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A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing
Published on: July 3, 2016
Interactions between residues in staphylococcal alpha-hemolysin revealed by reversion mutagenesis
1Worcester Foundation for Biomedical Research, Shrewsbury, Massachusetts 01545, USA.
Abstract:
alpha-Hemolysin (alpha HL), a pore-forming polypeptide of 293 amino acids, is secreted by Staphylococcus aureus as a water-soluble monomer. Residues that play key roles in the formation of functional heptameric pores on rabbit red blood cells (rRBC) have been identified previously by site-directed mutagenesis. alpha HL-H35N, in which the histidine at position 35 of the wild-type sequence is replaced with asparagine, is nonlytic and is arrested in assembly as a heptameric prepore. In this study, second-site revertants of H35N that have the ability to lyse rRBC were generated by error-prone PCR under conditions designed to produce single base changes. The analysis of 22 revertants revealed new codons clustered predominantly in three distinct regions of the H35N gene. One cluster includes amino acids 107-111 (four revertants) and another residues 144-155 (five revertants). These two clusters flank the central glycine-rich loop of alpha HL, which previously has been implicated in formation of the transmembrane channel, and encompass residues Lys-110 and Asp-152 that, like His-35, are crucial for lytic activity. The third cluster lies in the region spanning amino acids 217-228 (eight revertants), a region previously unexplored by mutagenesis. Single revertants were found at amino acid positions 84 and 169. When compared with H35N, the heptameric prepores formed by the revertants underwent more rapid conversion to fully assembled pores, as determined by conformational analysis by limited proteolysis. The rate of conversion to the fully assembled pore was strongly correlated with hemolytic activity. Previous work has suggested that the N terminus of alpha HL and the central loop cooperate in the final step of assembly. The present study suggests that the key N-terminal residue His-35 operates in conjunction with residues flanking the loop and C-terminal residues in the region 217-228. Hence, reversion mutagenesis extends the linear analysis that has been provided by direct point mutagenesis.
Insights
Staphylococcus aureus alpha-hemolysin (alpha HL) pore formation involves key residues. Reversion mutagenesis identified new critical sites, revealing His-35 works with flanking and C-terminal residues for lytic activity.
Area of Science:
- Microbiology
- Molecular Biology
- Protein Structure and Function
Background:
- Alpha-hemolysin (alpha HL) from Staphylococcus aureus is a pore-forming toxin.
- Previous studies identified key residues for alpha HL pore formation via site-directed mutagenesis.
- The alpha HL-H35N mutant is nonlytic and forms a stable prepore.
Purpose of the Study:
- To identify novel amino acid residues involved in alpha HL pore formation and lytic activity.
- To investigate the functional roles of second-site revertants of the alpha HL-H35N mutant.
- To elucidate the cooperative mechanisms in the final steps of alpha HL assembly.
Main Methods:
- Error-prone PCR was used to generate second-site revertants of the alpha HL-H35N mutant.
- Revertant mutants were analyzed for their ability to lyse rabbit red blood cells (rRBC).
- Conformational analysis by limited proteolysis assessed the conversion rate of prepores to fully assembled pores.
Main Results:
- Twenty-two revertants were identified, with mutations clustering in three regions: amino acids 107-111, 144-155, and 217-228.
- Residues Lys-110 and Asp-152, flanking the glycine-rich loop, were found to be crucial for lytic activity, similar to His-35.
- The third cluster of mutations in the 217-228 region, previously unexplored, also contributed to lytic function.
- Revertant prepores showed a more rapid conversion to fully assembled pores, correlating with increased hemolytic activity.
Conclusions:
- The N-terminal residue His-35 cooperates with residues flanking the central loop and C-terminal residues (217-228) in alpha HL pore assembly.
- Reversion mutagenesis provides a complementary approach to direct mutagenesis for mapping functional residues.
- These findings expand the understanding of the molecular mechanisms underlying alpha HL pore formation and its lytic function.
