Mapping the Complement Factor H-Related Protein 1 (CFHR1):C3b/C3d Interactions

<div><p>Complement factor H-related protein 1 (CFHR1) is a complement regulator which has been reported to regulate complement by blocking C5 convertase activity and interfering with C5b surface association. CFHR1 also competes with complement factor H (CFH) for binding to C3b, and may act as an antagonist of CFH-directed regulation on cell surfaces. We have employed site-directed mutagenesis in conjunction with ELISA-based and functional assays to isolate the binding interaction that CFHR1 undertakes with complement components C3b and C3d to a single shared interface. The C3b/C3d:CFHR1 interface is identical to that which occurs between the two C-terminal domains (SCR19-20) of CFH and C3b. Moreover, we have been able to corroborate that dimerization of CFHR1 is necessary for this molecule to bind effectively to C3b and C3d, or compete with CFH. Finally, we have established that CFHR1 competes with complement factor H-like protein 1 (CFHL-1) for binding to C3b. CFHL-1 is a <i>CFH</i> gene splice variant, which is almost identical to the N-terminal 7 domains of CFH (SCR1-7). CFHR1, therefore, not only competes with the C-terminus of CFH for binding to C3b, but also sterically blocks the interaction that the N-terminus of CFH undertakes with C3b, and which is required for CFH-regulation.</p></div

C3b TED/C3d-binding sites on CFH SCR19-20 and their conservation within the CFHR proteins.

<p><b>(A)</b> Ribbon representations of CFH SCR19-20 in complex with C3d (corresponding to the C3b TED) and a separate C3d interaction. Shown is a superposition of the available structures of wild-type CFH SCR19-20 in complex with C3d and a D1119G/Q1139A double mutant form of CFH SCR19-20 in complex with C3d (PDB ID codes: 3OXU and 2XQW, respectively) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166200#pone.0166200.ref006" target="_blank">6</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166200#pone.0166200.ref009" target="_blank">9</a>]. Wild-type CFH SCR19-20 is represented in magenta, and the C3d molecule most likely to approximate to that of the C3b TED is shown in red (both taken from 3OXU). A second proposed interaction with C3d is also shown, with the C3d molecule in this case being colored cyan (taken from 2XQW). For the purposes of this representation, the structure of D1119G/Q1139A CFH SCR19-20 is not shown, nor is a similar but not identical second C3d interaction observed in the 3OXU structure. Gln1013, which is the point of covalent attachment of C3 moieties to activating surfaces, is indicated in orange for both C3d molecules. All structural representations have been generated using the PyMOL Molecular Graphics System, Version 1.7.0.1 (Schrödinger, LLC). <b>(B)</b> Sequence alignment of the two C-terminal SCR domains of CFH with the corresponding two C-terminal SCR modules of each of the CFHR proteins (CFHR1-5). Indicated within red boxes are those residues of CFH which are involved in interface-formation with a C3d molecule which can be approximated to the C3b TED. Red boxes which are shaded in tan indicate those residues of CFH SCR19-20 which contribute side-chains to intermolecular hydrogen bonds [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166200#pone.0166200.ref009" target="_blank">9</a>]. Indicated in blue boxes are those residues which contribute to the interface of an additional proposed physiologically important interaction that CFH SCR20 undertakes with a separate C3d molecule. Blue boxes which are shaded in tan indicate those residues of CFH SCR20 which contribute side-chains to intermolecular hydrogen bonds and/or salt-bridges [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166200#pone.0166200.ref006" target="_blank">6</a>].</p

Mutagenesis data targeting the C3b TED and C3d interfaces.

<p><b>(A)</b> Close-up of the CFH SCR19-20:C3d/C3b TED interface generated from the wild-type CFH SCR19-20:C3d crystal structure [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166200#pone.0166200.ref009" target="_blank">9</a>]. The complex is in the same orientation as that represented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166200#pone.0166200.g002" target="_blank">Fig 2</a>. CFH SCR19-20 and C3d are colored magenta and red, respectively. Intermolecular hydrogen bonds are indicated by black dashed lines. This structure was used to direct alanine substitutions in CFHR1 targeting residues Asn216 and Asp218, corresponding to Asn1117 and Asp1119 in CFH (CFHR1 Mut1: N216A/D218A) and also residues Gln238 and Tyr241, corresponding to Gln1139 and Tyr1142 in CFH (CFHR1 Mut2: Q238A/Y241A). Labels for CFH residues are shown in yellow boxes, and labels for C3d residues are shown in grey boxes. For clarity, residues within CFHR1 and C3d which were selected for mutagenesis studies are delineated by orange boxes. <b>(B)</b> Close-up of a second possible CFH SCR20:C3d interaction site generated from the CFH SCR19-20 D1119G/Q1139A:C3d crystal structure [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166200#pone.0166200.ref006" target="_blank">6</a>]. In this case, the additional C3d moiety is colored cyan, with labels for residues from this molecule highlighted in green boxes. Again, residues which were targeted for mutagenesis are highlighted within orange boxes. The complex has been rotated by 20 degrees in the y-axis to that represented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166200#pone.0166200.g002" target="_blank">Fig 2</a>. Intermolecular hydrogen bonds and salt-bridges are indicated by black dashed lines. <b>(C)</b> and <b>(D)</b> The abilities of wild-type CFHR1 and mutant CFHR proteins to bind to plate-bound recombinant C3d and serum-derived C3b, respectively. Obtained OD₄₀₅ readings were normalized to values obtained for wild-type CFHR1 binding at a concentration of 1.04 μM to C3d or C3b. <b>(E)</b> The ability of wild-type and mutant forms of CFHR1 to compete with CFH-binding to plate-bound serum-derived C3b are shown. CFH-binding is inhibited in the presence of wild-type CFHR1 in a dose dependent manner. However, in the presence of the CFHR1 Mut1 and CFHR1 Mut2 proteins, no inhibition of CFH-binding to C3b can be observed. OD values at 405 nm were expressed as a percentage of CFH binding where CFH incubated in the absence of wild-type or mutant CFHR1 proteins was considered 100%.</p

Domain composition of the <i>CFH</i> gene family.

