title: The Nucleotide-Free State of the Multidrug Resistance ABC Transporter LmrA: Sulfhydryl Cross-Linking Supports a Constant Contact, Head-to-Tail Configuration of the Nucleotide-Binding Domains.
abstract: ABC transporters are integral membrane pumps that are responsible for the import or export of a diverse range of molecules across cell membranes. ABC transporters have been implicated in many phenomena of medical importance, including cystic fibrosis and multidrug resistance in humans. The molecular architecture of ABC transporters comprises two transmembrane domains and two ATP-binding cassettes, or nucleotide-binding domains (NBDs), which are highly conserved and contain motifs that are crucial to ATP binding and hydrolysis. Despite the improved clarity of recent structural, biophysical, and biochemical data, the seemingly simple process of ATP binding and hydrolysis remains controversial, with a major unresolved issue being whether the NBD protomers separate during the catalytic cycle. Here chemical cross-linking data is presented for the bacterial ABC multidrug resistance (MDR) transporter LmrA. These indicate that in the absence of nucleotide or substrate, the NBDs come into contact to a significant extent, even at 4°C, where ATPase activity is abrogated. The data are clearly not in accord with an inward-closed conformation akin to that observed in a crystal structure of V. cholerae MsbA. Rather, they suggest a head-to-tail configuration 'sandwich' dimer similar to that observed in crystal structures of nucleotide-bound ABC NBDs. We argue the data are more readily reconciled with the notion that the NBDs are in proximity while undergoing intra-domain motions, than with an NBD 'Switch' mechanism in which the NBD monomers separate in between ATP hydrolysis cycles.
authors.affiliation: School of Life Sciences, University of Technology Sydney, P.O. Box 123, Broadway, NSW 2007, Australia.
authors.forename: Peter M
authors.initials: PM
authors.lastname: Jones
authors.affiliation: School of Life Sciences, University of Technology Sydney, P.O. Box 123, Broadway, NSW 2007, Australia.
authors.forename: Anthony M
authors.initials: AM
authors.lastname: George
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D024901:Drug Resistance, Multiple, Bacterial / Q000187:drug effects; D008958:Models, Molecular; D008969:Molecular Sequence Data; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry* / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D009711:Nucleotides / Q000378:metabolism*; D010084:Oxidation-Reduction / Q000187:drug effects; D055503:Protein Multimerization / Q000187:drug effects; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000378:metabolism; D016415:Sequence Alignment; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D014700:Verapamil / Q000494:pharmacology
chemical_list: D000251:Adenosine Triphosphatases; D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D003545:Cysteine; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009711:Nucleotides; D021122:Protein Subunits; D013438:Sulfhydryl Compounds; D014700:Verapamil; C101824:LmrA protein, Lactococcus lactis