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The crystal structure of the TonB-dependent transporter YncD reveals a positively charged substrate-binding site.

ABSTRACT: The outer membrane of Gram-negative bacteria is highly impermeable to hydrophilic molecules of larger than 600?Da, protecting these bacteria from toxins present in the environment. In order to transport nutrients across this impermeable membrane, Gram-negative bacteria utilize a diverse family of outer-membrane proteins called TonB-dependent transporters. The majority of the members of this family transport iron-containing substrates. However, it is becoming increasingly clear that TonB-dependent transporters target chemically diverse substrates. In this work, the structure and phylogenetic distribution of the TonB-dependent transporter YncD are investigated. It is shown that while YncD is present in some enteropathogens, including Escherichia coli and Salmonella spp., it is also widespread in Gammaproteobacteria and Betaproteobacteria of environmental origin. The structure of YncD was determined, showing that despite a distant evolutionary relationship, it shares structural features with the ferric citrate transporter FecA, including a compact positively charged substrate-binding site. Despite these shared features, it is shown that YncD does not contribute to the growth of E. coli in pure culture under iron-limiting conditions or with ferric citrate as an iron source. Previous studies of transcriptional regulation in E. coli show that YncD is not induced under iron-limiting conditions and is unresponsive to the ferric uptake regulator (Fur). These observations, combined with the data presented here, suggest that YncD is not responsible for the transport of an iron-containing substrate.

SUBMITTER: Grinter R

PROVIDER: S-EPMC7193533 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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The crystal structure of the TonB-dependent transporter YncD reveals a positively charged substrate-binding site.

Grinter Rhys R Lithgow Trevor T

Acta crystallographica. Section D, Structural biology 20200427 Pt 5

The outer membrane of Gram-negative bacteria is highly impermeable to hydrophilic molecules of larger than 600 Da, protecting these bacteria from toxins present in the environment. In order to transport nutrients across this impermeable membrane, Gram-negative bacteria utilize a diverse family of outer-membrane proteins called TonB-dependent transporters. The majority of the members of this family transport iron-containing substrates. However, it is becoming increasingly clear that TonB-dependen ...[more]

PMID: 32355044

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Project description:ATP-binding cassette (ABC) transport systems comprise two transmembrane domains/subunits that form a translocation path and two nucleotide-binding domains/subunits that bind and hydrolyze ATP. Prokaryotic canonical ABC import systems require an extracellular substrate-binding protein for function. Knowledge of substrate-binding sites within the transmembrane subunits is scarce. Recent crystal structures of the ABC importer Art(QN)2 for positively charged amino acids of Thermoanerobacter tengcongensis revealed the presence of one substrate molecule in a defined binding pocket in each of the transmembrane subunits, ArtQ (J. Yu, J. Ge, J. Heuveling, E. Schneider, and M. Yang, Proc Natl Acad Sci U S A 112:5243-5248, 2015, https://doi.org/10.1073/pnas.1415037112). This finding raised the question of whether both sites must be loaded with substrate prior to initiation of the transport cycle. To address this matter, we first explored the role of key residues that form the binding pocket in the closely related Art(MP)2 transporter of Geobacillus stearothermophilus, by monitoring consequences of mutations in ArtM on ATPase and transport activity at the level of purified proteins embedded in liposomes. Our results emphasize that two negatively charged residues (E153 and D160) are crucial for wild-type function. Furthermore, the variant Art[M(L67D)P]2 exhibited strongly impaired activities, which is why it was considered for construction of a hybrid complex containing one intact and one impaired substrate-binding site. Activity assays clearly revealed that one intact binding site was sufficient for function. To our knowledge, our study provides the first biochemical evidence on transmembrane substrate-binding sites of an ABC importer.IMPORTANCE Canonical prokaryotic ATP-binding cassette importers mediate the uptake of a large variety of chemicals, including nutrients, osmoprotectants, growth factors, and trace elements. Some also play a role in bacterial pathogenesis, which is why full understanding of their mode of action is of the utmost importance. One of the unsolved problems refers to the chemical nature and number of substrate binding sites formed by the transmembrane subunits. Here, we report that a hybrid amino acid transporter of G. stearothermophilus, encompassing one intact and one impaired transmembrane binding site, is fully competent in transport, suggesting that the binding of one substrate molecule is sufficient to trigger the translocation process.

Dataset Information

The crystal structure of the TonB-dependent transporter YncD reveals a positively charged substrate-binding site.

Publications

The crystal structure of the TonB-dependent transporter YncD reveals a positively charged substrate-binding site.

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