Disease-associated Sequence Variations Congregate in a Polyanion Recognition Patch on Human Factor H Revealed in Three-dimensional Structure
2006; Elsevier BV; Volume: 281; Issue: 24 Linguagem: Inglês
10.1074/jbc.m513611200
ISSN1083-351X
AutoresAndrew P. Herbert, Dušan Uhrı́n, Malcolm Lyon, Michael K. Pangburn, Paul N. Barlow,
Tópico(s)Blood groups and transfusion
ResumoMutations and polymorphisms in the regulator of complement activation, factor H, have been linked to atypical hemolytic uremic syndrome (aHUS), membranoproliferative glomerulonephritis, and age-related macular degeneration. Many aHUS patients carry mutations in the two C-terminal modules of factor H, which normally confer upon this abundant 155-kDa plasma glycoprotein its ability to selectively bind self-surfaces and prevent them from inappropriately triggering the complement cascade via the alternative pathway. In the current study, the three-dimensional solution structure of the C-terminal module pair of factor H has been determined. A binding site for a fully sulfated heparin-derived tetrasaccharide has been delineated using chemical shift mapping and the C3d/C3b-binding site inferred from sequence comparisons and computational docking. The resultant information allows assessment of the likely consequences of aHUS-associated amino acid substitutions in this critical region of factor H. It is striking that, excepting those likely to perturb the three-dimensional structure, aHUS-associated missense mutations congregate in the polyanion-binding site delineated in this study, thus potentially disrupting a vital mechanism for control of complement on self-surfaces in the microvasculature of the kidney. It is intriguing that a single nucleotide polymorphism predisposing to age-related macular degeneration occupies another region of factor H that harbors a polyanion-binding site. Mutations and polymorphisms in the regulator of complement activation, factor H, have been linked to atypical hemolytic uremic syndrome (aHUS), membranoproliferative glomerulonephritis, and age-related macular degeneration. Many aHUS patients carry mutations in the two C-terminal modules of factor H, which normally confer upon this abundant 155-kDa plasma glycoprotein its ability to selectively bind self-surfaces and prevent them from inappropriately triggering the complement cascade via the alternative pathway. In the current study, the three-dimensional solution structure of the C-terminal module pair of factor H has been determined. A binding site for a fully sulfated heparin-derived tetrasaccharide has been delineated using chemical shift mapping and the C3d/C3b-binding site inferred from sequence comparisons and computational docking. The resultant information allows assessment of the likely consequences of aHUS-associated amino acid substitutions in this critical region of factor H. It is striking that, excepting those likely to perturb the three-dimensional structure, aHUS-associated missense mutations congregate in the polyanion-binding site delineated in this study, thus potentially disrupting a vital mechanism for control of complement on self-surfaces in the microvasculature of the kidney. It is intriguing that a single nucleotide polymorphism predisposing to age-related macular degeneration occupies another region of factor H that harbors a polyanion-binding site. Inappropriate or disproportionate activation of the complement system underlies the debilitating symptoms of a long list of autoimmune, degenerative, and iatrogenic diseases (1Morgan B.P. Harris C.L. Mol. Immunol. 