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

Abstract

The tripartite ATP-independent periplasmic (TRAP) transporters use an extra cytoplasmic substrate binding protein (SBP) to transport a wide variety of substrates in bacteria and archaea. The SBP can adopt an ‘open’ or ‘closed’ state depending on the presence of substrate. The two transmembrane domains of TRAP transporters form a monomeric elevator whose function is strictly dependent on the presence of a sodium ion gradient. Insights from experimental structures, structural predictions and molecular modeling have suggested a conformational coupling between the membrane elevator and the substrate binding protein. Here, we use a disulfide engineering approach to lock the TRAP transporter HiSiaPQM fromHaemophilus influenzaein different conformational states. The SBP, HiSiaP, was locked in its substrate-bound form and the transmembrane elevator, HiSiaQM, was locked in either its predicted inward- or outward-facing states. We characterized the disulfide-locked variants and used single-molecule total internal reflection fluorescence (TIRF) microscopy to study their interactions. Our experiments demonstrate that the SBP and the transmembrane elevator are indeed ‘conformationally coupled’, meaning that the open and closed state of the SBP recognize specific conformational states of the transporter and vice versa.

Structure, substrate selectivity determinants and membrane interactions of a Glutamate-specific TAXI TRAP binding protein fromVibrio cholerae

“…Structural analysis of HiSiaQM from H. influenzae and P. profundum has revealed important details regarding the interaction between the membrane component and the SBPs and the overall architecture of the membrane component. As previously predicted 29 , TRAP transporters share the same fold as members of the divalent anion Na + symporter (DASS) family 30–33 , and both employ an elevator-like mechanism to transport substrate across the membrane 31,34,35 .…”

On the function of TRAP substrate-binding proteins: conformational variation of the sialic acid binding protein SiaP

“…We speculate that it is more likely that substrate release is governed by SiaP-SiaQM surface (49). How the fully open state observed here fits in with the mechanism is unclear, though we speculate that this state should have the lowest affinity for SiaQM, and that this promotes dissociation from the transporter.…”

“…This is consistent with findings from PELDOR spectroscopy experiments on frozen Vc SiaP (36) and atomistic MD simulations based on this PELDOR data, which suggest that Vc SiaP in solution may also be more open than in the open-state crystal structure (48). More recently, rare closure events have been reported for an apo disulfide engineered Hi SiaP construct, where residues within the latch region of SiaP are substituted (S15C/A194C) (49). In this experiment, disulfide formation between these residues only occurs when SiaP closes, and the residues were in close contact in the required geometry and was detected as a distinct band shift by non-reducing SDS-PAGE in the absence of oxidising agents.…”

“…Exactly how SiaP interacts with the OFS needs investigation, and in particular whether or not SiaP is fully open during this interaction. It may be possible that SiaP adopts an intermediate open state while bound to the OFS, considering that the closed SiaP could still occasionally interact with the OFS (49). How the fully open state observed here fits in with the mechanism is unclear, though we speculate that this state should have the lowest affinity for SiaQM, and that this promotes dissociation from the transporter.…”