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• W3085403609 abstract "The human complement Factor H–related 5 protein (FHR5) antagonizes the main circulating complement regulator Factor H, resulting in the deregulation of complement activation. FHR5 normally contains nine short complement regulator (SCR) domains, but a FHR5 mutant has been identified with a duplicated N-terminal SCR-1/2 domain pair that causes CFHR5 nephropathy. To understand how this duplication causes disease, we characterized the solution structure of native FHR5 by analytical ultracentrifugation and small-angle X-ray scattering. Sedimentation velocity and X-ray scattering indicated that FHR5 was dimeric, with a radius of gyration (Rg) of 5.5 ± 0.2 nm and a maximum protein length of 20 nm for its 18 domains. This result indicated that FHR5 was even more compact than the main regulator Factor H, which showed an overall length of 26–29 nm for its 20 SCR domains. Atomistic modeling for FHR5 generated a library of 250,000 physically realistic trial arrangements of SCR domains for scattering curve fits. Only compact domain structures in this library fit well to the scattering data, and these structures readily accommodated the extra SCR-1/2 domain pair present in CFHR5 nephropathy. This model indicated that mutant FHR5 can form oligomers that possess additional binding sites for C3b in FHR5. We conclude that the deregulation of complement regulation by the FHR5 mutant can be rationalized by the enhanced binding of FHR5 oligomers to C3b deposited on host cell surfaces. Our FHR5 structures thus explained key features of the mechanism and pathology of CFHR5 nephropathy. The human complement Factor H–related 5 protein (FHR5) antagonizes the main circulating complement regulator Factor H, resulting in the deregulation of complement activation. FHR5 normally contains nine short complement regulator (SCR) domains, but a FHR5 mutant has been identified with a duplicated N-terminal SCR-1/2 domain pair that causes CFHR5 nephropathy. To understand how this duplication causes disease, we characterized the solution structure of native FHR5 by analytical ultracentrifugation and small-angle X-ray scattering. Sedimentation velocity and X-ray scattering indicated that FHR5 was dimeric, with a radius of gyration (Rg) of 5.5 ± 0.2 nm and a maximum protein length of 20 nm for its 18 domains. This result indicated that FHR5 was even more compact than the main regulator Factor H, which showed an overall length of 26–29 nm for its 20 SCR domains. Atomistic modeling for FHR5 generated a library of 250,000 physically realistic trial arrangements of SCR domains for scattering curve fits. Only compact domain structures in this library fit well to the scattering data, and these structures readily accommodated the extra SCR-1/2 domain pair present in CFHR5 nephropathy. This model indicated that mutant FHR5 can form oligomers that possess additional binding sites for C3b in FHR5. We conclude that the deregulation of complement regulation by the FHR5 mutant can be rationalized by the enhanced binding of FHR5 oligomers to C3b deposited on host cell surfaces. Our FHR5 structures thus explained key features of the mechanism and pathology of CFHR5 nephropathy. Complement activation and regulation is of major importance in enabling clearance of pathogens, while preventing complement-mediated host cell damage. Complement factor H–related 5 protein (FHR5) was first identified co-localized with C3 in glomerular immune deposits from patients with glomerulonephritis and is a member of a family of structurally related proteins comprising the major serum complement regulator Factor H and five complement Factor H–related proteins. Factor H, comprising 20 short complement regulator (SCR) domains, has