<p>CFH comprises 20 SCR modules, and the <i>CFH</i> gene splice variant, CFHL-1, encompasses 7 SCR modules. CFHL-1 is invariant from the N-terminal seven SCRs of CFH apart from the C-terminal four residues (indicated). CFHR1-5 range from 4 to 9 SCRs. The SCR domains of each CFHR protein are aligned with the most homologous domains of CFH. C3b/C3d and GAG/sialic-binding regions are indicated. Two forms of CFHR4 are found in circulation, a long 9 SCR form of the protein, CFHR4a, and a shorter 5 SCR form, CFHR4b.</p

Mapping the Complement Factor H-Related Protein 1 (CFHR1):C3b/C3d Interactions - Fig 4

<p><b>(A)</b> Capacity of wild-type and mutant forms of CFHR1 to inhibit CFH-mediated protection of guinea pig erythrocytes from complement-driven cell lysis. CFH-binding to C3b deposited on guinea pig erythrocytes, with concomitant protection from hemolysis, is reduced by the presence of wild-type CFHR1. However, CFH-mediated protection of erythrocytes is maintained in the presence of CFHR1 Mut1 and Mut2 proteins, and to a lesser extent in the presence of CFHR Mut3. <b>(B)</b> Summary of wild-type CFHR1 binding to mutant forms of C3d targeting both the C3b TED-binding site (indicated within a red box) and the second possible C3d-binding site (indicated within a blue box). Given are percentage values of CFHR1-binding to plate-bound mutant C3d relative to that with wild-type C3d at a concentration of 1.04 μM.</p

CFHR1 blocks other CFH-interaction sites on C3b.

<p><b>(A)</b> The capacity of CFHR1 to compete with CFHL-1-binding to plate-bound C3b is shown. OD values at 405 nm were expressed as a percentage of CFHL-1 binding where CFHL-1 incubated in the absence of CFHR1 was calculated as 100%. <b>(B)</b> Heparin sepharose is able to pull-down CFHR1 (Left), but is unable to pull-down C3d on its own (Centre). However, when both CFHR1 and C3d are present, heparin-conjugated beads are able to pull-down both CFHR1 and C3d (Right). <b>(C)</b> Proposed mechanism of CFH regulation of C3b on a non-activating surface and CFHR1-driven deregulation of CFH on an activating surface. (Left) CFH engages C3b at two sites located within SCR1-4 and SCR19-20, while simultaneously binding to surface anionic markers at sites located within SCR6-8 and SCR20. SCR20 of CFH has also been reported to additionally bind to C3d. (Right) CFHR1 can also form a ternary complex by dually engaging C3d/C3b and GAGs. CFHR1 competes with CFH SCR19-20 for binding to the TED, and also sterically inhibits engagement of SCR1-7. CFHR1 is represented in its various characterized physiologic oligomeric states, but additionally forms hetero-dimers with CFHR2 and CFHR5 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166200#pone.0166200.ref022" target="_blank">22</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166200#pone.0166200.ref023" target="_blank">23</a>].</p

Population characteristics by high-risk autoantibody profile (HRP) phenotype in 257 serum/plasma samples from clinic visits of 144 FDRs from the studies of the Etiology of rheumatoid arthritis (SERA) cohort.

<p>Population characteristics by high-risk autoantibody profile (HRP) phenotype in 257 serum/plasma samples from clinic visits of 144 FDRs from the studies of the Etiology of rheumatoid arthritis (SERA) cohort.</p

Median (25th, 75th Percentile)^* levels of adiponectin and inflammatory markers by high-risk autoantibody profile (HRP) phenotype in 257 serum/plasma samples from clinic visits of 144 FDRs from the studies of the Etiology of rheumatoid arthritis (SERA) cohort.

<p>Median (25th, 75th Percentile)^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199578#t002fn001" target="_blank">*</a>} levels of adiponectin and inflammatory markers by high-risk autoantibody profile (HRP) phenotype in 257 serum/plasma samples from clinic visits of 144 FDRs from the studies of the Etiology of rheumatoid arthritis (SERA) cohort.</p

Modification of the association between adiponectin and inflammatory markers (A through H) by HRP status using linear mixed models in the studies of the Etiology of rheumatoid arthritis.

<p>This figure presents the interaction between adiponectin and High-Risk profile autoantibody (HRP) status in 257 serum and plasma samples from clinic visits of 144 first degree-relatives of RA patients in the Studies of the Etiology of Rheumatoid Arthritis. All analyses were adjusted for age, sex, ethnicity, BMI, pack-years of smoking, and current use of cholesterol-lowering medications.</p

Association between adiponectin and markers of inflammation in 257 serum/plasma samples from clinic visits of 144 FDRs from the studies of the Etiology of rheumatoid arthritis (SERA) cohort.

<p>Association between adiponectin and markers of inflammation in 257 serum/plasma samples from clinic visits of 144 FDRs from the studies of the Etiology of rheumatoid arthritis (SERA) cohort.</p