ATPASE ACTIVITY AND ATP ADP-INDUCED CONFORMATIONAL CHANGE IN THE SOLUBLE DOMAIN OF THE BACTERIAL PROTEIN TRANSLOCATOR HLYB/

The haemolysin exporter HlyB and its homologues are central to the unc onventional signal-peptide-independent secretion of toxins, proteases and nodulation proteins by bacteria. HlyB is a member of the ATP-bindi ng cassette (ABC) or traffic ATPase superfamily, and resembles closely in structure and function mammalian exporters such as the multidrug-r esistance P-glycoprotein, combining both integral membrane and cytosol ic domains. Overproduction of the HlyB cytoplasmic domain as a C-termi nal peptide fused to glutathione S-transferase allowed the direct affi nity purification and concentration of 30-50 mg ml-1 of soluble protei n (GST-Bctp) in an apparently dimeric form possessing both transferase and ATPase activity. GST-Bctp bound to ADP-agarose and was eluted spe cifically by ATP and ADP, affinity behaviour which was confirmed in bo th the full-length HlyB and the unfused HlyB cytoplasmic domain synthe sized in vitro. The stoichiometry of binding to MgATP and MgADP was cl ose to equimolar and both ligands induced substantial conformational c hange in the protein. Mg2+-dependent ATPase activity of GST-Bctp (V(ma x) 1 mumol min-1 mg-1, K(m) 0.2 mM) was comparable with the activity o f the bacterial importer Malk and human P-glycoprotein reconstituted i nto proteoliposomes, and over an order of magnitude higher than in vit ro measurements of disaggregated MalK purified from inclusion bodies. Activity was unaffected by inhibitors of F- and V-type ATPases, non-hy drolysable ATP analogues, or translocation substrate, but was severely inhibited by inhibitors of E1E2 (P-type) ATPases, and the acidic phos pholipid phosphatidyl glycerol.