Localization and molecular modelling of the membrane-inserted domain of the ninth component of human complement and perforin.
Upon interaction with the membrane-bound C5b-8 complex, the ninth component of complement (C9) unfolds and inserts into the membrane of cells on which surface complement has been activated. Consequently C9 oligomerization occurs and transmembrane channels of varying sizes are formed. The domain of the unfolded protein interacting with the cell membrane has so far not been identified since, unlike many integral membrane proteins, the C9 sequence does not contain a continuous stretch of hydrophobic amino acids. We studied the interaction of C9 with the lipid bilayer using the membrane-restricted photoaffinity label 3-(trifluoromethyl)-3-(m[125I]iodophenyl)diazirine (125I-TID). C9 was assembled on liposomes and after photoactivation, several labeled and non-labeled peptides, obtained by chemical and enzymatic cleavage or the 125I-TID-labeled C9, were analyzed. The segment from 176 to 345 was identified as the region containing the membrane-interacting structure. By means of secondary structure predictions, we identified two amphipathic alpha-helices (292-308 and 313-333) separated by a turn (309-312). Based on these results, we constructed a molecular model for the membrane-spanning region of C9. By analogy, we also constructed a model for this domain in perforin/cytolysin, a pore-forming protein found in the cytoplasmic granules of cytotoxic T-lymphocytes.
Institute of Mathematical Biology, National Cancer Institute, Frederick, MD 21701.
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Peitsch
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Amiguet
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Guy
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Brunner
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Maizel
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Tschopp
D000345:Affinity Labels; D000595:Amino Acid Sequence; D002308:Cardiolipins; D003186:Complement C9 / Q000648:ultrastructure*; D006801:Humans; D066298:In Vitro Techniques; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000648:ultrastructure*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D054353:Perforin; D010713:Phosphatidylcholines; D052899:Pore Forming Cytotoxic Proteins; D011487:Protein Conformation; D012995:Solubility
D000345:Affinity Labels; D002308:Cardiolipins; D003186:Complement C9; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D052899:Pore Forming Cytotoxic Proteins; D054353:Perforin