been well characterized, both in terms of its structure and function, binding to activated C3b and its fragment C3d and regulating excess C3 activation (1Murphy B. Georgiou T. Machet D. Hill P. McRae J. Factor H-related protein-5: a novel component of human glomerular immune deposits.Am. J. Kidney Dis. 2002; 39 (11774097): 24-2710.1053/ajkd.2002.29873Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). However, the principal physiological function of FHR5 is poorly understood. FHR5 circulates in plasma in extremely low concentrations of 3–6 μg/ml (2McRae J.L. Duthy T.G. Griggs K.M. Ormsby R.J. Cowan P.J. Cromer B.A. McKinstry W.J. Parker M.W. Murphy B.F. Gordon D.L. Human factor H-related protein 5 has cofactor activity, inhibits C3 convertase activity, binds heparin and C-reactive protein, and associates with lipoprotein.J. Immunol. 2005; 174 (15879123): 6250-625610.4049/jimmunol.174.10.6250Crossref PubMed Scopus (111) Google Scholar), which is ∼100-fold lower than Factor H. It is also the least abundant of the FHR proteins, yet its structure is the longest of these proteins, with a linear sequence of nine SCR domains (Fig. 1). The SCR domain (3Soares D. Barlow P.N. Complement control protein modules in the regulators of complement activators.in: Morikis D. Lambris J.D. Structural Biology of the Complement System. Taylor & Francis, Boca Raton, FL2005: 19-62Crossref Google Scholar) is the major domain type found in the complement regulators. An SCR domain is characterized by a consensus sequence of ∼61 amino acids, with four invariant cysteine residues that form two disulfide bridges (I–III and II–IV) and a conserved tryptophan residue. It folds compactly, with a hydrophobic core, in a β-sandwich arrangement of six hydrogen-bonded β-strands. The key C-terminal C3b/C3d recognition sites are conserved between SCR-19/20 of Factor H and SCR-8/9 of FHR5 (Fig. 1). FHR5 also interacts with heparin (2McRae J.L. Duthy T.G. Griggs K.M. Ormsby R.J. Cowan P.J. Cromer B.A. McKinstry W.J. Parker M.W. Murphy B.F. Gordon D.L. Human factor H-related protein 5 has cofactor activity, inhibits C3 convertase activity, binds heparin and C-reactive protein, and associates with lipoprotein.J. Immunol. 2005; 174 (15879123): 6250-625610.4049/jimmunol.174.10.6250Crossref PubMed Scopus (111) Google Scholar); however, FHR5 has no complement regulatory domains equivalent to SCR-1/4 of Factor H. FHR5 forms native homodimers via its two N-terminal domains SCR-1/2 that exhibit increased avidity for C3b/C3d compared with the monovalent Factor H, and, although early studies using supraphysiological concentrations of FHR5 showed evidence of weak (compared with Factor H) complement regulating activity (2McRae J.L. Duthy T.G. Griggs K.M. Ormsby R.J. Cowan P.J. Cromer B.A. McKinstry W.J. Parker M.W. Murphy B.F. Gordon D.L. Human factor H-related protein 5 has cofactor activity, inhibits C3 convertase activity, binds heparin and C-reactive protein, and associates with lipoprotein.J. Immunol. 2005; 174 (15879123): 6250-625610.4049/jimmunol.174.10.6250Crossref PubMed Scopus (111) Google Scholar), more recent work has shown that, at physiological concentrations, FHR5 competitively antagonizes Factor H, thus deregulating complement (4Goicoechea de Jorge E. Caesar J.J. Malik T.H. Patel M. Colledge M. Johnson S. Hakobyan S. Morgan B.P. Harris C.L. Pickering M.C. Lea S.M. Dimerization of complement factor H-related proteins modulates complement activation in vivo.Proc. Natl. Acad. Sci. U. S. A. 2013; 110 (23487775): 4685-469010.1073/pnas.1219260110Crossref PubMed Scopus (177) Google Scholar, 5Jokiranta T.S. Jaakola V.P. Lehtinen M.J. Pärepalo M. Meri S. Goldman A. Structure of complement factor H carboxyl-terminus reveals molecular basis of atypical haemolytic uremic syndrome.EMBO J. 2006; 25 (16601698): 1784-179410.1038/sj.emboj.7601052Crossref PubMed Scopus (129) Google Scholar). Conflicting data exist on whether FHR5 forms heterodimers with other FHRs in vivo (6van Beek A.E. Pouw R.B. Brouwer M.C. van Mierlo G. Rispens T. Kuijpers T.W. Wouters D. Dimerization of complement factor H-related (FHR) proteins: FHR-5 forms homodimers whereas FHR-1 and FHR-2 both homodimerize and heterodimerize with each other.Immunobiology. 