A specific interaction between the NBD of the ABC-transporter HlyB and a C-terminal fragment of its transport substrate haemolysin A.
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.
Benabdelhak
Houssain
H
Institut de Génétique et Microbiologie, Bât. 409, Université de Paris XI, 91405, Orsay, France.
Kiontke
Stephan
S
Horn
Carsten
C
Ernst
Robert
R
Blight
Mark A
MA
Holland
I Barry
IB
Schmitt
Lutz
L
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
D001426:Bacterial Proteins; D002352:Carrier Proteins; D006460:Hemolysin Proteins; C050837:Hlyb protein, Bacteria