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

Abstract

Abstract␣-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 ␤-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.

Anti-Alpha-Hemolysin Monoclonal Antibodies Mediate Protection againstStaphylococcus aureusPneumonia

“…Thus, the similar signal intensities observed for the monomer in lanes treated with the MAb and in lanes without antibody treatment or in the presence of control antibody strongly suggest that the MAbs do not function merely by preventing toxin binding to target cells. The monomeric form of Hla produced in vitro migrated as two bands, as seen in previous studies (13). Each MAb, however, prevented the assembly of Hla into an SDS-stable oligomer (Hla 7 ) in a close-dependent fashion (Fig.…”

“…This chromosomally encoded toxin is secreted as a water-soluble monomer by the majority of S. aureus strains (22). Membrane binding of the monomer permits a series of well-defined intermolecular interactions between neighboring monomers, resulting in the formation of a barrel-shaped oligomeric pore that penetrates the membrane (9,13). Residues located at the N terminus of the mature toxin are essential for assembly of the lytic oligomer, as point mutations or truncations within this region disrupt the formation of an active toxin (21,27,28).…”

Role of the Amino Latch of Staphylococcal α-Hemolysin in Pore Formation

“…The central axis of the prepore lies perpendicular to the membrane surface (9,50,51), and the residues that will become the transmembrane ␤ barrel have not yet undergone the required conformational change (25,26,46,48). In the case of ␣HL, the prepore was believed to feature a protease-sensitive N terminus ( Table 2) that was needed to form a protease-resistant latch upon transformation to the fully assembled pore (22).…”

Bacterial Two-component and Hetero-heptameric Pore-forming Cytolytic Toxins: Structures, Pore-forming Mechanism, and Organization of the Genes

“…Recently, Kawate and Gouaux determined the essential residues of -hemolysin, D108 and K154 for the conformational rearrangements from homo-heptameric pre-pore intermediate to pore, by creating a double cysteine mutant, D108C/K154C, 48) according to our method for -hemolysin which was described above. 37) 5.…”