2016; 221: 1182-118310.1016/j.imbio.2016.06.130Crossref Google Scholar, 7Ruseva M.M. Malik T.H. Pickering M.C. Insights into the role of FHR5 in C3 glomerulopathy.Immunobiology. 2016; 221: 116710.1016/j.imbio.2016.06.098Crossref Google Scholar). CFHR5 nephropathy, a monogenic cause of kidney failure endemic in Cypriots (individuals residing in or with ancestry from the island of Cyprus), is characterized in almost all affected individuals by persistent microscopic hematuria and, in a proportion of patients, episodes of kidney damage and visible blood in the urine that occur at times of otherwise trivial mucosal infections, with repeated episodes typically resulting in progressive kidney damage and eventually end-stage kidney failure occurring in >80% of affected males and <20% of affected females by the age of 55 years. Kidney biopsy shows predominantly mesangial-based glomerular inflammation with deposition of C3 but not Igs in the mesangium and, under electron microscopic examination, the subendothelial part of the glomerular basement membrane—appearances termed C3 glomerulopathy that suggest defective regulation of the complement system. The disease is a highly penetrant autosomal dominant disorder that is caused by heterozygosity for an in-frame duplication of exons 2 and 3 of the CFHR5 gene that results in production of an elongated FHR5 protein with an extra two N-terminal SCR-1/2 domains in tandem. No extrarenal features of the disease have been reported, despite the review of clinical data from over 100 affected individuals of all ages (8Gale D.P. Goicoechea de Jorge E. Cook H.T. Martinez-Barricarte R. Hadjisavvas A. McLean A.G. Pusey C.D. Pierides A. Kyriacou K. Athanasiou Y. Voskarides K. Deltas C. Palmer A. Frémeaux-Bacchi V. de Cordoba S.R. et al.Identification of a mutation in complement factor H-related protein 5 in patients of Cypriot origin with glomerulonephritis.Lancet. 2010; 376 (20800271): 794-80110.1016/S0140-6736(10)60670-8Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 9Athanasiou Y. Voskarides K. Gale D.P. Damianou L. Patsias C. Zavros M. Maxwell P.H. Cook H.T. Demosthenous P. Hadjisavvas A. Kyriacou K. Zouvani I. Pierides A. Deltas C. Familial C3 glomerulopathy associated with CFHR5 mutations: clinical characteristics of 91 patients in 16 pedigrees.Clin. J. Am. Soc. Nephrol. 2011; 6 (21566112): 1436-144610.2215/CJN.09541010Crossref PubMed Scopus (104) Google Scholar). The molecular mechanisms that make the kidney susceptible to complement-mediated damage in CFHR5 nephropathy and other common causes of glomerulonephritis (e.g. lupus nephritis and IgA nephropathy, in which flares of disease triggered by mucosal infections also occur) are not well-understood. Protein structural studies of full-length FHR5 are complicated by its large size and its eight potentially flexible interdomain linkers of lengths between three and eight residues (Fig. 1), both of which make it difficult to crystallize to determine its three-dimensional appearance. To date, atomic-level structures have not been determined for any small FHR5 fragments. However, alternative methods can be used for structural studies. Previously for full-length factor H, EM, small-angle X-ray scattering (SAXS), analytical ultracentrifugation (AUC), and molecular modeling showed that full-length factor H has a partially folded-back structure that is relevant to its regulatory function (10DiScipio R.G. Ultrastructures and interactions of complement factors H and I.J. Immunol. 