Functional Reconstitution of HlyB, a Type I Secretion ABC Transporter, in Saposin-A Nanoparticles

Type I secretion systems (T1SS) are ubiquitous transport machineries in Gram-negative bacteria. They comprise a relatively simple assembly of three membrane-localised proteins: an inner-membrane complex composed of an ABC transporter and a membrane fusion protein, and a ToIC-like outer membrane component. T1SS transport a wide variety of substrates with broad functional diversity. The ABC transporter hemolysin B (HlyB), for example, is part of the hemolysin A-T1SS in Escherichia coli. In contrast to canonical ABC transporters, an accessory domain, a C39 peptidase-like domain (CLD), is located at the N-terminus of HlyB and is essential for secretion. In this study, we have established an optimised purification protocol for HlyB and the subsequent reconstitution employing the saposin nanoparticle system. We point out the negative influence of free detergent on the basal ATPase activity of HlyB, studied the influence of a lysolipid or lipid matrix on activity and present functional studies with the full-length substrate proHlyA in its folded and unfolded states, which both have a stimulatory effect on the ATPase activity.