2003; 40: 159-170Crossref PubMed Scopus (149) Google Scholar). The role of the complement system is to rid the body of infectious agents and clear the bloodstream of immune complexes, apoptotic cells, and other debris (2Walport M.J. N. Engl. J. Med. 2001; 344: 1058-1066Crossref PubMed Scopus (2375) Google Scholar, 3Walport M.J. N. Engl. J. Med. 2001; 344: 1140-1144Crossref PubMed Scopus (1253) Google Scholar). Following its activation, deposition of complement proteins onto target surfaces ensues, marking the target for destruction and clearance, accompanied by release of mediators of inflammation and activators of the acquired immune response. Tight regulation and targeting of the complement system is thus essential to good health. The recent association of genetic variations in complement regulatory proteins with pathologies of the kidney (4Buddles M.R. Donne R.L. Richards A. Goodship J. Goodship T.H. Am. J. Hum. Genet. 2000; 66: 1721-1722Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 5Warwicker P. Goodship T.H. Donne R.L. Pirson Y. Nicholls A. Ward R.M. Turnpenny P. Goodship J.A. Kidney Int. 1998; 53: 836-844Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar) and eye (6Edwards A.O. Ritter R. III Abel Manning K.J. Panhuysen A. Farrer C.L.A. Science. 2005; 308: 421-424Crossref PubMed Scopus (2071) Google Scholar, 7Hageman G.S. Anderson D.H. Johnson L.V. Hancox L.S. Taiber A.J. Hardisty L.I. Hageman J.L. Stockman H.A. Borchardt J.D. Gehrs K.M. Smith R.J. Silvestri G. Russell S.R. Klaver C.C. Barbazetto I. Chang S. Yannuzzi L.A. Barile G.R. Merriam J.C. Smith R.T. Olsh A.K. Bergeron J. Zernant J. Merriam J.E. Gold B. Dean M. Allikmets R. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 7227-7232Crossref PubMed Scopus (1685) Google Scholar, 8Klein R.J. Zeiss C. Chew E.Y. Tsai J.Y. Sackler R.S. Haynes C. Henning A.K. SanGiovanni J.P. Mane S.M. Mayne S.T. Bracken M.B. Ferris F.L. Ott J. Barnstable C. Hoh J. Science. 2005; 308: 385-389Crossref PubMed Scopus (3494) Google Scholar, 9Zareparsi S. Branham K.E. Li M. Shah S. Klein R.J. Ott J. Hoh J. Abecasis G.R. Swaroop A. Am. J. Hum. Genet. 2005; 77: 149-153Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar) is an important development. It not only offers additional insights into the role played by complement in pathophysiological mechanisms, but it opens up the possibility of tailoring prevention and intervention according to genotype. The ability to consider such sequence variations within the context of a three-dimensional structure in which key functional regions have been identified significantly enhances these opportunities. In this report, we describe the structure of a region of factor H implicated in atypical hemolytic uremic syndrome (HUS). 2The abbreviations used are: HUS, hemolytic uremic syndrome; aHUS, atypical HUS; RCA, regulators of complement activation; CCP, complement control protein module; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; HSQC, heteronuclear single quantum correlation; GAG, glycosaminoglycan; fH, factor H.2The abbreviations used are: HUS, hemolytic uremic syndrome; aHUS, atypical HUS; RCA, regulators of complement activation; CCP, complement control protein module; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; HSQC, heteronuclear single quantum correlation; GAG, glycosaminoglycan; fH, factor H. Hemolytic uremic syndrome is a thrombotic microangiopathy that occurs primarily in the kidneys. It is characterized by hemolytic anemia and thrombocytopenia and is a leading cause of acute renal failure in children. Typical HUS is associated with infection by Escherichia coli (10Tarr P.I. Gordon C.A. Chandler W.L. Lancet. 2005; 365: 1073-1086Abstract Full Text Full Text PDF PubMed Scopus (1369) Google Scholar) and has a good prognosis (11Renaud C. Niaudet P. Gagnadoux M.F. Broyer M. Habib R. Pediatr. Nephrol. 1995; 9: 24-29Crossref PubMed Scopus (61) Google Scholar). The rarer atypical variant of HUS (aHUS) (12Neuhaus T.J. Calonder S. Leumann E.P. Arch. Dis. Child. 1997; 76: 518-521Crossref PubMed Scopus (61) Google Scholar) is recurrent and displays high mortality and morbidity even following renal transplantation (13Olie K.H. Florquin S. Groothoff J.W. Verlaak R. Strain L. Goodship T.H. Weening J.J. Davin J.C. Pediatr. Nephrol. 