1992; 149 (1401896): 2592-2599PubMed Google Scholar, 11Okemefuna A.I. Nan R. Gor J. Perkins S.J. Electrostatic interactions contribute to the folded-back conformation of wild type human factor H.J. Mol. Biol. 2009; 391 (19505476): 98-11810.1016/j.jmb.2009.06.010Crossref PubMed Scopus (51) Google Scholar, 12Osborne A.J. Nan R. Miller A. Bhatt J.S. Gor J. Perkins S.J. Two distinct conformations of factor H regulate discrete complement-binding functions in the fluid phase and at cell surfaces.J. Biol. Chem. 2018; 293 (30217822): 17166-1718710.1074/jbc.RA118.004767Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). This combination of analytical ultracentrifugation, X-ray solution scattering, and atomistic modeling has been effective in determining many macromolecular structures in solution (13Perkins S.J. Nan R. Li K. Khan S. Abe Y. Analytical ultracentrifugation combined with X-ray and neutron scattering: experiment and modelling.Methods. 2011; 54 (21256219): 181-19910.1016/j.ymeth.2011.01.004Crossref PubMed Scopus (29) Google Scholar, 14Perkins S.J. Wright D.W. Zhang H. Brookes E.H. Chen J. Irving T.C. Krueger S. Barlow D.J. Edler K.J. Scott D.J. Terrill N.J. King S.M. Butler P.D. Curtis J.E. Atomistic modelling of scattering data in the Collaborative Computational Project for Small Angle Scattering (CCP-SAS).J. Appl. Crystallogr. 2016; 49 (27980506): 1861-187510.1107/S160057671601517XCrossref PubMed Scopus (42) Google Scholar, 15Perkins S.J. Nan R.D. Li K.Y. Khan S. Miller A. Complement factor H-ligand interactions: self-association, multivalency and dissociation constants.Immunobiology. 2012; 217 (22137027): 281-29710.1016/j.imbio.2011.10.003Crossref PubMed Scopus (58) Google Scholar). Many of the first structural explanations for factor H–associated diseases, such as atypical hemolytic uremic syndrome, were based on homology models for the SCR domains (16Perkins S.J. Goodship T.H. Molecular modelling of the C-terminal domains of factor H of human complement: a correlation between haemolytic uraemic syndrome and a predicted heparin binding site.J. Mol. Biol. 2002; 316 (11851332): 217-22410.1006/jmbi.2001.5337Crossref PubMed Scopus (59) Google Scholar, 17Saunders R.E. Abarrategui-Garrido C. Frémeaux-Bacchi V. Goicoechea de Jorge E. Goodship T.H.J. López Trascasa M. Noris M. Ponce Castro I.M. Remuzzi G. Rodríguez de Córdoba S. Sánchez-Corral P. Skerka C. Zipfel P.F. Perkins S.J. The interactive Factor H—atypical haemolytic uraemic syndrome mutation database and website: update and integration of membrane cofactor protein and Factor I mutations with structural models.Hum. Mutat. 2007; 28 (17089378): 222-23410.1002/humu.20435Crossref PubMed Scopus (138) Google Scholar, 18Rodriguez E. Rallapalli P.M. Osborne A.J. Perkins S.J. New functional and structural insights from updated mutational databases for complement factor H, factor I, membrane cofactor protein and C3.Biosci. Rep. 2014; 34: 635-649Crossref Scopus (56) Google Scholar). Here, these solution structural and modeling approaches were applied to determine the solution conformation of full-length FHR5 to explain its role in healthy individuals and how CFHR5 nephropathy may arise through the SCR-1/2 duplication. Following SAXS and AUC data collection, full-length FHR5 was modeled using molecular dynamics, followed by Monte Carlo simulations to generate a large library of physically realistic trial atomistic structures for the FHR5 dimer (14Perkins S.J. Wright D.W. Zhang H. Brookes E.H. Chen J. Irving T.C. Krueger S. Barlow D.J. Edler K.J. Scott D.J. Terrill N.J. King S.M. Butler P.D. Curtis J.E. Atomistic modelling of scattering data in the Collaborative Computational Project for Small Angle Scattering (CCP-SAS).J. Appl. Crystallogr. 