Abstract
Bacterial ABC importers catalyze the uptake of essential nutrients including transition metals and metal-containing co-factors. Recently, an IgG antibody targeting the external binding protein of the Staphylococcus aureus Mn(II) ABC importer was reported to inhibit transport activity and reduce bacterial cell growth. We here explored the possibility of using alpaca-derived nanobodies to inhibit the vitamin B12 transporter of Escherichia coli, BtuCD-F, as a model system by generating nanobodies against the periplasmic binding protein BtuF. We isolated six nanobodies that competed with B12 for binding to BtuF, with inhibition constants between 10−6 and 10−9 M. Kinetic characterization of the nanobody-BtuF interactions revealed dissociation half-lives between 1.6 and 6 minutes and fast association rates between 104 and 106 M−1s−1. For the tightest-binding nanobody, we observed a reduction of in vitro transport activity of BtuCD-F when an excess of nanobody over B12 was used. The structure of BtuF in complex with the most effective nanobody Nb9 revealed the molecular basis of its inhibitory function. The CDR3 loop of Nb9 reached into the substrate-binding pocket of BtuF, preventing both B12 binding and BtuCD-F complex formation. Our results suggest that nanobodies can mediate ABC importer inhibition, providing an opportunity for novel antibiotic strategies.
Highlights
ATP-binding cassette (ABC) importers are multi-subunit membrane protein complexes mediating the uptake of essential nutrients[1]
An approach to inhibit transport by an ABC importer was established by blocking the interaction of the substrate binding protein (SBP) with the transporter using a Fab fragment of an IgG antibody that bound to the SBP and restricted the interaction with the transporter by steric hindrance
ABC transporters share a common architecture of two transmembrane domains and two nucleotide binding domains (NBDs)[8,9], but large scale mechanistic diversity has been observed[10]
Summary
ATP-binding cassette (ABC) importers are multi-subunit membrane protein complexes mediating the uptake of essential nutrients[1]. They contain a soluble or membrane-anchored substrate binding protein (SBP) that recognizes the substrate and delivers it to the transporter located in the cytoplasmic membrane[2]. Single chain variable domain antibody fragments derived from heavy chain only antibodies of camelids, might be able to accomplish similar blocking[4] This would offer additional possibilities in developing novel antibiotic strategies, because nanobodies are less immunogenic and smaller than antibodies, offering certain advantages for therapeutic approaches. Our data showed that a nanobody, which is much smaller than a Fab fragment, could mediate inhibition of B12 import by competing with substrate binding and transport
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