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.

Structural and biophysical characterization of the tandem substrate-binding domains of the ABC importer GlnPQ

“…Both crystallography and single-molecule Förster-resonance energy transfer (smFRET) experiments on the single SBD1 and SBD2 showed that substrate binding was linked to a conformational change of the corresponding SBD from an open apo conformation to a closed liganded conformation [18][19][20][21]. The results also implied an induced-fit-type ligand-binding mechanism, where conformational dynamics are induced by ligand-SBD interactions similar as later also demonstrated for other SBPs [22][23][24][25][26]. Additionally, it was shown that the opening of the SBDs and ligand release was the rate-limiting step in the transport cycle and that the closed conformation triggers ATP-hydrolysis and transport [18].…”

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

“…The conformational dynamics between the open-and closed-states of SBPs have only recently been studied for ABC-transporter related SBPs 14,16,31 and were completely unknown for TRAP transporter SBPs. Our above described FRET experiments and kinetic analysis reveal that VcSiaP has a ligand dependent substrate-binding rate.…”

“…Here, we used single-molecule Förster-resonance energy transfer (smFRET) spectroscopy to investigate this important step of the mechanism by real-time measurements of VcSiaP dynamics under more physiological conditions. Such experiments should allow comparisons to SBPs from the ABC transporter family 14,17 . For this purpose, we employed the Q54C/L173C double mutant of VcSiaP that was used for the PELDOR/DEER experiments 18 .…”

“…All sample solutions were measured with µl drop on a coverslip with concentration of around 50 pM in buffer A. Data analysis was performed using home written software package as described in 14 .…”

“…The closing motion and the binding mechanism of the SBP have often been compared to a Venus flytrap 12,13 . A PELDOR spectroscopy study (pulsed electron double resonance, also known as DEER) on spin labelled VcSiaP indicated that in frozen solution and without substrate, the P-domain "lurks" in the open state, similar to some ABC transporter SBPs [14][15][16][17][18][19][20] . The substrate binding cleft has two highly conserved arginine residues (Arg125 and Arg145 in VcSiaP), which coordinate the carboxylic acid group of the substrate 10,11,21 .…”