Biology subjects

Jungnickel, K. E. J.

Publications and source records attributed to Jungnickel, K. E. J..

4 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↗

bioRxiv2024

Structural basis of substrate transport and drug recognition by the human thiamine transporter SLC19A3

Thiamine (vitamin B1) functions as an essential coenzyme in cells. Humans and other mammals cannot synthesise this vitamin de novo and thus have to take it up from their diet. Eventually, every cell needs to import thiamine across its plasma membrane which is mainly mediated by two specific thiamine transporters SLC19A2 and SLC19A3. Loss of function mutations in either of these transporters leads to detrimental, life-threatening metabolic disorders. SLC19A3 is furthermore a major site of drug interactions. Many medications, including antidepressants, antibiotics and chemotherapeutics are known to inhibit this transporter, with potentially fatal consequences for patients. Despite a thorough functional characterisation over the past two decades, the structural basis of its transport mechanism and drug interactions has remained elusive. Here, we report eight cryo-electron microscopy (cryo-EM) structures of the human thiamine transporter SLC19A3 in complex with various ligands. Conformation-specific nanobodies enabled us to capture different states of SLC19A3s transport cycle, revealing the molecular details of thiamine recognition and transport. We identified nine novel drug interactions of SLC19A3 and determined structures of the transporter in complex with the inhibitors fedratinib, hydroxychloroquine, amprolium and amitriptyline. These data allow us to develop an understanding of the transport mechanism and ligand recognition of SLC19A3.

biochemistry↗

bioRxiv2023

MFSD1 in complex with its accessory subunit GLMP functions as a general dipeptide uniporter in lysosomes

Lysosomal degradation of macromolecules in lysosomes produces diverse small metabolites exported by specific transporters for reuse in biosynthetic pathways. Here, we deorphanized the Major Facilitator Superfamily Domain Containing 1 (MFSD1) protein, which forms a tight complex with the Glycosylated Lysosomal Membrane Protein (GLMP) in the lysosomal membrane. Untargeted metabolomics analysis of MFSD1-deficient mouse lysosomes revealed an increase in cationic dipeptides. Purified MFSD1 selectively bound diverse dipeptides, while electrophysiological, isotope tracer, and fluorescence-based studies in Xenopus oocytes and proteoliposomes showed that MFSD1/GLMP acts as a uniporter for cationic and neutral dipeptides. Cryo-EM structure of the dipeptide-bound MFSD1/GLMP complex in outward-open conformation characterized the heterodimer interface and, in combination with molecular dynamics simulations, provided a structural basis for its selectivity towards diverse dipeptides. Together, our data identify MFSD1 as a general lysosomal dipeptide uniporter, providing an alternative route to recycle lysosomal proteolysis products when lysosomal amino acid exporters are overloaded.

biochemistry↗

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↗