Structural and biophysical analysis of aHaemophilus influenzaetripartite ATP-independent periplasmic (TRAP) transporter

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-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 theHaemophilus 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 previousHiSiaQM 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, theHiSiaQM 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 (K_D) for the complex between the solubleHiSiaP protein andHiSiaQM is in the micromolar range and that a related SiaP can bindHiSiaQM. This work provides key data that enhances our understanding of the ‘elevator-with-an-operator’ mechanism of TRAP transporters.