1. Deleting two C-terminal alpha-helices is effective to crystallize the bacterial ABC transporter Escherichia coli MsbA complexed with AMP-PNP.
- Author
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Terakado K, Kodan A, Nakano H, Kimura Y, Ueda K, Nakatsu T, and Kato H
- Subjects
- ATP-Binding Cassette Transporters metabolism, Adenylyl Imidodiphosphate metabolism, Amino Acid Sequence, Bacterial Proteins metabolism, Crystallography, X-Ray, Escherichia coli metabolism, Models, Molecular, Molecular Sequence Data, Protein Binding, Protein Structure, Quaternary, Protein Structure, Secondary, Protein Structure, Tertiary, Sequence Alignment, ATP-Binding Cassette Transporters chemistry, Adenylyl Imidodiphosphate chemistry, Bacterial Proteins chemistry, Escherichia coli chemistry
- Abstract
An MsbA deletion mutant DeltaC21 that lacks the two C-terminal alpha-helices was expressed in Escherichia coli strain C41 and purified by metal-affinity and gel-filtration chromatography. Purified DeltaC21 retained 26% of the activity of the wild-type ATPase and had a similar binding affinity to fluorescent nucleotide derivatives. Although crystals of wild-type MsbA complexed with adenosine 5'-(beta,gamma-imido)triphosphate could not be obtained, crystals of DeltaC21 that diffracted to 4.5 A resolution were obtained. The preliminary DeltaC21 structure had the outward-facing conformation, in contrast to the previously reported E. coli MsbA structure. This result suggests that deletion of the C-terminal alpha-helices may play a role in facilitating the outward-facing nucleotide-bound crystal structure of EcMsbA.
- Published
- 2010
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