Structural and mechanistic analysis of a tripartite ATP-independent periplasmic TRAP transporter

Martin F. Peter, Jan A. Ruland, Peer Depping, Niels Schneberger, Emmanuele Severi, Jonas Moecking, Karl Gatterdam, Sarah Tindall, Alexandre Durand, Veronika Heinz, Jan Peter Siebrasse, Paul-Albert Koenig, Matthias Geyer, Christine Ziegler, Ulrich Kubitscheck, Gavin H. Thomas and Gregor Hagelueken ()
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Martin F. Peter: University of Bonn
Jan A. Ruland: University of Bonn
Peer Depping: University of Bonn
Niels Schneberger: University of Bonn
Emmanuele Severi: University of York
Jonas Moecking: University of Bonn
Karl Gatterdam: University of Bonn
Sarah Tindall: University of York
Alexandre Durand: Institut de Génétique et de Biologie Molecule et Cellulaire
Veronika Heinz: University of Regensburg
Jan Peter Siebrasse: University of Bonn
Paul-Albert Koenig: University of Bonn
Matthias Geyer: University of Bonn
Christine Ziegler: University of Regensburg
Ulrich Kubitscheck: University of Bonn
Gavin H. Thomas: University of York
Gregor Hagelueken: University of Bonn

Nature Communications, 2022, vol. 13, issue 1, 1-15

Abstract: 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.

Date: 2022
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DOI: 10.1038/s41467-022-31907-y

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