella pneumoniae near physiolog- ical temperatures. By utilizing serial synchrotron crystallography, we demonstrate the acyl-enzyme intermediates of the catalytically impaired CTX-M-14 mutant E166A in complex with three isoxazolyl penicillins: oxacillin, cloxacillin and dichloxacillin. While the three derivatives differ only by one and two Cl atoms, respectively, they show marked differences in their binding mode.

biochemistry↗

bioRxiv2024

Model-free photon analysis of diffusion-based single-molecule FRET experiments

Photon-by-photon analysis tools for diffusion-based single-molecule Forster resonance energy transfer (smFRET) experiments often describe protein dynamics with Markov models. However, FRET efficiencies are only projections of the conformational space such that the measured dynamics can appear non-Markovian. Model-free methods to quantify FRET efficiency fluctuations would be desirable in this case. Here, we present such an approach. We determine FRET efficiency correlation functions free of artifacts from the finite length of photon trajectories or the diffusion of molecules through the confocal volume. We show that these functions capture the dynamics of proteins from micro-to milliseconds both in simulation and experiment, which provides a rigorous validation of current model-based analysis approaches.

biophysics↗

bioRxiv2023

Plasticity of the binding pocket in peptide transporters underpins promiscuous substrate recognition

Proton-coupled oligopeptide transporters (POTs) are promiscuous transporters of the Major Facilitator Superfamily, that constitute the main route of entry for a wide range of dietary peptides and orally administrated peptidomimetic drugs. Given their clinical and pathophysiological relevance, several bacterial and mammalian POT homologs have been extensively studied on a structural and molecular level. However, the molecular basis of recognition and transport of the wide range of peptide substrates has remained elusive. Here we present 14 X-ray structures of the bacterial POT DtpB in complex with chemically diverse di- and tripeptides, providing novel insights into the plasticity of the conserved central binding cavity. We analyzed binding affinities for more than 80 peptides and monitored uptake by a fluorescence-based transport assay. To probe if all natural 8400 di- and tripeptides can bind to DtpB, we employed state-of-the-art molecular docking and machine learning and conclude that peptides of a specific subset with compact hydrophobic residues are the best DtpB binders.

biophysics↗

bioRxiv2020

The conformational distribution of a major facilitator superfamily peptide transporter is modulated by the membrane composition

While structural biology aims at explaining the biological function of membrane proteins with their structure, it is unclear how these proteins are modulated by the complex lipid composition of membranes. Here, we address this question by mapping the conformational distribution of the bacterial oligopeptide transporter DtpA using single-molecule fluorescence spectroscopy. We show that DtpA populates ensembles of conformers that respond sensitively to the environment. Detergents trap the transporter in an inward-open ensemble in which the substrate binding site faces the cytosol. However, re-constitution in Saposin nanoparticles with different lipid compositions, reveal a plethora of alternative conformations, including a fully inward-open ensemble whose existence had not been anticipated before. The relative abundance of these ensembles depends on the lipid composition of the nanoparticles. Our results therefore demonstrate that membranes sensitively affect the structural distribution of DtpA and we expect this to be a general property of membrane proteins.

biophysics↗

bioRxiv2019

Leukocyte-derived High-mobility group box 1 controls innate immune responses against Listeria monocytogenes.

High-mobility group box 1 (HMGB1) is a damage-associated molecular pattern with key proinflammatory functions following tissue injury. Moreover, HMGB1 neutralization was shown to alleviate LPS-induced shock, suggesting a role for the protein as a master therapeutic target for inflammatory and infectious diseases. Here, we report that HMGB1 neutralization impedes immune responses to Listeria monocytogenes, a wide-spread bacterium with pathogenic relevance for humans and rodents. Using genetic deletion strategies and neutralizing antibodies, we demonstrate that hepatocyte HMGB1, a major driver of post-necrotic inflammation in the liver, is dispensable for pathogen defense during moderately severe infection with listeria. In contrast, antibody-mediated HMGB1 neutralization and HMGB1 deficiency in myeloid cells effectuate rapid and uncontrolled bacterial dissemination in mice despite preserved basic leukocyte functionality and autophagy induction. During overwhelming infection, hepatocyte injury may contribute to increased HMGB1 serum levels and excessive inflammation in the liver, supporting context-dependent roles for HMGB1 from different cellular compartments during infection. We provide mechanistic evidence that HMGB1 from circulating immune cells contributes to the timely induction of hepatic immune regulatory gene networks, early inflammatory monocyte recruitment to the liver and promotion of neutrophil survival, which are mandatory for pathogen control. In summary, our data establish HMGB1 as a critical co-factor in the immunological clearance of listeria, and argue against HMGB1 neutralization as a universal therapeutic strategy for sepsis.\n\nAuthor summaryHigh-mobility group box 1 (HMGB1) is an abundantly expressed nucleoprotein with signaling properties following secretion or release into the extracellular space. Given its central immune-regulatory roles during tissue injury and LPS-induced septic shock, interventions aimed at HMGB1 signaling have been advocated as therapeutic options for various disease conditions. Here, we show that antibody-mediated HMGB1 neutralization interferes with immunological defense against Listeria monocytogenes, a gram-positive bacterium with high pathogenic relevance for rodents and humans, effectuating uncontrolled bacterial growth and inflammation. Using conditional knockout animals, we demonstrate that while leukocyte functionality is preserved in HMGB1-deficient myeloid cells, HMGB1 released in response to Listeria triggers hepatic inflammatory monocyte recruitment and activation of transcriptional immune networks required for the early control of bacterial dissemination. Hepatocyte HMGB1, a key driver of post-necrotic inflammation in the liver, is dispensable for the immune response during moderately severe infection, but likely contributes to excessive hepatitis when infection is uncontrolled and cellular injury is high. We demonstrate a critical and non-redundant role for HMGB1 in the immune-mediated clearance of listeriosis and argue against HMGB1 neutralization as a universal therapeutic option in the context of infection.

immunology↗