Triggering Closure of a Sialic Acid TRAP Transporter Substrate Binding Protein through Binding of Natural or Artificial Substrates

Abstract

The pathogens Vibrio cholerae and Haemophilus influenzae use tripartite ATPindependent periplasmic transporters (TRAPs) to scavenge sialic acid from host tissues.They use it as a nutrient or to evade the innate immune system by sialylating surface lipopolysaccharides. An essential component of TRAP transporters is a periplasmic substrate binding protein (SBP). Without substrate, the SBP has been proposed to rest in an open-state, which is not recognised by the transporter. Substrate binding induces a conformational change of the SBP and it is thought that this closed state is recognised by the transporter, triggering substrate translocation. Here we use real time single molecule FRET experiments and crystallography to investigate the open-to closed-state transition of VcSiaP, the SBP of the sialic acid TRAP transporter from V. cholerae. We show that the conformational switching of VcSiaP is strictly substrate induced, confirming an important aspect of the proposed transport mechanism. Two new crystal structures of VcSiaP provide insights into the closing mechanism. While the first structure contains the natural ligand, sialic acid, the second structure contains an artificial peptide in the sialic acid binding site. Together, the two structures suggest that the ligand itself stabilises the closed state and that SBP closure is triggered by physically bridging the gap between the two lobes of the SBP. Finally, we demonstrate that the affinity for the artificial peptide substrate can be substantially increased by varying its amino acid sequence and by this, serve as a starting point for the development of peptide-based inhibitors of TRAP transporters.

The structure of HiSiaQM defines the architecture of tripartite ATP-independent periplasmic (TRAP) transporters

“…As known from several previous studies, the SBP binds tightly (KD 20-300 nM) to its substrate and switches from an opened-to a closed conformation (Figure 4, steps 1-2) 22,24,31 . As the P-domain only closes when a substrate is bound, empty transport cycles are prevented 25,31 . In the next step, the substrate-bound SBP binds to the transporter in its inward open resting state, the structure of which has been determined in this work.…”

“…TRAPs and TTTs have so far proved recalcitrant to experimental attempts at elucidating their structures. To date, only structures of the soluble Pdomains, mainly from the family of DctP-like SBPs [22][23][24][25][26][27][28] have been determined. Features such as the role of a conserved arginine residue for high-affinity substrate binding and specificity, and the conformational rearrangement of the protein upon substrate binding have been characterized in detail [29][30][31] .…”

“…The SBP was previously cloned into a pBADHisTEV vector which fuses the HiSiaP protein to an N-terminal His6-tag and TEV-cleavage site. The protein was expressed and purified as described in Peter et al 25 for the homologous protein VcSiaP from V. cholerae. The protein was expressed in M9 minimal medium to avoid contamination with sialic acid and the His-tag was removed in an overnight digestion with TEV protease.…”

Metabolic fate of dietary sialic acid and its influence on gut and oral bacteria

“…Despite the lack of sialidase, Haemophilus can still utilize sialic acid secreted by other bacteria or free sialic acid from host cells. For instance, Haemophilus influenzae realizes the sialylation of LPS by its own Sia transporting system to escape the host's immune response [63].…”

Selective Nutrient Transport in Bacteria: Multicomponent Transporter Systems Reign Supreme

“…A model of transport by TRAP transporters has been proposed and is based upon the alternating-mechanism seen in Type I ABC transporters ( Mulligan et al, 2009 ). Recent work has started to uncover potential “scoop loops” in the membrane domains of TRAP transporters ( Peter et al, 2021 ). Additionally, Darby et al (2019) have found that disruption of the ordered waters within the binding cleft of an SBP can dramatically alter substrate binding affinity.…”