Abstract
The ATP-binding cassette (ABC) transporter BtuCD mediates the uptake of vitamin B12 across the inner membrane of Escherichia coli. Previous structures have shown the conformations of apo states, but the transport mechanism has remained unclear. Here we report the 3.5 Å crystal structure of the transporter-binding protein complex BtuCD–BtuF (BtuCD–F) trapped in an β-γ-imidoadenosine 5′-phosphate (AMP-PNP)-bound intermediate state. Although the ABC domains (BtuD subunits) form the expected closed sandwich dimer, the membrane-spanning BtuC subunits adopt a new conformation, with the central translocation pathway sealed by a previously unrecognized cytoplasmic gate. A fully enclosed cavity is thus formed approximately halfway across the membrane. It is large enough to accommodate a vitamin B12 molecule, and radioligand trapping showed that liposome-reconstituted BtuCD–F indeed contains bound B12 in the presence of AMP-PNP. In combination with engineered disulphide crosslinking and functional assays, our data suggest an unexpected peristaltic transport mechanism that is distinct from those observed in other ABC transporters.
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Acknowledgements
We thank the beamline staff at the Swiss Light Source for assistance with data collection. We are grateful to R. Schibli and M. Badertscher for access to the gamma counter. This research was supported by the NCCR Structural Biology Zurich and Swiss National Science Foundation (grant SNF 31003A-131075/1).
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V.M.K., S.A.M. and K.P.L. designed the experiments, V.M.K. and S.A.M. performed the experiments, V.M.K. and K.P.L. performed crystallographic calculations and all authors analysed the data. V.M.K. and K.P.L. wrote the manuscript.
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Korkhov, V., Mireku, S. & Locher, K. Structure of AMP-PNP-bound vitamin B12 transporter BtuCD–F. Nature 490, 367–372 (2012). https://doi.org/10.1038/nature11442
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DOI: https://doi.org/10.1038/nature11442
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