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The architectural transition of human complement component C9 to poly(C9)
1La Jolla Institute for Experimental Medicine, CA 92037, USA. richard@ljiem.org
Insights
The study reveals how C9 protein polymerization exposes its middle sections, forming a U-shape. Thrombospondin (TS) and LDL modules remain exposed, aiding structural integrity during C9 polymerization.
Area of Science:
- Biochemistry
- Structural Biology
- Molecular Biology
Background:
- C9 protein's role in biological processes is not fully understood.
- Understanding C9 polymerization is crucial for elucidating its function.
- Previous studies have identified key domains within the C9 polypeptide chain.
Purpose of the Study:
- To investigate the structural changes and domain exposure of C9 during polymerization.
- To identify the specific regions of C9 involved in the polymerization process.
- To elucidate the overall structure of polymerized C9 (poly(C9)).
Main Methods:
- Bacterial expression of C9 protein fragments and domains.
- Antibody production against specific C9 segments.
- Enzyme-linked immunosorbent assays (ELISAs) to probe epitope exposure.
- Immuno-electron microscopy to visualize poly(C9) structure.
- Computer-assisted image enhancement of electron micrographs.
Main Results:
- Thrombospondin (TS) and low-density lipoprotein receptor (LDL) modules are exposed in both monomeric and polymerized C9.
- The middle region of the C9 polypeptide chain is buried in monomers but exposed in polymers.
- Fab fragments targeting the middle region and EDGF module inhibited C9 polymerization.
- Immuno-electron microscopy revealed a 'U' shape for poly(C9), with TS and LDL modules on the upper rim.
- Subunits of poly(C9) exhibit a volute shape, with the midsection forming the tubule barrel.
Conclusions:
- TS and LDL modules do not directly participate in C9 polymerization but shield hydrophobic regions in monomers.
- Polymerization exposes the midsection of the C9 polypeptide chain, facilitating tubule formation.
- The 'U' shape and volute structure of poly(C9) subunits are key to its assembly and function.
Abstract:
Several regions of C9 including three cysteine-rich modules homologous to those in thrombospondin (TS), the low density lipoprotein receptor (LDL), the epidermal growth factors (EDGF), as well as two middle sections of the polypeptide chain were expressed in bacteria. Antibodies derived from these segments were used to probe the relative exposure of epitopes in C9 and poly(C9) using ELISAs. The results indicated that the TS and LDL modules are fully exposed in both monomer and polymer; however, the middle region of the polypeptide chain is buried in the monomer but external in the polymer. Using specified conditions, Fab fragments to the TS and LDL modules did not block C9 polymerization, but those to the middle region of the polypeptide chain and to some extent to the EDGF module did so. Immuno-electron microscopy of poly(C9) indicated that the C9 polypeptide chain assumes a 'U' shape, in which the TS and LDL modules are located on the upper rim. The EDGF module is located on the lower edge of the upper rim, and midsection of the polypeptide chain constructs the barrel of the tubule. Computer assisted contrast enhancement of select electron micrograph images of poly(C9) allowed the clear visualization of each subunit. These were seen to have a volute shape. The upper rim is composed of whorls that are apparently not in lateral contact. It is concluded that the TS and LDL modules do not participate directly in polymerization but cover the hydrophobic central region of the polypeptide chain in the monomer. As a consequence of circular polymerization the midsection of the polypeptide chain becomes exposed as each C9 lengths to fashion a volute form. reserved.