2004; 19: 1173-1176Crossref PubMed Scopus (35) Google Scholar). It is triggered by a range of factors (14Esparza-Gordillo J. Goicoechea de Jorge E. Buil A. Carreras Berges L. Lopez-Trascasa M. Sanchez-Corral P. Rodriguez de Cordoba S. Hum. Mol. Genet. 2005; 14: 703-712Crossref PubMed Scopus (237) Google Scholar) but generally presents in the absence of infections. Many aHUS patients carry mutations in the regulators of complement activation (RCA) gene cluster of chromosome 1q32. Between 10 and 30% of aHUS patients are heterozygous for mutations in the complement regulatory protein, factor H (fH) (4Buddles M.R. Donne R.L. Richards A. Goodship J. Goodship T.H. Am. J. Hum. Genet. 2000; 66: 1721-1722Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 5Warwicker P. Goodship T.H. Donne R.L. Pirson Y. Nicholls A. Ward R.M. Turnpenny P. Goodship J.A. Kidney Int. 1998; 53: 836-844Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar), most of which map to its C-terminal region (15Caprioli J. Bettinaglio P. Zipfel P.F. Amadei B. Daina E. Gamba S. Skerka C. Marziliano N. Remuzzi G. Noris M. J. Am. Soc. Nephrol. 2001; 12: 297-307Crossref PubMed Google Scholar, 16Sanchez-Corral P. Perez-Caballero D. Huarte O. Simckes A.M. Goicoechea E. Lopez-Trascasa M. de Cordoba S.R. Am. J. Hum. Genet. 2002; 71: 1285-1295Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 17Perez-Caballero D. Gonzalez-Rubio C. Gallardo M.E. Vera M. Lopez-Trascasa M. Rodriguez de Cordoba S. Sanchez-Corral P. Am. J. Hum. Genet. 2001; 68: 478-484Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). Many of the remaining patients carry mutations elsewhere in the RCA gene cluster (18Fremeaux-Bacchi V. Kemp E.J. Goodship J.A. Dragon-Durey M.A. Strain L. Loirat C. Deng H.W. Goodship T.H. J. Med. Genet. 2005; 22: 22Google Scholar). All such mutations should be considered against a background of single nucleotide polymorphisms in the RCA gene cluster that appear to further enhance disease susceptibility (14Esparza-Gordillo J. Goicoechea de Jorge E. Buil A. Carreras Berges L. Lopez-Trascasa M. Sanchez-Corral P. Rodriguez de Cordoba S. Hum. Mol. Genet. 2005; 14: 703-712Crossref PubMed Scopus (237) Google Scholar). A different single nucleotide polymorphism in the factor H gene predisposes to age-related macular degeneration (6Edwards A.O. Ritter R. III Abel Manning K.J. Panhuysen A. Farrer C.L.A. Science. 2005; 308: 421-424Crossref PubMed Scopus (2071) Google Scholar, 7Hageman G.S. Anderson D.H. Johnson L.V. Hancox L.S. Taiber A.J. Hardisty L.I. Hageman J.L. Stockman H.A. Borchardt J.D. Gehrs K.M. Smith R.J. Silvestri G. Russell S.R. Klaver C.C. Barbazetto I. Chang S. Yannuzzi L.A. Barile G.R. Merriam J.C. Smith R.T. Olsh A.K. Bergeron J. Zernant J. Merriam J.E. Gold B. Dean M. Allikmets R. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 7227-7232Crossref PubMed Scopus (1685) Google Scholar, 8Klein R.J. Zeiss C. Chew E.Y. Tsai J.Y. Sackler R.S. Haynes C. Henning A.K. SanGiovanni J.P. Mane S.M. Mayne S.T. Bracken M.B. Ferris F.L. Ott J. Barnstable C. Hoh J. Science. 2005; 308: 385-389Crossref PubMed Scopus (3494) Google Scholar, 9Zareparsi S. Branham K.E. Li M. Shah S. Klein R.J. Ott J. Hoh J. Abecasis G.R. Swaroop A. Am. J. Hum. Genet. 2005; 77: 149-153Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar) and to membranoproliferative glomerulonephritis (7Hageman G.S. Anderson D.H. Johnson L.V. Hancox L.S. Taiber A.J. Hardisty L.I. Hageman J.L. Stockman H.A. Borchardt J.D. Gehrs K.M. Smith R.J. Silvestri G. Russell S.R. Klaver C.C. Barbazetto I. Chang S. Yannuzzi L.A. Barile G.R. Merriam J.C. Smith R.T. Olsh A.K. Bergeron J. Zernant J. Merriam J.E. Gold B. Dean M. Allikmets R. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 7227-7232Crossref PubMed Scopus (1685) Google Scholar), both of which are characterized by formation of fatty plaques, or deposits, rich in immune molecules. Factor H is an abundant plasma glycoprotein (155 kDa) (19Sim R.B. DiScipio R.G. Biochem. J. 1982; 205: 285-293Crossref PubMed Scopus (104) Google Scholar) that controls the alternative pathway of complement activation (20Whaley K. Ruddy S. Science. 1976; 193: 1011-1013Crossref PubMed Scopus (113) Google Scholar). The alternative pathway of complement is triggered on any surface (self or foreign) not protected by specialized regulatory proteins. Self-cells carry membrane-associated regulators to prevent complement activation via the alternative pathway, most of which, like fH, are encoded by the RCA gene cluster (21Hourcade D. Holers V.M. Atkinson J.P. Adv. Immunol. 1989; 45: 381-416Crossref PubMed Scopus (374) Google Scholar). Factor H is a fluid phase protein that normally prevents complement amplification on all self-surfaces. It is, therefore, important for blocking complement at host surfaces not enclosed by a cell membrane carrying other RCAs. Factor H acts on a key bimolecular enzymatic complex in the complement cascade, C3 convertase (C3b.Bb). Factor H competes with C3b for binding of factor B, is a cofactor for proteolytic cleavage of C3b, and accelerates the decay of the C3 convertase into its components (22Pangburn M.K. Schreiber R.D. Muller-Eberhard H.J. J. Exp. Med. 1977; 146: 257-270Crossref PubMed Scopus (523) Google Scholar, 23Weiler J.M. Daha M.R. Austen K.F. Fearon D.T. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 3268-3272Crossref PubMed Scopus (445) Google Scholar). Factor H consists of 20 tandem, ∼60-residue-long, complement control protein modules (CCPs). The cofactor and decay acceleration activities of fH map to the N-terminal four CCPs (24Alsenz J. Lambris J.D. Schulz T.F. Dierich M.P. Biochem. J. 1984; 224: 389-398Crossref PubMed Scopus (92) Google Scholar, 25Kuhn S. Skerka C. Zipfel P.F. J. Immunol. 1995; 155: 5663-5670PubMed Google Scholar). There are three C3b-binding sites in fH (26Sharma A.K. Pangburn M.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10996-11001Crossref PubMed Scopus (200) Google Scholar): CCPs 1–4, CCPs 8–15, and finally, CCPs 19–20, which also binds C3d generated during complement activation (27Jokiranta T.S. Hellwage J. Koistinen V. Zipfel P.F. Meri S. J. Biol. Chem. 2000; 275: 27657-27662Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Factor H interacts with polyanions via three sites, in CCPs 7, 9, or 13 and in CCP 20 (28Pangburn M.K. Atkinson M.A. Meri S. J. Biol. Chem. 1991; 266: 16847-16853Abstract Full Text PDF PubMed Google Scholar, 29Blackmore T.K. Sadlon T.A. Ward H.M. Lublin D.M. Gordon D.L. J. Immunol. 1996; 157: 5422-5427PubMed Google Scholar, 30Blackmore T.K. Hellwage J. Sadlon T.A. Higgs N. Zipfel P.F. Ward H.M. Gordon D.L. J. Immunol. 1998; 160: 3342-3348PubMed Google Scholar). Indeed, the capacity of fH to distinguish self from non-self depends upon a sophisticated recognition capability for specific polyanionic markers on host surfaces (31Meri S. Pangburn M.K. Biochem. Biophys. Res. Commun. 1994; 198: 52-59Crossref PubMed Scopus (112) Google Scholar). The primary region for host recognition has been mapped to the four C-terminal modules (32Pangburn M.K. J. Immunol. 2002; 169: 4702-4706Crossref PubMed Scopus (113) Google Scholar). Thus, it is noteworthy that the two C-terminal modules of fH harbor a binding site for polyanions and one for C3b/C3d, in addition to being a hotspot of aHUS-associated mutations (15Caprioli J. Bettinaglio P. Zipfel P.F. Amadei B. Daina E. Gamba S. Skerka C. Marziliano N. Remuzzi G. Noris M. J. Am. Soc. Nephrol. 2001; 12: 297-307Crossref PubMed Google Scholar). This polyanion-binding site appears to also represent a site for attachment to endothelial cell surfaces (33Jokiranta T.S. Cheng Z.Z. Seeberger H. Jozsi M. Heinen S. Noris M. Remuzzi G. Ormsby R. Gordon D.L. Meri S. Hellwage J. Zipfel P.F. Am. J. Pathol. 