Assembly of the Bi-component leukocidin pore examined by truncation mutagenesis.
Staphylococcal leukocidin (Luk) and alpha-hemolysin (alphaHL) are members of the same family of beta barrel pore-forming toxins (betaPFTs). Although the alphaHL pore is a homoheptamer, the Luk pore is formed by the co-assembly of four copies each of the two distantly related polypeptides, LukF and LukS, to form an octamer. Here, we examine N- and C-terminal truncation mutants of LukF and LukS. LukF subunits missing up to nineteen N-terminal amino acids are capable of producing stable, functional hetero-oligomers with WT LukS. LukS subunits missing up to fourteen N-terminal amino acids perform similarly in combination with WT LukF. Further, the simultaneous truncation of both LukF and LukS is tolerated. Both Luk subunits are vulnerable to short deletions at the C terminus. Interestingly, the N terminus of the LukS polypeptide becomes resistant to proteolytic digestion in the fully assembled Luk pore while the N terminus of LukF remains in an exposed conformation. The results from this work and related experiments on alphaHL suggest that, although the N termini of betaPFTs may undergo reorganization during assembly, they are dispensable for the formation of functional pores.
Miles
George
G
Department of Medical Biochemistry & Genetics, Texas A&M University System Health Science Center, College Station, TX 77843-1114, USA.
Jayasinghe
Lakmal
L
Bayley
Hagan
H
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D002462:Cell Membrane / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism; D017353:Gene Deletion; D006461:Hemolysis; D007956:Leukocidins / Q000737:chemistry*; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D014176:Protein Biosynthesis; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014357:Trypsin / Q000494:pharmacology
D001426:Bacterial Proteins; D007956:Leukocidins; D010455:Peptides; C108483:leukocidin S-component protein, Staphylococcus; C074300:LukF protein, Staphylococcus aureus; D014357:Trypsin