Complement Factor H C-terminus and its significance : a structural narrative

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Title: Complement Factor H C-terminus and its significance : a structural narrative
Author: Bhattacharjee, Arnab
Contributor organization: University of Helsinki, Faculty of Medicine, Haartman Institute, Dept. of Bacteriology and Immunology
Institute of Biotechnology, Viikki
Helsingin yliopisto, lääketieteellinen tiedekunta, kliinisteoreettinen laitos
Helsingfors universitet, medicinska fakulteten, Haartman institutet
Publisher: Helsingin yliopisto
Date: 2014-04-11
Language: eng
Thesis level: Doctoral dissertation (article-based)
Abstract: Complement is comprised of a cascade of proteins that recognizes and attacks the invading microbes and thus is the first line of defense for the human body against invading pathogens. It is initiated via different activation pathways that lead to C3b deposition on the target and sequentially to the formation of lytic membrane attack complexes (MAC). One of the complement activation pathways the alternative pathway (AP) can be activated on any surface, self or non-self, and is therefore tightly regulated. Complement Factor H (CFH) is the most important complement down-regulator and it mediates target discrimination between self and non-self cells. Several point mutations in CFH and/or autoantibodies (AA) against it are found to be directly associated with atypical haemolytic uremic syndrome (aHUS), a severe and often fatal disease triggered by the impaired regulation of AP on self surfaces leading to complement attack. ----- CFH is composed of 20 homologous complement control protein domains (CCP). The N-terminal domains 1 to 4 (CFH1-4) mediate inactivation of C3b on the self-surfaces and the C-terminal domains 19 and 20 (CFH19-20) are critical for the ability of CFH to discriminate between self and non-self structures. Self-surfaces are rich in anionic sialic acids (SA) and glycosaminoglycans (GAGs) that are not present on pathogenic microbes. CFH19-20 contains binding sites for both the C3d part of C3b and self-surface polyanions that enhance avidity of CFH to C3b on self surfaces and thus enhance C3b inactivation. CFH mutations that have been found in aHUS patients are mostly located in CFH19-20. ----- The previously solved X-ray crystal structure of CFH19-20 illuminated the location of aHUS related mutations. The aims of this thesis work were to study the functionality of CFH on a molecular level by studying the molecular structure of CFH C-terminus and its mutants along with structures of CFH in complex with its different interacting partners, as well as the structure of the domains of CFH-related protein-1 highly homologous to CFH19-20. The structures solved and the their relevance are described in the four articles attached to this thesis. ------ In the first article, stability of the CFH C-terminus fold by aHUS mutation(s) was studied by analyzing the binding of the CFH19-20 mutant proteins to C3d/C3b using radioligand assays and affinity chromatography. The X-ray crystal structures of CFH19-20 with two different point mutations (of residues indicated to be involved in binding C3d/C3b) were solved. It was shown that these mutations did not result in the disruption of the basic fold of CFH19-20, but maintained the same fold with a prominent difference in the surface charge distribution in the zone of the residues. The results clearly indicated that the aHUS mutations on CFH do not disrupt the basic structural fold, but induce anomaly in the charge distribution of the molecule, explaining the effects of the mutations to its C3b/heparin binding abilities suggested to be critical for the pathogenesis of the disease. In the second article, we revealed the rationale of the molecular mechanism of CFH19-20 mediated self nonself discrimination and showed why point mutations in CFH19-20 lead to aHUS. The CFH19-20 :C3d structure reported in this article revealed two independent binding interfaces between CFH19-20 and C3d, namely the CFH19 site and the CFH20 site . The results of deeper analysis of this structure also showed that the simultaneous binding of the CFH19-20 via the CFH19 site to C3b and via CFH20 site to C3d was possible. In the third article, the X-ray crystal structure of CFH19-20 was solved in complex with the outer surface protein E (OspE) from Borrelia burgdorferi in order to understand the molecular mechanism of sequestering CFH by microbes which results in complement evasion. The nucleomagnetic resonance (NMR) structure of the OspE protein from Borrelia burgdorferi reported in this paper was required for solving the structure of OspE in complex with CFH19-20. Chemical shift perturbations studies using NMR also confirmed the physiological viability of the complex structure. This complex structure was the first structure of CFH19-20 in complex with any microbial protein and thus answered the puzzle of the molecular mimicry used by microbes involving CFH C-terminus in order to evade complement. ----- In the fourth article the structure of the CFHR1 domains 4 and 5 (CFHR14-5) was solved and used to explain why AA bind to a common epitope on CFH domain 20, which is highly homologous to domain 5 of CFHR1. The CFHR14-5 structure revealed an important structural bigamy of CFH and its related proteins that can be used to understand why CFHR-1 deficiency and formation of AA against CFH (CFH- AA) lead to autoimmune aHUS (AI-aHUS). We extensively studied CFH-AA from more than a dozen of patients and their binding behavior to CFH and CFHR1. The results suggest a novel hypothesis on the pathogenesis of AI-aHUS. In conclusion, the results have revealed the molecular mechanisms beneath different functionalities of the C-terminus of CFH. It is not only the most important molecule to facilitate the target discrimination by the AP, but is also a prominent tool used by the microbes in order to evade complement attack. Furthermore, the CFH C- terminus also houses the AA binding epitope and thus also plays a role in the pathogenesis of AI-aHUS. We were also quite surprised to find out that the molecular structure of CFH C-terminus is extremely stable, and hardly undergoes any changes in its conformation in the presence of other ligands. Cumulatively, the structures of the CFH in presence of its different partners of interactions contributed greatly to the knowledge pool of the understanding of the molecular mechanisms associated with CFH in complement activation and regulation in health and disease.
Subject: immunology and Biophysics
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