title: Vanadate-catalyzed photocleavage of the signature motif of an ATP-binding cassette (ABC) transporter.
abstract: The maltose transport complex of Escherichia coli, a member of the ATP-binding cassette (ABC) superfamily, is made up of two nucleotide-binding subunits, MalK(2), which hydrolyze ATP with positive cooperativity, and two transmembrane subunits, MalF and MalG. The ABC family is defined in part by the canonical signature motif LSGGQ whose exact function remains controversial. Taking advantage of the dual function of vanadate as a transition state analogue and as a photoactive chemical, we demonstrate that vanadate catalyzes the UV-dependent cleavage of the polypeptide backbone at both the LSGGQ motif and the nucleotide-binding, or Walker A, motif when it is trapped in the nucleotide-binding site of the bacterial maltose transporter. This highly specific cleavage pattern indicates that residues in both motifs are immediately adjacent to ATP during hydrolysis, and are therefore likely to participate directly in ATP-binding and/or hydrolysis. Because the LSGGQ motif is too distant from the nucleotide in the structure of an ABC monomer for cleavage to occur, these data support a model in which the LSGGQ motif contacts the nucleotide across the interface of a MalK dimer, as seen in the crystal structure of Rad50. This architecture provides a basis for the cooperativity observed in the nucleotide-binding domains of ABC transporters and a function for this highly conserved family signature motif.
authors.affiliation: Department of Molecular Virology and Microbiology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
authors.forename: Erin E
authors.initials: EE
authors.lastname: Fetsch
authors.forename: Amy L
authors.initials: AL
authors.lastname: Davidson
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006868:Hydrolysis; D007700:Kinetics; D008320:Maltose / Q000378:metabolism; D058619:Maltose-Binding Proteins; D008958:Models, Molecular; D009004:Monosaccharide Transport Proteins; D010766:Phosphorylation; D010777:Photochemistry; D011487:Protein Conformation; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014638:Vanadates / Q000494:pharmacology*
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C079123:MalG protein, E coli; C068021:MalK protein, Bacteria; C489398:MalK protein, E coli; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; C461599:maltose transport system, E coli; D014638:Vanadates; D008320:Maltose; D017868:Cyclic AMP-Dependent Protein Kinases