The molecular basis for perforin oligomerization and transmembrane pore assembly.
Perforin, a pore-forming protein secreted by cytotoxic lymphocytes, is indispensable for destroying virus-infected cells and for maintaining immune homeostasis. Perforin polymerizes into transmembrane channels that inflict osmotic stress and facilitate target cell uptake of proapoptotic granzymes. Despite this, the mechanism through which perforin monomers self-associate remains unknown. Our current study establishes the molecular basis for perforin oligomerization and pore assembly. We show that after calcium-dependent membrane binding, direct ionic attraction between the opposite faces of adjacent perforin monomers was necessary for pore formation. By using mutagenesis, we identified the opposing charges on residues Arg213 (positive) and Glu343 (negative) to be critical for intermolecular interaction. Specifically, disrupting this interaction had no effect on perforin synthesis, folding, or trafficking in the killer cell, but caused a marked kinetic defect of oligomerization at the target cell membrane, severely disrupting lysis and granzyme B-induced apoptosis. Our study provides important insights into perforin's mechanism of action.
Baran
Katherine
K
Cancer Immunology Program, Peter MacCallum Cancer Centre, St Andrew's Place, East Melbourne, Victoria 3002, Australia.
Dunstone
Michelle
M
Chia
Jenny
J
Ciccone
Annette
A
Browne
Kylie A
KA
Clarke
Christopher J P
CJ
Lukoyanova
Natalya
N
Saibil
Helen
H
Whisstock
James C
JC
Voskoboinik
Ilia
I
Trapani
Joseph A
JA
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000502:physiology; D022082:Cellular Structures / Q000378:metabolism; D003185:Complement C8 / Q000378:metabolism*; D004912:Erythrocytes / Q000502:physiology; D053804:Granzymes / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D009154:Mutation / Q000235:genetics; D054353:Perforin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016062:Porosity; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012756:Sheep
D003185:Complement C8; D011994:Recombinant Proteins; D054353:Perforin; D053804:Granzymes