A Specific Interaction Between the NBD of the ABC- transporter HlyB and a C-Terminal Fragment of its Transport Substrate Haemolysin A Houssain Benabdelhak 1 †, Stephan Kiontke 2 †, Carsten Horn 2 Robert Ernst 2 , Mark A. Blight 1 , I. Barry Holland 1 and Lutz Schmitt 2 * 1 Institut de Ge ´ne ´tique et Microbiologie Ba ˆt. 409, Universite ´ de Paris XI, 91405 Orsay France 2 Institute of Biochemistry Biocenter N210 University of Frankfurt Marie-Curie Str. 9 60439 Frankfurt, Germany 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 23 kDa 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 mM. 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 pro- pose 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 dis- placement of HlyA from HlyB by ATP. q 2003 Elsevier Science Ltd. All rights reserved Keywords: ABC-transporters; protein secretion; protein – protein interaction; surface plasmon resonance *Corresponding author Introduction Haemolysin A (HlyA), a 110 kDa toxin, is a member of the RTX (repeats in toxins) family. 1 These proteins share repetitive glycine-rich sequences, which bind Ca 2þ in the folded molecule. HlyA and all other members of this family are secreted by type 1 secretion systems. 2,3 Type 1 secretion is independent of the Sec pathway and depends on the presence of an uncleaved secretion signal at the C terminus of the toxin. 4 Translocation through the cell envelope proceeds without a periplasmic intermediate, resulting in a one-step transfer of the HlyA toxin across both membranes of the cell envelope to the medium. 5 The secretion signal is localized in the last 50 to 60 C-terminal amino acid residues of HlyA. 6,7 Mutational studies have indicated a large functional redundancy in the primary sequence of HlyA and other type 1 transport substrates. 8–13 Mutagenesis, complemen- tation and competition studies resulted in the identification of an important functional region extending at least from residues 2 15 to 2 46 with respect to the C terminus. This region, which, although containing very few conserved residues, even compared with close relatives of HlyA, included at least three residues that were appar- ently involved in recognition of the translocator. 9 Different genetic studies have provided evidence for 10,13 and against 8,11 a specific secondary structure 0022-2836/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved † H.B. and S.K. contributed equally to this work. Present address: S. Kiontke, Institute of Physiological Chemistry, Philipps-University Marburg, Karl-von- Frisch Str. 1, 35033 Marburg, Germany. E-mail address of the corresponding author: lschmitt@em.uni-frankfurt.de Abbreviations used: ABC, ATP-binding cassette; GST, glutathione-S-transferase; Hly, haemolysin; MFP, membrane fusion protein; NTA, N-nitrilo-triacetic acid; OpuAA, osmoprotectant uptake system A ATPase; pmf, proton motive force; SPR, surface plasmon resonance; RTX, repeat in toxins; RU, resonance unit; TMD, transmembrane domain; TMS, transmembrane segments. doi:10.1016/S0022-2836(03)00204-3 J. Mol. Biol. (2003) 327, 1169–1179