Calcium-dependent plasma membrane binding and cell lysis by perforin are mediated through its C2 domain: A critical role for aspartate residues 429, 435, 483, and 485 but not 491.
The lymphocyte pore-forming protein perforin is essential for maintaining immune homeostasis and for effective defense against intracellular pathogens. To date, there have been no reported structure-function studies to substantiate the function of any putative domains of perforin, which have been postulated totally on primary sequence similarities with domains in other proteins. In this report, we have used recently developed modalities for expressing full-length perforin and robust functional assays to investigate one of the hallmarks of perforin function: its absolute dependence on calcium for lipid binding and cell lysis. We provide, for the first time, experimental evidence that the predicted C-terminal C2 motif constitutes a functional domain that is responsible for membrane binding of perforin. Whereas conserved aspartate residues at positions 429, 435, 483, and 485 were essential for calcium-dependent plasma membrane binding and cell lysis, the contribution of Asp-491 was limited. Finally, after experimentally verifying an optimized three-dimensional model, we have made predictions on the impact of two inherited perforin mutations of the C2 domain on calcium-dependent lipid binding and cell lysis.
Voskoboinik
Ilia
I
Cancer Immunology Program, Peter MacCallum Cancer Centre, Locked Bag 1, A'Beckett St. 8006 and The Walter and Eliza Hall Institute for Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia.
Thia
Marie-Claude
MC
Fletcher
Jamie
J
Ciccone
Annette
A
Browne
Kylie
K
Smyth
Mark J
MJ
Trapani
Joseph A
JA
D020816:Amino Acid Motifs; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry*; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004912:Erythrocytes / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007313:Insecta; D019169:Jurkat Cells; D008024:Ligands; D050356:Lipid Metabolism; D008214:Lymphocytes / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000502:physiology*; D008958:Models, Molecular; D009154:Mutation; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D012756:Sheep; D013329:Structure-Activity Relationship; D013997:Time Factors; D014162:Transfection
D018076:DNA, Complementary; D008024:Ligands; D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D011994:Recombinant Proteins; D054353:Perforin; D001224:Aspartic Acid; D002118:Calcium