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

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.