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Literature summary for 7.6.2.8 extracted from
- Single-molecule visualization of conformational changes and substrate transport in the vitamin B12 ABC importer BtuCD-F (2017), Nat. Commun., 8, 1652 .
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| genes btuC, btuD, and btuF, recombinant expression of wild-type and mutant BtuCD-F complex components | Escherichia coli |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| D141C | site-directed mutagenesis of BtuF on a residue pointing outward in the middle of the alpha-helix connecting the two lobes, the mutation allows for specific coupling of fluorescent labels to each of these proteins | Escherichia coli |
| E159Q | site-directed mutagenesis of BtuD, an ATPase impaired mutant | Escherichia coli |
| additional information | the mutations and labels on BtuF and BtuC have no critical effect on ATP hydrolysis and transport activity | Escherichia coli |
| Q111C | site-directed mutagenesis of BtuC on the periplasmic loop connecting transmembrane (TM) helix 3 and 4 | Escherichia coli |
| W115L | site-directed mutagenesis of BtuC to remove any quenching effects of this tryptophan on the fluorescent probes | Escherichia coli |
General Stability
| General Stability | Organism |
|---|---|
| evaluation of the stability of the liposome-reconstituted BtuCD-F complex during ATP hydrolysis | Escherichia coli |
Localization
| Localization | Comment | Organism | GeneOntology No. | Textmining |
|---|---|---|---|---|
| membrane | - |
Escherichia coli | 16020 | - |
| periplasm | - |
Escherichia coli | - |
- |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Mg2+ | required | Escherichia coli |  |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| ATP + H2O + vitamin B12-[cobalamin-binding protein][side 1] | Escherichia coli | - |
ADP + phosphate + vitamin B12[side 2] + [cobalamin-binding protein][side 1] | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Escherichia coli | P06609 AND P06611 AND P37028 | genes btuC, btuD, and btuF encoding for vitamin B12 import system permease protein BtuC, vitamin B12 import ATP-binding protein BtuD, and vitamin B12-binding protein BtuF | - |
Reaction
| Reaction | Comment | Organism | Reaction ID |
|---|---|---|---|
| ATP + H2O + vitamin B12-[cobalamin-binding protein][side 1] = ADP + phosphate + vitamin B12[side 2] + [cobalamin-binding protein][side 1] | the substrate resides in a different place than the BtuF-binding pocket. Upon binding and hydrolysis of ATP, the complex returns to state III. The transporter can cycle multiple times between states III and IV without releasing vitamin B12, resulting in a much higher ATPase rate than transport rate. This cycling is equivalent to the cycling between states I and II in the absence of substrate. Occasionally, a vitamin B12 molecule is released on the cytoplasmic side of the membrane via a proposed intermediate state. When the binding location becomes vacant, a new vitamin B12 molecule is bound rapidly. ATPase activity and transport are uncoupled and ATP hydrolysis is continuously required to (unproductively) reset the transporter | Escherichia coli |
Renatured (Commentary)
| Renatured (Comment) | Organism |
|---|---|
| purified BtuCD-F complex is functionally reconstituted in liposomes | Escherichia coli |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| ATP + H2O + vitamin B12-[cobalamin-binding protein][side 1] | - |
Escherichia coli | ADP + phosphate + vitamin B12[side 2] + [cobalamin-binding protein][side 1] | - |
? | |
| additional information | a vitamin B12 molecule remains bound to the liposome-reconstituted transporter complex for tens of seconds, during which several ATP hydrolysis cycles can take place, before it is being transported across the membrane. Measurements of fluorescence changes on BtuCD induced by BtuF and ATP, transport of single vitamin B12 molecules, overview | Escherichia coli | ? | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| More | the homodimer BtuC spans the membrane and the two identical cytosolic ATPase domains BtuD form a sandwich dimer that couple chemical energy of two ATP molecules into structural changes of the full complex. A single substrate-binding protein (SBP) BtuF completes the transporter. BtuCD-F forms a stable complex in the ground state, analysis of crystal structures, PDB ID 2QI9 and 4DBL | Escherichia coli |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| BtuC | - |
Escherichia coli |
| BtuCD-F | - |
Escherichia coli |
| BtuD | - |
Escherichia coli |
| BtuF | - |
Escherichia coli |
| vitamin B12 ABC importer | - |
Escherichia coli |
| vitamin B12 import ATP-binding protein | - |
Escherichia coli |
| vitamin B12 import system permease protein | - |
Escherichia coli |
| vitamin B12-binding protein | - |
Escherichia coli |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| ATP | - |
Escherichia coli | |
General Information
| General Information | Comment | Organism |
|---|---|---|
| evolution | Escherichia coli vitamin B12 transporter BtuCD-F is a type II importer and belongs to the ATP-binding cassette (ABC) transporter superfamily | Escherichia coli |
| additional information | the homodimer BtuC spans the membrane and the two identical cytosolic ATPase domains BtuD form a sandwich dimer that couple chemical energy of two ATP molecules into structural changes of the full complex. A single substrate-binding protein (SBP) completes the transporter. The SBP belongs to cluster A or class III and exhibits relatively small conformational changes upon substrate binding. Modeling of the transport mechanism of BtuCD-F transporters embedded in lipid bilayers at the single molecule level, overview | Escherichia coli |
| physiological function | ATP-binding cassette (ABC) transporters form the largest class of active membrane transport proteins. Binding and hydrolysis of ATP by their highly conserved nucleotide-binding domains drive conformational changes of the complex that mediate transport of substrate across the membrane. The transporter complex of vitamin B12 importer BtuCD-F from Escherichia coli is consisting of a periplasmic soluble binding protein BtuF that binds the ligand and the transmembrane and ATPase domains BtuCD mediating translocation | Escherichia coli |
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