2016; 49 (27980506): 1861-187510.1107/S160057671601517XCrossref PubMed Scopus (42) Google Scholar, 19Curtis J.E. Raghunandan S. Nanda H. Krueger S. SASSIE: A program to study intrinsically disordered biological molecules and macromolecular ensembles using experimental scattering restraints.Comput. Phys. Commun. 2012; 183: 382-38910.1016/j.cpc.2011.09.010Crossref Scopus (85) Google Scholar). The theoretical scattering profiles of this library were compared with the experimental SAXS curves to determine best-fit FHR5 structures. We thus defined a small subset of compact folded-back solution structures. The extra SCR-1/2 domain pair in mutant FHR5 was readily added to these structures, their presence leading to the formation of multivalent oligomers of FHR5. Our work explains how FHR5 regulates complement activation in the kidney and how CFHR5 nephropathy arises. Human FHR5 SCR-1/9 purchased from Creative Biolabs was subjected to gel-filtration chromatography to ensure monodispersity and removal of aggregates prior to SAXS experiments. The protein eluted as a single symmetrical peak at ∼15-ml elution volume (Fig. 2A). This was preceded by a broader peak that was eluted between 10 and 14 ml, which was attributed to protein aggregates. Only the protein fractions between 14.3 and 16.3 ml (red in Fig. 2A) were retained. By SDS-PAGE (Fig. 2B), a single band was seen at 60–66 kDa (nonreduced) that corresponds well to the expected monomer molecular mass of 62.4 kDa. Reducing conditions resulted in another single band but at a slightly lower mass, this difference being attributed to the presence of glycan chains on FHR5. SEC-MALLS was used to determine the mass and self-association of FHR5 in our Tris-150 purification buffer, as in previous work (4Goicoechea de Jorge E. Caesar J.J. Malik T.H. Patel M. Colledge M. Johnson S. Hakobyan S. Morgan B.P. Harris C.L. Pickering M.C. Lea S.M. Dimerization of complement factor H-related proteins modulates complement activation in vivo.Proc. Natl. Acad. Sci. U. S. A. 2013; 110 (23487775): 4685-469010.1073/pnas.1219260110Crossref PubMed Scopus (177) Google Scholar). FHR5 from a size-exclusion column was detected by UV (blue, Fig. 3) and refractive index (green) measurements, in parallel with multi-angle light scattering (red) to analyze size distributions. Three peaks were observed in the elution profile. Peak 1 at 2.7–4.2 min was assigned as aggregated material, because this had a lower UV and refractive index, but high light scattering intensities that indicated very large sizes. Its molecular mass was calculated to be above 5,400 kDa. Peak 2 at 4.9–5.2 min was the FHR5 dimer that eluted with higher UV and refractive index values but with lower light scattering. Its molecular mass was estimated as 162 kDa, this being consistent with FHR5 dimer formation, given that the mass of the monomer was 62.4 kDa from its composition (20Perkins S.J. Protein volumes and hydration effects: the calculation of partial specific volumes, neutron scattering matchpoints and 280-nm coefficients for proteins and glycoproteins from amino acid sequences.Eur. J. Biochem. 