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Substrate Binding Stabilizes a Pre-translocation Intermediate in the ATP-binding Cassette Transport Protein MsbA*

R. Doshi,H. V. van Veen

Published 2013 in Journal of Biological Chemistry

ABSTRACT

Background: During substrate transport, ATP-binding cassette exporters switch between an inward-facing and an outward-facing state in a nucleotide-dependent fashion. Results: Substrate binding to bacterial MsbA initiates dimerization of nucleotide-binding domains without opening the membrane domains at their external side. Conclusion: Substrate binding to MsbA stabilizes an “inward-facing closed” pre-translocation state that binds ATP. Significance: Our observations suggest a fundamental mechanism by which substrates stimulate ATP hydrolysis. ATP-binding cassette (ABC) transporters belong to one of the largest protein superfamilies that expands from prokaryotes to man. Recent x-ray crystal structures of bacterial and mammalian ABC exporters suggest a common alternating access mechanism of substrate transport, which has also been biochemically substantiated. However, the current model does not yet explain the coupling between substrate binding and ATP hydrolysis that underlies ATP-dependent substrate transport. In our studies on the homodimeric multidrug/lipid A ABC exporter MsbA from Escherichia coli, we performed cysteine cross-linking, fluorescence energy transfer, and cysteine accessibility studies on two reporter positions, near the nucleotide-binding domains and in the membrane domains, for transporter embedded in a biological membrane. Our results suggest for the first time that substrate binding by MsbA stimulates the maximum rate of ATP hydrolysis by facilitating the dimerization of nucleotide-binding domains in a state, which is markedly distinct from the previously described nucleotide-free, inward-facing and nucleotide-bound, outward-facing conformations of ABC exporters and which binds ATP.

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