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

Sci Rep. 2019 Jun 10;9(1):8436. doi: 10.1038/s41598-019-44812-0.

Abstract

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 TolC-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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP-Binding Cassette Transporters / isolation & purification
  • ATP-Binding Cassette Transporters / metabolism*
  • Adenosine Triphosphatases / metabolism
  • Escherichia coli / metabolism
  • Hemolysin Proteins / isolation & purification
  • Hemolysin Proteins / metabolism*
  • Kinetics
  • Lipoproteins / metabolism
  • Nanoparticles / chemistry*
  • Protein Folding
  • Saposins / metabolism*

Substances

  • ATP-Binding Cassette Transporters
  • Hemolysin Proteins
  • Lipoproteins
  • Saposins
  • Adenosine Triphosphatases
  • hemolysin B