The Role of the Amino Terminus in the Kinetics and Assembly of α-Hemolysin of Staphylococcus aureus

Abstract

The nature of the involvement of an intact NH 2 terminus in the assembly of ␣-hemolysin of Staphylococcus aureus was reinvestigated. For the first time, a deletion of the first four amino acids at the NH 2 terminus of ␣-hemolysin yielded a novel mutant that undergoes all of the conformational changes to form a lytic pore. The experimental evidence shows unequivocally that the mutant toxin forms heat-and sodium dodecyl sulfatestable heptameric oligomers. The concentration required to achieve 50% lysis of red blood cells is around 58 -116 ng/ml, and the time taken to achieve lysis to the same extent as that of intact toxin is considerably longer. Transmission electron microscopic studies also suggest that the pores formed by this deletion mutant are similar to those by the full-length toxin. This is in contrast to the previously reported 2-and 11-amino acid deletions that failed to proceed further from a presumed prefinal nonlytic pore to a lytic pore. Studies on the kinetics of assembly indicate that this mutant can form heat-and sodium dodecyl sulfate-stable oligomers as fast as full-length ␣-hemolysin but that pore opening is slowed down. The data strongly suggest that these amino acids (Ala-Asp-Ser-Asp) are involved in the final stages of assembly of ␣-hemolysin in target membranes.The assembly of proteins into membranes is an intricate phenomenon in which these proteins undergo a large degree of conformational changes in exerting their biological functions. Ion channels (1), integral membrane proteins (2), and other membrane-associated proteins (3) are a few well known examples. Pore-forming toxins such as ␣-hemolysin of Staphylococcus aureus, streptolysin O of Streptococcus pyogenes, aerolysin of Aeromonas hydrophila, and ␣-toxin of Clostridium septicum, secreted by several bacteria in water-soluble forms, also assemble spontaneously in target cell membranes to form transmembrane channels (4 -6). Unlike ion channels, membrane-associated proteins, and cell surface receptors, these pore-forming toxins do not have well defined stretches of hydrophobic amino acids in their sequences. The localized environment experienced by stretches of amino acids that insert into membranes appears to control the assembly and function of some of these toxins. Hence, a detailed study of such toxins would provide valuable information regarding the spatial and structural organization of the polypeptide chain segments that span the membrane bilayer. This would eventually lead to a better understanding of their mode of action as well as pave the way for the rational design of novel lytic molecules (7). Among the molecules available for the study of such pore-forming toxins, ␣-hemolysin (␣-HL) 1 of S. aureus is a relatively small protein.It is therefore amenable for redesign of its functions (8). ␣-HL is a 293-amino acid polypeptide that binds to the target cells as a monomer, and the cell-bound monomers undergo extensive conformational changes to form a transmembrane pore. The pore formation takes place through a series of intermedi...

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

“…1B). However, a later study suggested that truncated polypeptides have limited pore-forming activity (36). In the present work, we resolve this issue through an examination of the original truncation mutants and a new series in which up to 22 residues are deleted.…”

“…The mutation has no effect on the activity of the full-length protein as discussed in the report (22). Nevertheless, a later study from another laboratory suggested that truncated polypeptides might have limited activity (36). Further, Panchal and coworkers (43) from our laboratory had discovered that a mutation at position 217 could partially reactivate the non-lytic mutant ␣HL-H35N.…”

Assembly of the Bi-component Leukocidin Pore Examined by Truncation Mutagenesis

“…L1130). The complete 1 mM amino acid mixture minus methionine (2.5 l) or the complete 1 mM amino acid mixture minus cysteine (2.5 l) that was supplied in the kit was mixed with premix solution (10 l), [L- 35 S]methionine (1 l, ICN Biomedicals, Inc., Irvine, CA, 1175 Ci/mmol) or [ 35 S]cysteine (1 l, Amersham Biosciences, 1200 Ci/mmol), plasmid DNA (4 l, 400 ng/l), and T7-S30 extract (7.5 l) supplemented with rifampicin (20 g/ml final) to generate "hot" radiolabeled polypeptides (25 l) (24). Synthesis was carried out for 60 min at 30°C, and reactions were terminated with chloramphenicol (100 M final).…”

“…The complete amino acid mixture minus cysteine and the complete amino acid mixture minus methionine were mixed in equal volumes and replaced the individual amino acid mixture minus methionine (or cysteine) (2.5 l) to yield "warm" radiolabeled proteins. Unlabeled (cold) proteins were generated by replacing the [L- 35 (27) in MBSA (10 mM 3-[Nmorpholino]propane sulfonic acid (MOPS), 150 mM NaCl, pH 7.4, containing 1 mg/ml bovine serum albumin), in a final volume of 100…”

Membrane Insertion of the Heptameric Staphylococcal α-Toxin Pore

“…This leads us to the notion that, in membrane insertion, the central loop receives dual activation both from the NH 2 terminus in cis and from another central loop in trans. This model readily accommodates the second cluster of breakthrough mutations located within that loop, and it can also account for previous results obtained with an NH 2 -terminal deletion mutant (12). The latter protein exhibits impaired hemolytic activity, which it also imparts on wild type toxin within hybrid heptamers (Fig.…”