2005; 167: 1173-1181Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Therefore one hypothesis is that aHUS-linked mutations induce pathogenesis by interfering with a recognition site on fH for polyanionic self-markers. Additional explanations are that the mutations disrupt structure, that they interfere with the C3b/C3d-binding site, or that they compromise the ability of fH to form quaternary structure. Affinity chromatography and related techniques, utilizing for the most part commercially available preparations of heparin, have been employed previously to provide an overall assessment of how well mutant forms of fH bind polyanionic carbohydrates, but they do not provide a clear picture of the potential contribution of individual amino acid residues to the more sophisticated in vivo recognition process. Thus, as described below, elucidation of the three-dimensional structure of the CCPs 19 and 20 (fH∼19–20) and experimental identification of the residues that comprise a polyanion recognition site represent a powerful means of investigating these hypotheses further. Protein Production—The fragment containing human fH residues 1107–1231 (native sequence numbering) was cloned into the Pichia pastoris expression vector pPICZα. The expressed fH∼19–20 was directed to the secretory pathway by placing the coding sequence behind the Saccharomyces cerevisiae α-factor secretion sequence. Inefficient cleavage of this secretion signal combined with cloning artifacts resulted in the additional sequence EAEF at the N terminus. Cation-exchange chromatography was used to purify the fH∼19–20 collected from the culture supernatant. NMR Data Collection—NMR spectra were acquired on Bruker AVANCE 600 and 800 MHz spectrometers using 5-mm triple resonance probes. Data were collected at 37 °C on a 1 mm sample of 13C, 15N fH∼19–20 in 20 mm sodium acetate (deuterated), 200 mm NaCl at pH 4.0. 15N-edited and 13C-edited NOESY-HSQC experiments were acquired with mixing times of 100 ms. NMR data were processed using the AZARA suite of programs, provided by Wayne Boucher and the Department of Biochemistry, University of Cambridge with maximum entropy processing used for the F1 and F2 dimensions of the three-dimensional experiments. Heteronuclear (1H–15N) nuclear Overhauser effects (NOEs), at 600 MHz, were calculated from the ratio of the intensities of the cross-peaks in the reference spectra to those recorded with saturation of the 1H signal. Resonance Assignment and Structure Calculation—Processed spectra were viewed and nuclei were assigned using ANSIG (34Kraulis P.J. J. Mol. Biol. 1994; 243: 696-718Crossref PubMed Scopus (54) Google Scholar). Resolved peaks in 15N-NOESY and 13C-NOESY spectra were picked and, where possible, assigned unambiguously. Ambiguous restraints were generated by matching the chemical shifts of NOESY cross-peaks with the resonance assignments. The "connect" routine within the AZARA suite was used to convert normalized intensities into four distance categories: <2.7 Å, <3.3 Å, <5Å, and <6 Å. Relatively slowly exchanging amide protons were identified by analysis of two-dimensional heteronuclear water exchange experiments. Structure calculations were carried out using the crystallography and NMR system (CNS)-based protocols (35Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16948) Google Scholar). The first round of structure calculations was performed using only the unambiguous restraints list, whereas all subsequent rounds were performed using both unambiguous and ambiguous restraints (36Nilges M. J. Mol. 