Structural and mechanistic analysis of a tripartite ATP-independent periplasmic TRAP transporter

Abstract

Tripartite ATP-independent periplasmic (TRAP) transporters are found widely in bacteria and archaea and consist of three structural domains, a soluble substrate-binding protein (P-domain), and two transmembrane domains (Q- and M-domains). HiSiaPQM and its homologs are TRAP transporters for sialic acid and are essential for host colonization by pathogenic bacteria. Here, we reconstitute HiSiaQM into lipid nanodiscs and use cryo-EM to reveal the structure of a TRAP transporter. It is composed of 16 transmembrane helices that are unexpectedly structurally related to multimeric elevator-type transporters. The idiosyncratic Q-domain of TRAP transporters enables the formation of a monomeric elevator architecture. A model of the tripartite PQM complex is experimentally validated and reveals the coupling of the substrate-binding protein to the transporter domains. We use single-molecule total internal reflection fluorescence (TIRF) microscopy in solid-supported lipid bilayers and surface plasmon resonance to study the formation of the tripartite complex and to investigate the impact of interface mutants. Furthermore, we characterize high-affinity single variable domains on heavy chain (VHH) antibodies that bind to the periplasmic side of HiSiaQM and inhibit sialic acid uptake, providing insight into how TRAP transporter function might be inhibited in vivo.

Structure and mechanism of a tripartite ATP-independent periplasmic TRAP transporter

“…It is possible that the larger scaffold may have arisen to accommodate the interaction with SiaP, which could have become hindered upon an oligomeric assembly of SiaM. This motion and mechanism are consistent with that proposed by Peter et al 39 , who similarly used a model of VcINDY in the outward state. Here the authors also present a 4.7 Å cryo-EM structure of the fused Hi SiaQM system.…”

“…This arm helix links the scaffold to the transport domain and is likely important for the recognition and docking of SiaP and potentially for conformational transitions of SiaM. This finding is supported by recent work by Peter et al, who show that mutation of the equivalent residues in HiSiaPQM (R484 and S60) affects growth in an E. coli complementation experiment 39 .…”

“…Here, potential interactors on SiaM are less clear, although there is a patch of positive charged residues on the corresponding surface of TM3a-TM3b adjacent to D50. This result is supported by a charge swap mutation of the equivalent residue in Hi SiaP (E172) to an arginine, which gives a significant growth defect 39 . Together, the tripartite model and functional data suggest that multiple sites play a role in the recognition and allosteric interactions necessary for transport.…”

“…1 ), but the D50A mutant did not disrupt assembly of SiaQM and the complex was stable throughout purification. Analysis of this region by Peter et al only showed a minor growth defect with N150D in an in vivo assay, perhaps suggesting this mutation is not strong enough to fully lock the SiaP in a closed conformation 39 . Finally, at this central interaction point, mutation of the conserved E170A (SiaP, α5) results in greatly reduced activity (Fig.…”

Conformational coupling of the sialic acid TRAP transporter HiSiaQM with its substrate binding protein HiSiaP

“…It was predicted that the closed-state SBP forms a complex with the IFS of the elevator and that the open-state SBP structurally matches and interacts with its OFS. While the predicted binding interfaces could be confirmed by mutagenesis 14 , a direct observation of this (which was not certified by peer review) is the author/funder. All rights reserved.…”