Structural and biophysical analysis of aHaemophilus influenzaetripartite 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-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 (KD) for the complex between the solubleHiSiaP protein andHiSiaQM is in the micromolar range and that a related SiaP can bindHiSiaQM, highlighting features of the complex interface. This work provides key data that enhances our understanding of the ‘elevator-with-an-operator’ mechanism of TRAP transporters.