The objective of this project is to obtain a detailed understanding of the formation and function of human C5b-9, the cytolytic complex of complement referred to as the """"""""membrane attack complex"""""""" or MAC. The MAC is a product of the innate immune system. The MAC provides protection against bacterial infection but it also contributes to the pathogenesis of inflammatory diseases. Assembly of the MAC occurs on target cell membranes by a poorly understood mechanism of protein-protein and protein-lipid interactions between complement components C5b, C6, C7, C8 and C9. This project will provide new insight into the molecular basis of these interactions and lay the foundation for designing inhibitors of MAC formation.
Specific aims are: 1) Determine the crystal structure of human C8, a 151-kDa protein composed of three genetically distinct subunits, C81, C82, and C83, which are arranged as a disulfide-linked C81-3 heterodimer that is noncovalently associated with C82. Recombinant forms of the central 40-kDa MACPF segment of C81 (1MACPF), and 1MACPF linked to C83 (1MACPF-3), have been crystallized and their structures partially solved. These data will facilitate solving the crystal structure of whole C8 for which X-ray diffraction data (3.0 E resolution) have recently been obtained. 2) Identify and characterize functionally important binding sites on C8. Proposed studies will complement Aim 1 and allow C8 structural features to be correlated with function. Sites in 1MACPF that are involved in binding C82 and C9, and those in the MACPF segment of C82 (2MACPF) which are involved in binding C81 and C5b, will be identified and characterized. The functional significance of C83 binding to C81 will also be examined. 3) Identify structure-function relationships in human C9.
This aim will focus on characterizing the C81 binding site on C9 and the site(s) that mediates C9-C9 interaction and polyC9 formation. The mechanism of poly C9 formation will also be examined. Attempts will be made to produce a recombinant C9 MACPF segment to be used for detailed studies of C9 binding interactions. Efforts will also be made to crystallize C9 for structure determination. NARRATIVE This project focuses on a group of human blood proteins that interact and form a complex which disrupts cell membranes and contributes to the killing of pathogenic organisms, e.g. bacteria. However, it can also alter human cell membranes and contribute to the pathogenesis of inflammatory diseases. The interaction of these proteins will be studied in detail and the results could lead to the development of drugs that control the formation and function of the complex.
|Weiland, Mitch H; Qian, Yu; Sodetz, James M (2014) Membrane pore formation by human complement: functional importance of the transmembrane ?-hairpin (TMH) segments of C8? and C9. Mol Immunol 57:310-6|
|Lovelace, Leslie L; Cooper, Christopher L; Sodetz, James M et al. (2011) Structure of human C8 protein provides mechanistic insight into membrane pore formation by complement. J Biol Chem 286:17585-92|
|Slade, Daniel J; Lovelace, Leslie L; Chruszcz, Maksymilian et al. (2008) Crystal structure of the MACPF domain of human complement protein C8 alpha in complex with the C8 gamma subunit. J Mol Biol 379:331-42|
|Lovelace, Leslie L; Chiswell, Brian; Slade, Daniel J et al. (2008) Crystal structure of complement protein C8gamma in complex with a peptide containing the C8gamma binding site on C8alpha: implications for C8gamma ligand binding. Mol Immunol 45:750-6|
|Chiswell, Brian; Lovelace, Leslie L; Brannen, Charity et al. (2007) Structural features of the ligand binding site on human complement protein C8gamma: a member of the lipocalin family. Biochim Biophys Acta 1774:637-44|
|Brannen, Charity L; Sodetz, James M (2007) Incorporation of human complement C8 into the membrane attack complex is mediated by a binding site located within the C8beta MACPF domain. Mol Immunol 44:960-5|
|Slade, Daniel J; Chiswell, Brian; Sodetz, James M (2006) Functional studies of the MACPF domain of human complement protein C8alpha reveal sites for simultaneous binding of C8beta, C8gamma, and C9. Biochemistry 45:5290-6|
|Chiswell, Brian; Slade, Daniel J; Sodetz, James M (2006) Binding of the lipocalin C8gamma to human complement protein C8alpha is mediated by loops located at the entrance to the C8gamma ligand binding site. Biochim Biophys Acta 1764:1518-24|
|Ortlund, Eric; LaCount, Michael W; Lebioda, Lukasz (2003) Crystal structures of human prostatic acid phosphatase in complex with a phosphate ion and alpha-benzylaminobenzylphosphonic acid update the mechanistic picture and offer new insights into inhibitor design. Biochemistry 42:383-9|
|Ortlund, Eric; Parker, Chasta L; Schreck, Steven F et al. (2002) Crystal structure of human complement protein C8gamma at 1.2 A resolution reveals a lipocalin fold and a distinct ligand binding site. Biochemistry 41:7030-7|
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