1986; 157 (3709531): 169-18010.1111/j.1432-1033.1986.tb09653.xCrossref PubMed Scopus (528) Google Scholar). Despite a large inherent error associated with light scattering, no evidence of an FHR5 monomer peak was detectable. A small peak 3 at 7.6–7.9 min was assigned to fragments below 30 kDa. AUC sedimentation velocity experiments on FHR5 studied its oligomerization and shape using size distribution c(s) analyses to determine its molecular mass and sedimentation coefficient s20,w. Absorbance data for FHR5 at 0.16 mg/ml in PBS were collected for five different salt concentrations between 20 and 250 mm NaCl. SEDFIT analyses involved as many as 500 absorbance scans. The experimental sedimentation boundaries (left, Fig. 4) gave good fits to the Lamm equation to give the size-distribution c(s) profiles (right, Fig. 4), despite the low concentrations in use. These fits were obtained by floating the meniscus, the bottom of the cell, the baseline, and the frictional ratio f/f0 of around 1.5. Protein aggregation was visible in the earliest boundaries that sedimented rapidly at the start of the runs, to leave behind the FHR5 dimer that sedimented more slowly (Fig. 4). This agreed with SEC-MALLS. A major c(s) peak at 6.0 S was observed for FHR5 in PBS-137 that corresponded to an average molecular mass of 134 kDa. This mass confirmed the presence of dimer in solution. The aggregates made little contribution to the c(s) analyses between 3 and 12 S, even though they contributed as much as half of the protein present. The molecular masses for the five buffers were between 133 and 139 kDa (Table 1), showing that the FHR5 dimer was stable between 20 and 350 mm NaCl. The c(s) analyses did not reveal any FHR5 monomer at lower s values. The reproducibility of these data was tested at two different rotor speeds of 40,000 and 50,000 rpm, to show no difference.Table 1Sedimentation velocity data for FHR5 SCR-1/9SampleaAll samples were measured at 0.16 mg/ml. The values represent the mean ± S.D. The data presented include those collected both at 40,000 and 50,000 rpm.s20,wMolecular massFrictional ratio (f/f0)SkDaFHR5 in PBS-206.48 ± 0.05139 ± 21.49 ± 0.0003FHR5 in PBS-506.29 ± 0.1133 ± 41.48 ± 0.02FHR5 in PBS-906.19 ± 0.1134 ± 61.53 ± 0.05FHR5 in PBS-1375.97 ± 0.2134 ± 41.59 ± 0.03FHR5 in PBS-2505.91 ± 0.02136 ± 81.58 ± 0.02FHR5 in PBS-3505.35bOnly a single run was carried out for FHR5 SCR-1/9 in PBS-350.1381.80a All samples were measured at 0.16 mg/ml. The values represent the mean ± S.D. The data presented include those collected both at 40,000 and 50,000 rpm.b Only a single run was carried out for FHR5 SCR-1/9 in PBS-350. Open table in a new tab The solution structure of FHR5 between 20 and 350 mm NaCl was monitored using the mean s20,w values (Table 1). A significant decrease of 0.9 S from 6.48 to 5.35 S was seen on going from 20 mm NaCl to 350 mm NaCl. This shift in the FHR5 dimer peak was visible in the c(s) distribution plots (vertical dashed lines, Fig. 4). This result indicated a conformational change in FHR5, where the smaller s20,w values at high NaCl concentration indicated a more elongated FHR5 domain structure that formed as the ionic strength was increased (Fig. 5). SAXS was used to study the solution structure of the FHR5 dimer in concentration series in three different buffers, two being physiological (PBS-137 and Tris-150) and one being low-salt (PBS-50). The FHR5 samples were purified by gel filtration (Fig. 2). In Tris-150, data were collected using 0.04–0.5 mg/ml FHR5. In PBS-137 and PBS-50, data were collected using 0.04–0.17 mg/ml FHR5. Guinier analyses of the solution structure gave high-quality linear plots in two distinct regions of the I(Q) curves that corresponded to the radius of gyration (Rg) and the cross-sectional radius of gyration (Rxs) from two distinct Q ranges (Fig. 6). These values are measures of the overall and the shorter dimensions of macromolecular elongation, respectively. Their values were deduced according to Equations 1 and 2, respectively, within satisfactory Q.Rg and Q.Rxs limits close to 1.0 as follows. (i) In the overall structural Guinier Rg analyses in a low Q range of 0.1–0.27 nm−1 (Fig. 7A), in Tris-150 and PBS-137 buffers with similar NaCl concentrations, the mean Rg values