Structural and biophysical analysis of a Haemophilus influenzae tripartite ATP-independent periplasmic (TRAP) transporter
Michael Currie, James S. Davies, Mariafrancesca Scalise, Ashutosh Gulati, J. D. Wright, Michael C. Newton-Vesty, Gayan S. Abeysekera, Ramaswamy Subramanian, Weixiao Yuan Wahlgren, Rosmarie Friemann, Jane R. Allison, Peter D. Mace, Michael D. W. Griffin, Borries Demeler, Soichi Wakatsuki, David Drew, Cesare Indiveri, Renwick C. J. Dobson, Rachel A. North
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 ( Hi SiaQM) at 2.99 Å resolution (extending to 2.2 Å at the core), revealing new features. The improved resolution (the previous Hi 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 Hi 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 ( K D ) for the complex between the soluble Hi SiaP protein and Hi SiaQM is in the micromolar range and that a related SiaP can bind Hi SiaQM. This work provides key data that enhances our understanding of the ‘elevator-with-an-operator’ mechanism of TRAP transporters.