EMD-41266
Cryo-EM structure of the Tripartite ATP-independent Periplasmic (TRAP) transporter SiaQM from Haemophilus influenzae (antiparallel dimer)
EMD-41266
Single-particle2.99 Å
Deposition: 16/07/2023Map released: 22/11/2023
Last modified: 28/02/2024
Sample Organism:
Haemophilus influenzae Rd KW20
Sample: HiSiaQM transporter protein solubilised in amphipol A8-35
Fitted models: 8thj (Avg. Q-score: 0.52)
Deposition Authors: Davies JS
,
Currie MC,
Dobson RCJ ,
North RA
Sample: HiSiaQM transporter protein solubilised in amphipol A8-35
Fitted models: 8thj (Avg. Q-score: 0.52)
Deposition Authors: Davies JS
,
Currie MC,
Dobson RCJ ,
North RA
Structural and biophysical analysis of a Haemophilus influenzae tripartite ATP-independent periplasmic (TRAP) transporter.
Currie MJ ,
Davies JS ,
Scalise M,
Gulati A,
Wright JD ,
Newton-Vesty MC,
Abeysekera GS,
Subramanian R,
Wahlgren WY ,
Friemann R,
Allison JR,
Mace PD ,
Griffin MDW,
Demeler B,
Wakatsuki S ,
Drew D ,
Indiveri C,
Dobson RCJ ,
North RA
(2024) eLife , 12
,
Davies JS ,
Scalise M,
Gulati A,
Wright JD ,
Newton-Vesty MC,
Abeysekera GS,
Subramanian R,
Wahlgren WY ,
Friemann R,
Allison JR,
Mace PD ,
Griffin MDW,
Demeler B,
Wakatsuki S ,
Drew D ,
Indiveri C,
Dobson RCJ ,
North RA
(2024) eLife , 12
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.
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.