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Localization and molecular modelling of the membrane-inserted domain of the ninth component of human complement and
M C Peitsch1, P Amiguet, R Guy
1Institute of Mathematical Biology, National Cancer Institute, Frederick, MD 21701.
Insights
The ninth component of complement (C9) forms cell membrane channels. Researchers identified the specific C9 protein region that interacts with cell membranes, revealing its structure and aiding in modeling similar proteins.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- Complement component 9 (C9) forms transmembrane channels upon complement activation.
- The membrane-interacting domain of C9 is not well-defined due to its lack of hydrophobic amino acid stretches.
- Understanding C9 membrane interaction is crucial for comprehending complement-mediated cell lysis.
Purpose of the Study:
- To identify the specific region of C9 that interacts with lipid bilayers.
- To elucidate the structural characteristics of the membrane-interacting domain of C9.
- To develop a molecular model for the membrane-spanning region of C9 and related proteins.
Main Methods:
- Utilized the photoaffinity label 125I-TID to study C9-lipid bilayer interactions.
- Assembled C9 on liposomes and performed photoactivation.
- Analyzed labeled and non-labeled peptides after chemical and enzymatic cleavage.
- Employed secondary structure predictions to identify helical regions.
Main Results:
- Identified amino acid segment 176-345 as the primary membrane-interacting region of C9.
- Discovered two amphipathic alpha-helices (residues 292-308 and 313-333) separated by a turn (309-312) within this segment.
- Constructed a molecular model of the membrane-spanning domain of C9.
Conclusions:
- The identified helical structures are key to C9's membrane insertion and channel formation.
- The study provides a structural basis for C9's interaction with cell membranes.
- The findings facilitate modeling of the membrane-spanning domain in homologous proteins like perforin/cytolysin.
Abstract:
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.