title: Deleting two C-terminal alpha-helices is effective to crystallize the bacterial ABC transporter Escherichia coli MsbA complexed with AMP-PNP.
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.
authors.affiliation: Department of Structural Biology, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan.
authors.forename: Kanako
authors.initials: K
authors.lastname: Terakado
authors.forename: Atsushi
authors.initials: A
authors.lastname: Kodan
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Nakano
authors.forename: Yasuhisa
authors.initials: Y
authors.lastname: Kimura
authors.forename: Kazumitsu
authors.initials: K
authors.lastname: Ueda
authors.forename: Toru
authors.initials: T
authors.lastname: Nakatsu
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Kato
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; C079726:MsbA protein, Bacteria; D000266:Adenylyl Imidodiphosphate