title: Nucleotide dependent packing differences in helical crystals of the ABC transporter MsbA.
abstract: Bacterial ATP binding cassette (ABC) exporters fulfill a wide variety of transmembrane transport roles and are homologous to the human multidrug resistance P-glycoprotein. Recent X-ray structures of the exporters MsbA and Sav1866 have begun to describe the conformational changes that accompany the ABC transport cycle. Here we present cryo-electron microscopy structures of MsbA reconstituted into a lipid bilayer. Using ATPase inhibitors, we captured three nucleotide transition states of the transporter that were subsequently reconstituted into helical arrays. The enzyme-substrate complex (trapped by ADP-aluminum fluoride or AMPPNP) crystallized in a different helical lattice than the enzyme-product complex (trapped by ADP-vanadate). Approximately 20A resolution maps were calculated for each state and revealed MsbA to be a dimer with a large channel between the membrane spanning domains, similar to the outward facing crystal structures of MsbA and Sav1866. This suggests that while there are likely structural differences between the nucleotide transition states, membrane embedded MsbA remains in an outward facing conformation while nucleotide is bound.
authors.affiliation: Department of Cell Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd, CB227, La Jolla, CA 92037, USA. abward@scripps.edu
authors.forename: Andrew
authors.initials: A
authors.lastname: Ward
authors.forename: Sheila
authors.initials: S
authors.lastname: Mulligan
authors.forename: Bridget
authors.initials: B
authors.lastname: Carragher
authors.forename: Geoffrey
authors.initials: G
authors.lastname: Chang
authors.forename: Ronald A
authors.initials: RA
authors.lastname: Milligan
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry*; D000227:Adenine Nucleotides / Q000737:chemistry*; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000266:Adenylyl Imidodiphosphate / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry*; D020285:Cryoelectron Microscopy; D003460:Crystallization; D003461:Crystallography; D007091:Image Processing, Computer-Assisted; D008081:Liposomes / Q000737:chemistry; D008958:Models, Molecular; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D012486:Salmonella typhimurium / Q000737:chemistry / Q000235:genetics; D014638:Vanadates / Q000737:chemistry; D014734:Vibrio cholerae / Q000737:chemistry / Q000235:genetics
chemical_list: D018528:ATP-Binding Cassette Transporters; D000227:Adenine Nucleotides; D001426:Bacterial Proteins; D008081:Liposomes; C079726:MsbA protein, Bacteria; D011994:Recombinant Proteins; D000266:Adenylyl Imidodiphosphate; D014638:Vanadates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate