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

Abstract

AbstractTripartite ATP-independent periplasmic (TRAP) transporters are analogous to ABC transporters in that they use a substrate-binding proteins to scavenge metabolites (e.g.,N-acetylneuraminate) and deliver them to the membrane components for import. TRAP substrate-binding proteins are thought to bind the substrate using a two-state (open and closed) induced-fit mechanism. We solved the structure of the TRAPN-acetylneuraminate substrate-binding protein fromAggregatibacter actinomycetemcomitans(AaSiaP) in both the open ligand-free and closed liganded conformations. Surprisingly, we also observed an intermediate conformation, whereAaSiaP is mostly closed and is bound to a non-cognate ligand, acetate, which hints at howN-acetylneuraminate binding stabilises a fully closed state.AaSiaP preferentially bindsN-acetylneuraminate (KD= 0.4 µM) compared toN-glycolylneuraminate (KD= 4.4 µM), which is explained by the closed-N-acetylneuraminate bound structure. Small-angle X-ray scattering data alongside molecular dynamics simulations suggest theAaSiaP adopts a more open state in solution than in crystal. However, the open unliganded conformation can also sample closed conformations. Molecular dynamics simulations also demonstrate the importance of water molecules for stabilising the closed conformation. Although our data is consistent with an induced fit model of binding, it is likely that the open unliganded conformation encompasses multiple states capable of binding substrate. The mechanism by which the ligand is released for import remains to be determined.