were 5.36 ± 0.14 and 5.48 ± 0.17 nm, respectively. However, in the PBS-50 buffer with lower NaCl, the mean Rg value increased slightly to 5.91 ± 0.13 nm. This increase was attributed to trace aggregation in FHR5 that affected the lowest Q values (Fig. 7A). No concentration dependence was observed for the Rg values between 0.04 and 0.17 mg/ml; however, a slightly increased Rg value of up to 0.2 nm was seen at 0.2–0.5 mg/ml FHR5. (ii) In the cross-sectional Guinier Rxs analyses, using a Q range of 0.32–0.55 nm−1 (Fig. 7B), the mean Rxs values in each buffer were 2.41 ± 0.06, 2.29 ± 0.09, and 2.46 ± 0.14 nm for Tris-150, PBS-137, and PBS-50, respectively (Table 2). No significant changes in the Rxs values were seen between the data sets for these NaCl and protein concentrations, indicating that the cross-sectional structure of FHR5 was unchanged in conformation.Table 2X-ray scattering data for FHR5 SCR-1/9SampleRgaThe first Rg value is from Guinier analyses, and the second one is from the P(r) analyses.RgaThe first Rg value is from Guinier analyses, and the second one is from the P(r) analyses.RxsLMnm150 mm NaCl (Tris) 0.5 mg/ml5.525.852.4521.04.68 0.4 mg/ml5.565.922.5121.04.68 0.3 mg/ml5.56 ± 0.075.902.49 ± 0.0521.04.52 0.2 mg/ml5.635.942.3820.04.92 0.17 mg/ml5.28 ± 0.135.722.38 ± 0.0220.04.75 0.13 mg/ml5.40 ± 0.145.722.27 ± 0.0819.04.98 0.09 mg/ml5.37 ± 0.175.622.43 ± 0.1119.54.99 0.04 mg/ml5.30 ± 0.215.462.35 ± 0.2019.55.60137 mm NaCl (PBS) 0.17 mg/ml5.55 ± 0.055.672.28 ± 0.0621.04.98 0.13 mg/ml5.355.802.43 ± 0.0521.05.00 0.09 mg/ml5.61 ± 0.115.852.21 ± 0.1121.04.87 0.04 mg/ml5.635.882.7421.04.7550 mm NaCl (PBS) 0.17 mg/ml5.96 ± 0.096.212.41 ± 0.0420.05.25 0.13 mg/ml5.82 ± 0.146.112.42 ± 0.1120.05.12 0.09 mg/ml5.84 ± 0.056.262.37 ± 0.0819.05.38 0.04 mg/ml6.01 ± 0.276.332.63 ± 0.3319.55.77a The first Rg value is from Guinier analyses, and the second one is from the P(r) analyses. Open table in a new tab The distance distribution function P(r) in real space represents all of the distances between pairs of atoms in FHR5. This was calculated from Fourier transformation of the full I(Q) scattering curve following the specification of the maximum dimension Dmax (Equation 3; Fig. 8). The P(r) curve provided an independent Rg value for FHR5 for comparison with the Guinier value (Table 2). The Rg values from the P(r) analyses were in good agreement with those from the Guinier analyses (Table 2). The P(r) curve also gave the maximum length L of FHR5 from the value of r when P(r) = 0. The mean L values were 19.5 ± 0.4 nm in Tris-150 (Fig. 8C), 19.6 ± 0.5 nm in PBS-137 (Fig. 8B), and 21.0 nm in PBS-50 (Fig. 8A). The L value for PBS-50 was slightly higher than those in Tris-150 and PBS-137, most likely due to trace aggregation that resulted from the lower ionic strength used (see above). A single maximum M was observed in all of the P(r) curves. This corresponded to the most frequent interatomic distance within the FHR5 structure (Table 2). The mean M values were 4.9 ± 0.3, 4.9 ± 0.1, and 5.4 ± 0.3 nm for Tris-150, PBS-137, and PBS-50, respectively. The M values were relatively stable, although slightly higher for PBS-50 as the result of trace aggregates. Currently, there is no atomic level structural information on FHR5. To determine an atomistic-level solution structure for the FHR5 dimer, a starting model for the monomer was required. This was created by comparative modeling based on four known SCR crystal structures as structural templates (Fig. 1, B and C). Two used related crystal structures of the N-terminal FHR1 SCR-1/2 domains and the C-terminal FHR2 SCR-3/4 domains with high sequence identities of 85.2 and 61.7%, respectively, with SCR-1/2 and SCR-8/9 of FHR5. The SCR-3/7 domains of FHR5 share significant sequence similarities