Abstract
The ATP-binding cassette (ABC) transporter BtuCD mediates the uptake of vitamin B(12) 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 B(12) molecule, and radioligand trapping showed that liposome-reconstituted BtuCD-F indeed contains bound B(12) 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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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ATP-Binding Cassette Transporters / chemistry*
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ATP-Binding Cassette Transporters / genetics
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ATP-Binding Cassette Transporters / metabolism
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Adenosine Triphosphatases / chemistry
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Adenosine Triphosphatases / metabolism
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Adenosine Triphosphate / metabolism
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Adenylyl Imidodiphosphate / chemistry
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Adenylyl Imidodiphosphate / metabolism*
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Amino Acid Sequence
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Crystallography, X-Ray
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Cytoplasm / metabolism
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Disulfides / chemistry
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Disulfides / metabolism
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Escherichia coli / chemistry*
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Escherichia coli / genetics
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Escherichia coli Proteins / chemistry*
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Escherichia coli Proteins / genetics
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Escherichia coli Proteins / metabolism
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Magnesium / metabolism
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Models, Biological
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Models, Molecular
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Mutant Proteins / chemistry
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Mutant Proteins / genetics
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Mutant Proteins / metabolism
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Mutation
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Periplasmic Binding Proteins / chemistry*
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Periplasmic Binding Proteins / genetics
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Periplasmic Binding Proteins / metabolism
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Protein Binding
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Protein Conformation
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Protein Subunits / chemistry
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Protein Subunits / genetics
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Protein Subunits / metabolism
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Structure-Activity Relationship
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Vitamin B 12 / chemistry
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Vitamin B 12 / metabolism*
Substances
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ATP-Binding Cassette Transporters
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BtuC peptide, E coli
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BtuD peptide, E coli
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Disulfides
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Escherichia coli Proteins
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Mutant Proteins
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Periplasmic Binding Proteins
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Protein Subunits
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btuF protein, E coli
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Adenylyl Imidodiphosphate
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Adenosine Triphosphate
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Adenosine Triphosphatases
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Magnesium
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Vitamin B 12