Structural and biophysical analysis of a Haemophilus influenzae tripartite ATP-independent periplasmic (TRAP) transporter
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Date
2024
Authors
Currie, Michael J.
Davies, James S.
Scalise, Mariafrancesca
Gulati, Ashutosh
Wright, Joshua D.
Newton-Vesty, Michael C.
Abeysekera, Gayan S.
Subramanian, Ramaswamy
Wahlgren, Weixiao Y.
Friemann, Rosmarie
Journal Title
Journal ISSN
Volume Title
Publisher
eLife Sciences Publications
Abstract
Tripartite ATP-independent periplasmic (TRAP) transporters are secondary-active transporters that receive their substrates via a soluble-binding protein to move bioorganic acids across bacterial or archaeal cell membranes. Recent cryo-electron microscopy (cryo-EM) structures of TRAP transporters provide a broad framework to understand how they work, but the mechanistic details of transport are not yet defined. Here we report the cryo-EM structure of the Haemophilus influenzae N-acetylneuraminate TRAP transporter (HiSiaQM) at 2.99 Å resolution (extending to 2.2 Å at the core), revealing new features. The improved resolution (the previous HiSiaQM structure is 4.7 Å resolution) permits accurate assignment of two Na+ sites and the architecture of the substrate-binding site, consistent with mutagenic and functional data. Moreover, rather than a monomer, the HiSiaQM structure is a homodimer. We observe lipids at the dimer interface, as well as a lipid trapped within the fusion that links the SiaQ and SiaM subunits. We show that the affinity (KD) for the complex between the soluble HiSiaP protein and HiSiaQM is in the micromolar range and that a related SiaP can bind HiSiaQM. This work provides key data that enhances our understanding of the ‘elevator-with-an-operator’ mechanism of TRAP transporters.
Description
Open access article. Creative Commons Attribution 4.0 International license (CC BY 4.0) applies
Keywords
Neu5Ac , Membrane transport proteins , Molecular biophysics , Protein-protein interaction , Sialic acid , Structural biology , Transport mechanism
Citation
Currie, M. J., Davies, J. S., Scalise, M., Gulati, A., Wright, J. D., Newton-Vesty, M. C., Abeysekera, G. S., Subramanian, R., Wahlgren, W. Y., Friemann, R., Allison, J. R., Mace, P. D., Griffin, M. D. W., Demeler, B., Wakatsuki, S., Drew, D., Indiveri, C., Dobson, R. C. J., & North, R. A. (2024). Structural and biophysical analysis of a Haemophilus influenzae tripartite ATP-independent periplasmic (TRAP) transporter. eLife, 12, Article RP92307. https://doi.org/10.7554/eLife.92307.3