Abstract
Prokaryotic ATP-binding cassette (ABC) importers require a substrate-binding protein (SBP) for the capture and delivery of the cognate substrate to the transmembrane domain (TMD) of the transporter. Various biochemical compounds have been identified that bind to the SBP but are not transported. The mechanistic basis for the “non-cognate” substrates not being transported differs. Some non-cognate substrates fail to trigger the appropriate conformational change in the SBP, resulting in loss of affinity for the TMD or the inability to allosterically activate transport. In another mechanism, the SBP cannot release the bound non-cognate substrate. Here, we used rate equations to derive the steady-state transport rate of cognate substrates of an ABC importer and investigated how non-cognate substrates influence this rate. We found that under limiting non-cognate substrate concentrations, the transport rate remains unaltered for each of the mechanisms. In contrast, at saturating substrate and SBP concentrations, the effect of the non-cognate substrate depends heavily on the respective mechanism. For instance, the transport rate becomes zero when the non-cognate substrate cannot be released by the SBP. Yet it remains unaffected when substrate release is possible but the SBP cannot dock onto the TMDs. Our work shows how the different mechanisms of substrate inhibition impact the transport kinetics, which is relevant for understanding and manipulating solute fluxes and hence the propagation of cells in nutritionally complex milieus.
Highlights
ATP-binding cassette (ABC) transporters form a large family of membrane transport proteins[1,2,3] with a common domain architecture
In Gram-negative bacteria, most substrate-binding protein (SBP) are present as soluble protein in the periplasmic space, but some are lipid-anchored[15] or tethered to the transmembrane domain (TMD) of the ABC transporter[16], which is analogous to the surface association of SBPs in Gram-positive bacteria and archaea
We focus on Type I ABC importers, as they are structurally and mechanistically different from other ABC importers[38]
Summary
ATP-binding cassette (ABC) transporters form a large family of membrane transport proteins[1,2,3] with a common domain architecture. ABC importers are typical of prokaryotic organisms but recently a number of proteins with the ‘exporter fold’ have been discovered that mediate cellular uptake, both in prokaryotic and eukaryotic cells[9,10]. Both Type I and II ABC importers require a soluble extracellular or periplasmic substrate-binding protein (SBP) for their function[11,12]. These closed conformations productively interact with the translocator and activate transport and the ATPase activity[21,22,23]
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