Arresting and releasing Staphylococcal α-hemolysin at intermediate stages of pore formation by engineered disulfide bonds

Toshimitsu Kawate, Eric Gouaux

Research output: Contribution to journalArticle

54 Scopus citations

Abstract

α-Hemolysin (αHL) is secreted by Staphylococcus aureus as a water-soluble monomer that assembles into a heptamer to form a transmembrane pore on a target membrane. The crystal structures of the LukF water-soluble monomer and the membrane-bound α-hemolysin heptamer show that large conformational changes occur during assembly. However, the mechanism of assembly and pore formation is still unclear, primarily because of the difficulty in obtaining structural information on assembly intermediates. Our goal is to use disulfide bonds to selectively arrest and release αHL from intermediate stages of the assembly process and to use these mutants to test mechanistic hypotheses. To accomplish this, we created four double cysteine mutants, D108C/K154C (αHL-A), M113C/K147C (αHL-B), H48C/N121C (αHL-C), I5C/G130C (αHL-D), in which disulfide bonds may form between the pre-stem domain and the β-sandwich domain to prevent pre-stem rearrangement and membrane insertion. Among the four mutants, αHL-A is remarkably stable, is produced at a level at least 10-fold greater than that of the wild-type protein, is monomeric in aqueous solution, and has hemolytic activity that can be regulated by the presence or absence of reducing agents. Cross-linking analysis showed that αHL-A assembles on a membrane into an oligomer, which is likely to be a heptamer, in the absence of a reducing agent, suggesting that oxidized αHL-A is halted at a heptameric prepore state. Therefore, conformational rearrangements at positions 108 and 154 are critical for the completion of αHL assembly but are not essential for membrane binding or for formation of an oligomeric prepore intermediate.

Original languageEnglish (US)
Pages (from-to)997-1006
Number of pages10
JournalProtein Science
Volume12
Issue number5
DOIs
StatePublished - May 1 2003

Keywords

  • Assembly intermediate
  • Disulfide bonds
  • Double cysteine mutants
  • Pore-forming toxin
  • Staphylococcal α-hemolysin

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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