Abstract
Tripartite ATP-independent periplasmic (TRAP) transporters are found widely in bacteria and archaea and consist of three structural domains, a soluble substrate-binding protein (P-domain), and two transmembrane domains (Q- and M-domains). HiSiaPQM and its homologs are TRAP transporters for sialic acid and are essential for host colonization by pathogenic bacteria. Here, we reconstitute HiSiaQM into lipid nanodiscs and use cryo-EM to reveal the structure of a TRAP transporter. It is composed of 16 transmembrane helices that are unexpectedly structurally related to multimeric elevator-type transporters. The idiosyncratic Q-domain of TRAP transporters enables the formation of a monomeric elevator architecture. A model of the tripartite PQM complex is experimentally validated and reveals the coupling of the substrate-binding protein to the transporter domains. We use single-molecule total internal reflection fluorescence (TIRF) microscopy in solid-supported lipid bilayers and surface plasmon resonance to study the formation of the tripartite complex and to investigate the impact of interface mutants. Furthermore, we characterize high-affinity single variable domains on heavy chain (VHH) antibodies that bind to the periplasmic side of HiSiaQM and inhibit sialic acid uptake, providing insight into how TRAP transporter function might be inhibited in vivo.
Original language | English |
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Article number | 4471 |
Pages (from-to) | 4471 |
Journal | Nature Communications |
Volume | 13 |
Issue number | 1 |
Early online date | 4 Aug 2022 |
DOIs | |
Publication status | Published - 4 Aug 2022 |
Bibliographical note
© 2022. The Author(s). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you giveappropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
Funding Information:
Open Access funding enabled and organized by Projekt DEAL.
Keywords
- Adenosine Triphosphate/metabolism
- Archaea/metabolism
- Bacteria/metabolism
- Bacterial Proteins/metabolism
- Carrier Proteins/metabolism
- Membrane Transport Proteins/metabolism
- N-Acetylneuraminic Acid/metabolism