Deleting two C-terminal α-helices is effective to crystallize the bacterial ABC transporter Escherichia coli MsbA complexed with AMP-PNP

✍ Scribed by Terakado, Kanako (author);Kodan, Atsushi (author);Nakano, Hiroaki (author);Kimura, Yasuhisa (author);Ueda, Kazumitsu (author);Nakatsu, Toru (author);Kato, Hiroaki (author)

• Publisher: International Union of Crystallography
• Year: 2010
• Tongue: English
• Weight: 916 KB
• Volume: 66
• Category: Article
• ISSN: 0907-4449
• DOI: 10.1107/s0907444909055504

✦ Synopsis

An MsbA deletion mutant ΔC21 that lacks the two C-terminal α-helices was expressed in__Escherichia coli__strain C41 and purified by metal-affinity and gel-filtration chromatography. Purified ΔC21 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′-(β,γ-imido)triphosphate could not be obtained, crystals of ΔC21 that diffracted to 4.5 Å resolution were obtained. The preliminary ΔC21 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 α-helices may play a role in facilitating the outward-facing nucleotide-bound crystal structure of EcMsbA.