Molecular interactions of human C9: The pore forming protein of the complement system

The human complement system is part of our innate immunity that acts to eliminate pathogenic organisms. Activation of complement leads to the sequential, non-enzymatic assembly of C5b, C6, C7, C8, C9 forming C5b-9n (n=12-18), a cytolytically active pore-like structure known as the membrane attack complex (MAC). C8 is a heterotrimer comprised of a disulfide linked C8alpha-gamma dimer that is non-covalently associated with C8alpha. Following assembly of C5b-8, C8alpha-gamma facilitates the binding and polymerization of C9. C9 along with C6, C7, C8alpha, and C8beta constitute the MAC family of proteins. Members of this family have homologous N- and C-terminal modules along with a central ∼40 kDa segment called the membrane attack complex/perforin (MACPF) domain. A distinguishing feature of C9, compared to the other MAC family members, is its ability to self-polymerize. C9 polymerization may occur during MAC formation or insolution in the absence of C5b-8, the latter is referred to as "poly C9.";The goal of this research was to further identify the structure-function relationships in C9. The first aim was to identify binding sites within C9 for C8alpha-gamma and C9. One approach focused on the production of recombinant full-length and truncated C9 constructs to identify binding interactions. Another approach used phage display technology to further narrow the binding sites on C9 for C8alpha-gamma and C9. Two separate phage display systems were employed for accomplishing this goal: commercially available combinatorial libraries and a custom library containing peptides from the MACPF domain of C9.;The second aim was to address the long-standing question of whether the resistance of poly C9 to denaturation and proteolysis is due to strong non-covalent hydrophobic interactions or disulfide bond rearrangement and formation of intermolecular crosslinks. Efforts focused on resolving this issue by using mass spectrometry to identify Cys residues involved in disulfide shuffling during poly C9 formation.;The third aim was to produce crystals of C9 for structural determination by X-ray diffraction. Determination of the structure of C9 would provide significant insight into the role of C9 in MAC formation and could allow for a model of poly C9 to be built. The results from high throughput screening and in-house crystallization experiments performed using native and deglycosylated C9 are described.