Structural and biophysical analysis of a Haemophilus influenzae tripartite ATP-independent periplasmic (TRAP) transporter

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 <i>Haemophilus influenzae N</i>-acetylneuraminate TRAP transporter (<i>Hi</i>SiaQM) at 2.99 Å resolution (extending to 2.2 Å at the core), revealing new features. The improved resolution (the previous <i>Hi</i>SiaQM 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 <i>Hi</i>SiaQM 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 (<i>K</i>_D) for the complex between the soluble <i>Hi</i>SiaP protein and <i>Hi</i>SiaQM is in the micromolar range and that a related SiaP can bind <i>Hi</i>SiaQM. This work provides key data that enhances our understanding of the 'elevator-with-an-operator' mechanism of TRAP transporters.