Iqbal, Hina; (2022) Molecular insights into complement activation by multidisciplinary biophysical approaches. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The complement system is a key part of our innate immune defence and consists of about 30 components and their regulators. The three known complement activation pathways converge at one point to cleave complement C3 for opsonization. Mannan binding lectin (MBL) is an initiation molecule of the lectin pathway of complement activation whereas Factor H (FH) is a major regulator of activated C3 generated through the alternative pathway. MBL contains multiple carbohydrate recognition domains linked by homotrimeric collagen triple helices. MBL forms complexes with the serine protease (MASP-1, -2) in plasma through its collagen domain during complement activation. Low MBL levels in plasma is the most common human immunodeficiency and is associated with low oligomerization due to mutations in the collagen region. The binding site for MASPs is located near the ‘GQG’ interruption in the collagen helix. To understand structural flexibility in MBL during activation of complement, synthetic peptides based on MBL collagen sequence were studied using analytical ultracentrifugation (AUC) and small angle X-ray and neutron scattering (SAXS, SANS) followed by atomistic modelling using molecular dynamics (MD). Standard collagen (POG)n peptides have been shown to have rigid rod-like conformation from crystal structure. However, recently solution structures of (POG)n helices revealed variable conformational flexibility that increase with length. MBL collagen peptides were also compared with standard and mutated (POG)n peptides. The collagen peptide that corresponds to the native MBL sequence (MBL-N) showed maximum flexibility and thus may be key to the MBL collagen conformation in MBL-MASP complex. The activation of C3 via the alternative pathway results in the generation of C3u and C3b. The final cleavage products of sequential proteolytic processing of C3u and C3b by the regulators (FH, fI) are C3c and C3d. FHR5 is a complement activating plasma protein and routinely identified in kidney immune deposits in nephropathy. Its C-terminal domains SCR8-9 are almost identical to FH SCR-19/20 domains. FHR5 has been proposed to compete with FH for binding with C3b in a concentration dependent manner. The interactions of C3b with C3d, FH and FHR5 were explored with surface plasmon resonance in 137mM and 50mM NaCl. It was observed that the interaction between C3b and full-length FH is relatively weaker than SCR-16/20 or SCR-19/20 in 137mM NaCl. FHR5 showed a stronger affinity towards C3b than FH and its C-terminal region, thus clarifying how FHR5 performs its function. FH interacts through its C-terminal domains SCR-19/20, with C3d to regulate C3 activation. Genetic variations in C3d or SCR-19/20 are strongly associated with complement dysregulation. The stoichiometry of SCR-19/20 and C3d complexes is controversial to date between 1:1 and 2:1. The solution structures of recombinant C3d and SCR-19/20 were studied with AUC and SAXS. The investigation with multiwavelength AUC (MWL-AUC) revealed 1:1 stoichiometric SCR-19/20:C3d complex formation under physiological ionic conditions.

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