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Biol. 2004; 278: 379-402PubMed Google Scholar); the first three removed restraints contributing <1% of the total NOE, and the final one eliminated restraints contributing 4)469 IntermodularaFor the purpose of this table, module boundaries are defined as the 1st and 4th of the 4 consensus cysteines.11 Intralinker26 From CCP 20 to linker20 From CCP 19 to linker15For ambiguous NOEs (≥ 40 % of cross-peak) Sequential289 Short-range (2 ≤ |i—j| ≤ 4)152 Long range (|i—j| > 4)218 Intermodular6 Intralinker3 From CCP 20 to linker11 From CCP 19 to linker12Total (all structures) NOE violations > 0.5 Å24Percent of residues in regions of Ramachandran plot Most favored52.0 Additionally allowed38.1 Generously allowed6.8Root mean square deviations CCP 19 (all residues) All heavy atoms1.13 Backbone atoms Cα, N, CO0.69 Cα only0.73Root mean square deviations CCP 20 (all residues) All heavy atoms1.16 Backbone atoms Cα, N, CO0.67 Cα only0.71Root mean square deviations CCP 19-20 (all residues) All heavy atoms1.50 Backbone atoms Cα, N, CO1.17 Cα only1.20a For the purpose of this table, module boundaries are defined as the 1st and 4th of the 4 consensus cysteines. Open table in a new tab FIGURE 2Summary of NOE data as a function of residue number. A, the bars show the 1H, 15N NOE value for each residue. B, the bars show the number of 1H-1H NOEs per residue; long range, medium range, and sequential restraints are indicated by increasingly dense shading. The line and triangular symbols show the backbone root mean square deviation (r.m.s.d.) based on a superposition of the ensemble on the lowest total energy structure.View Large Image Figure ViewerDownload Hi-res image Download (PPT) CCP 19 adopts a classic CCP module fold (Fig. 3A), elongated (∼32 × ∼16 Å) with N and C termini at opposite ends. Two disulfide bonds, CysI–CysIII, CysII–CysIV, lie at opposite extremes of a hydrophobic core to which the consensus Trp (Trp1157) contributes. Its structure resembles those of other CCPs found elsewhere in factor H (45Norman D.G. Barlow P.N. Baron M. Day A.J. Sim R.B. Campbell I.D. J. Mol. 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Acad. Sci. U. S. A. 2004; 101: 1279-1284Crossref PubMed Scopus (96) Google Scholar). CCP 20 (Fig. 3A) has the same disulfide pattern, but the overall structure of this module differs from those of all other CCPs solved to date. Although adopting the CCP module fold, CCP 20 (∼28 × ∼20 Å) is shorter than CCP 19 and exhibits more prominent lateral bulges and a helical turn. Nearly all CCPs contain a structurally critical Trp residue between CysIII and CysIV; in CCP 20, Trp1219 occupies such a position in the sequence but is solvent-exposed. A second Trp residue in CCP 20, Trp1183, is part of a prominent hypervariable loop (Fig. 3A, and see below). The two modules are stacked in an elongated fashion (Fig. 3B), unlike the tilted arrangement observed in many other CCP module pairs (51Kirkitadze M.D. Barlow P.N. Immunol. Rev. 2001; 180: 146-161Crossref PubMed Scopus (174) Google Scholar), stabilized by interactions between the two modules and the bulky side chains of the short linker. Equivalent parts of the two modules are on the same face of the overall structure. An electrostatic surface representation (Fig. 3C) reveals that one face of fH∼19–20 is positively charged, whereas the other has a predominantly negative charge. A band of mainly positive charge running up one face of CCP 19 is contiguous with a positively charged area of the CCP 20 surface, which stretches to the C terminus of the protein. The five-membered ring of the indole side chain of Trp1183 is wedged between Arg1182 and Lys1186 within the hypervariable loop, leaving the six-membered aromatic ring exposed to solvent at the center of the positively charged CCP 20 surface patch. Determination of the Binding Sites in fH∼19,20—To characterize the binding of polyanions by fH∼19–20, sulfated heparin, a commonly used analogue of heparan sulfate (52Ganesh V.K. Smith S.A. Kotwal G.J. Murthy K.H. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8924-8929Crossref PubMed Scopus (56) Google Scholar, 53Delehedde M. 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