title: A specific interaction between the NBD of the ABC-transporter HlyB and a C-terminal fragment of its transport substrate haemolysin A.
abstract: A member of the family of RTX toxins, Escherichia coli haemolysin A, is secreted from Gram-negative bacteria. It carries a C-terminal secretion signal of approximately 50 residues, targeting the protein to the secretion or translocation complex, in which the ABC-transporter HlyB is a central element. We have purified the nucleotide-binding domain of HlyB (HlyB-NBD) and a C-terminal 23kDa fragment of HlyA plus the His-tag (HlyA1), which contains the secretion sequence. Employing surface plasmon resonance, we were able to demonstrate that the HlyB-NBD and HlyA1 interact with a K(D) of approximately 4 microM. No interaction was detected between the HlyA fragment and unrelated NBDs, OpuAA, involved in import of osmoprotectants, and human TAP1-NBD, involved in the export of antigenic peptides. Moreover, a truncated version of HlyA1, lacking the secretion signal, failed to interact with the HlyB-NBD. In addition, we showed that ATP accelerated the dissociation of the HlyB-NBD/HlyA1 complex. Taking these results together, we propose a model for an early stage of initiation of secretion in vivo, in which the NBD of HlyB, specifically recognizes the C terminus of the transport substrate, HlyA, and where secretion is initiated by subsequent displacement of HlyA from HlyB by ATP.
authors.affiliation: Institut de Génétique et Microbiologie, Bât. 409, Université de Paris XI, 91405, Orsay, France.
authors.forename: Houssain
authors.initials: H
authors.lastname: Benabdelhak
authors.forename: Stephan
authors.initials: S
authors.lastname: Kiontke
authors.forename: Carsten
authors.initials: C
authors.lastname: Horn
authors.forename: Robert
authors.initials: R
authors.lastname: Ernst
authors.forename: Mark A
authors.initials: MA
authors.lastname: Blight
authors.forename: I Barry
authors.initials: IB
authors.lastname: Holland
authors.forename: Lutz
authors.initials: L
authors.lastname: Schmitt
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry; D006460:Hemolysin Proteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D006460:Hemolysin Proteins; C050837:Hlyb protein, Bacteria