with the SCR-10/14 domains of Factor H. Although templates for individual SCR3/7 domains in FHR5 were searched for in PDB-BLAST, the best choices were these domain structures from Factor H due to their direct sequence similarities (Fig. 1C). FHR5 SCR-3/4 was represented by Factor H SCR-10/11 with a high sequence identity of 57.4%. FHR5 SCR-5/6 was represented by Factor H SCR-12/13, also with a high sequence identity of 53.9%. Although FHR5 SCR-7 is similar to Factor H SCR-14, no structure existed for Factor H SCR-14. Searches showed that the best template structure for FHR5 SCR-7 was that of SCR-11 of Factor H with a sequence identity of 34.5%. The individual template-target sequence alignments (Fig. 1C) showed no significant indels in the structure, because the numbers of residues in these were well-aligned. Thus, the FHR5 SCR-7 and SCR-8/9 sequences had only one gap inserted in each. The individual modeled domains satisfied validation checks using PROCHECK, where the Ramachandran plots showed that 70% of the residues were in the most favored steric regions. The FHR5 dimer was generated from its monomer structure by aligning its SCR-1/2 domains with the crystal structure of the FHR1 SCR-1/2 dimer (see “Experimental procedures”), followed by energy minimization to relax this starting structure. Atomistic modeling of the FHR5 scattering data established the best-fit FHR5 dimer structures, hence providing a molecular explanation for its solution structure. The scattering curves for 0.17 and 0.5 mg/ml FHR5 in Tris-150 were used to assess good quality curves with no traces of aggregation and better signal/noise ratios at 0.5 mg/ml (Fig. 9). Data for 0.5 mg/ml were not available in PBS-137 or PBS-50, and traces of aggregates were present in PBS-50 buffer; thus, these data sets were not used. The starting structure for the FHR5 dimer represented an extended conformation of the 18 SCR domains (Fig. 9). Each SCR domain was held fixed in conformation. Because as many as 14 linkers between the 18 domains were potentially variable, three different Monte Carlo conformational searches were set up. As detailed in Table 3, these varied all 14 linkers (Search 1) or eight linkers in which the crystal structure–observed linkers were kept fixed (Search 2) or four linkers after every third SCR domain (Search 3) (Fig. 1B) (see “Experimental procedures”). Initial Monte Carlo conformational simulations in Searches 1–3 gave many models that were too elongated with too large Rg values and few models with low Rg values close to the experimental Rg value of ∼5.5 nm. Thus, in further simulations, models were selected with Rg values closer to the experimental Rg value to generate further conformers, but now using an Rg cut-off of 6.0 nm as a constraint to generate more compact FHR5 dimers. This resulted in more structures with lower Rg values; however, many of these models were rejected by the workflow because the more compact shapes gave rise to physically disallowed steric clashes between the SCR domains.Table 3Three modeling fit searches for FHR5 using X-ray scattering curve fits and sedimentation coefficientsSearchFilterModelsaTotal number of models accepted after Monte Carlo simulations and after model filtering. The best fit model corresponds to that with the lowest R-factor in the filtered models.RgbThe first Rg value of the pair is from Gu" @default.
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• W3085403609 title "The solution structure of the complement deregulator FHR5 reveals a compact dimer and provides new insights into CFHR5 nephropathy" @default.
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• W3085403609 doi "https://doi.org/10.1074/jbc.ra120.015132" @default.
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