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-hemolysin at intermediate stages of pore formation by engineered disulfide bonds
1 Department of Biochemistry and Molecular Biophysics and
2 Howard Hughes Medical Institute, Columbia University, New York, New York 10032, USA
Reprint requests to: Eric Gouaux, Department of Biochemistry and Molecular Biophysics, Columbia University, 650 West 168th Street, New York, NY 10032, USA; e-mail: jeg52{at}columbia.edu; fax: (212) 305-8174.
-Hemolysin (HL) 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 -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 HL 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 (HL-A), M113C/K147C (HL-B), H48C/ N121C (HL-C), I5C/G130C (HL-D), in which disulfide bonds may form between the pre-stem domain and the ß-sandwich domain to prevent pre-stem rearrangement and membrane insertion. Among the four mutants, HL-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 HL-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 HL-A is halted at a heptameric prepore state. Therefore, conformational rearrangements at positions 108 and 154 are critical for the completion of HL assembly but are not essential for membrane binding or for formation of an oligomeric prepore intermediate.
Keywords: Double cysteine mutants; disulfide bonds; assembly intermediate; pore-forming toxin; Staphylococcal -hemolysin
Abbreviations: HL, -Hemolysin • Luk, leukocidin • 
HL, -hemolysin • SEC, size-exclusion chromatography • ßME, ß-mercaptoethanol • DTT, dithiothreitol • DiC8PC, dioctanoyl phosphatidyl choline • GA, glutaraldehyde • WT-His6, wild-type -hemolysin with a carboxyl terminal hexa-histidine tag • RBC, rabbit blood cells • SDS-PAGE, SDS polyacrylamide gel electrophoresis
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