Spontaneous oligomerization of a staphylococcal alpha-hemolysin conformationally constrained by removal of residues that form the transmembrane beta-barrel.
Staphylococcal alpha-hemolysin is a water soluble, monomeric, bacterial exotoxin, which forms heptameric pores in membranes. The rate determining step in assembly is the conversion of a heptameric prepore to the fully assembled pore in which the central glycine-rich domain of each subunit inserts into the membrane to form a 14 strand beta barrel. Barrel formation is accompanied by a conformational change in which each N terminus latches onto an adjacent subunit. In the monomer in solution, the central domain is loosely organized and exposed to solvent. In this study, 25 amino acids of the central domain were removed and replaced with the sequence Asp-Gly, which favors the formation of a type I' beta-turn, to yield a mutant devoid of hemolytic activity. Within minutes after synthesis in the absence of membranes, the mutant polypeptide spontaneously assembled into heptamers, as demonstrated by atomic force microscopy. Limited proteolysis suggested that the N termini of the subunits in the heptamers were in the fully assembled pore conformation rather than the prepore conformation. Based on these findings, the deletion is proposed to constrain the central domain and thereby force the creation of a shortened beta barrel, which in turn induces the additional structural changes that normally accompany pore formation. The truncated pore might make a useful framework for the construction of designed membrane active macromolecules.
Cheley
S
S
Department of Medical Biochemistry and Genetics, Texas A&M Health Science Center, College Station 77843-1114, USA.
Malghani
M S
MS
Song
L
L
Hobaugh
M
M
Gouaux
J E
JE
Yang
J
J
Bayley
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D019286:Endopeptidase K / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000378:metabolism; D005998:Glycine; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006461:Hemolysis; D046911:Macromolecular Substances; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011817:Rabbits; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D046911:Macromolecular Substances; C021091:staphylococcal alpha-toxin; D019286:Endopeptidase K; D005998:Glycine