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• W2078046435 abstract "Factor I-like modules (FIMs) of complement proteins C6, C7, and factor I participate in protein-protein interactions critical to the progress of a complement-mediated immune response to infections and other trauma. For instance, the carboxyl-terminal FIM pair of C7 (C7-FIMs) binds to the C345C domain of C5 and its activated product, C5b, during self-assembly of the cytolytic membrane-attack complex. FIMs share sequence similarity with follistatin domains (FDs) of known three-dimensional structure, suggesting that FIM structures could be reliably modeled. However, conflicting disulfide maps, inconsistent orientations of subdomains within FDs, and the presence of binding partners in all FD structures led us to determine the three-dimensional structure of C7-FIMs by NMR spectroscopy. The solution structure reveals that each FIM within C7 contains a small amino-terminal FOLN subdomain connected to a larger carboxyl-terminal KAZAL domain. The open arrangement of the subdomains within FIMs resembles that of first FDs within structures of tandem FDs but differs from the more compact subdomain arrangement of second or third FDs. Unexpectedly, the two C7-FIMs pack closely together with an approximate 2-fold rotational symmetry that is rarely seen in module pairs and has not been observed in FD-containing proteins. Interfaces between subdomains and between modules include numerous hydrophobic and electrostatic contributions, suggesting that this is a physiologically relevant conformation that persists in the context of the parent protein. Similar interfaces were predicted in a homology-based model of the C6-FIM pair. The C7-FIM structures also facilitated construction of a model of the single FIM of factor I. Factor I-like modules (FIMs) of complement proteins C6, C7, and factor I participate in protein-protein interactions critical to the progress of a complement-mediated immune response to infections and other trauma. For instance, the carboxyl-terminal FIM pair of C7 (C7-FIMs) binds to the C345C domain of C5 and its activated product, C5b, during self-assembly of the cytolytic membrane-attack complex. FIMs share sequence similarity with follistatin domains (FDs) of known three-dimensional structure, suggesting that FIM structures could be reliably modeled. However, conflicting disulfide maps, inconsistent orientations of subdomains within FDs, and the presence of binding partners in all FD structures led us to determine the three-dimensional structure of C7-FIMs by NMR spectroscopy. The solution structure reveals that each FIM within C7 contains a small amino-terminal FOLN subdomain connected to a larger carboxyl-terminal KAZAL domain. The open arrangement of the subdomains within FIMs resembles that of first FDs within structures of tandem FDs but differs from the more compact subdomain arrangement of second or third FDs. Unexpectedly, the two C7-FIMs pack closely together with an approximate 2-fold rotational symmetry that is rarely seen in module pairs and has not been observed in FD-containing proteins. Interfaces between subdomains and between modules include numerous hydrophobic and electrostatic contributions, suggesting that this is a physiologically relevant conformation that persists in the context of the parent protein. Similar interfaces were predicted in a homology-based model of the C6-FIM pair. The C7-FIM structures also facilitated construction of a model of the single FIM of factor I. The membrane attack complex (MAC) 2The abbreviations used are: MACmembrane attack complexFIMfactor I-like moduleFDfollistatin domainfIfactor IAPBSadaptive Poisson-Boltzmann solverRMSDroot mean square deviationHSQCheteronuclear single quantum coherenceNOEnuclear Overhauser effect. is the terminal product of the complement cascade and is therefore a fundamental component of mammalian innate immunity. The formation of this multi-protein complex is triggered by proteolytic cleavage of complement component C5. This is followed swiftly by a remarkable, although little understood, self-assembly process involving multiple sequential protein-protein recognition events. MAC assembly culminates in the formation of a pore traversing the targeted cell membrane (1.Podack E.R. Biesecker G. Kolb W.P. Müller-Eberhard H.J. J. Immunol. 1978; 121: 484-490PubMed Google Scholar). Accumulation of multiple MACs in a membrane triggers cell-dependent responses and may result in cell lysis (2.Bohana-Kashtan O. Ziporen L. Donin N. Kraus S. Fishelson Z. Mol. Immunol. 2004; 41: 583-597Crossref PubMed Scopus (134) Google Scholar). The key to progress in understanding MAC formation will be three-dimensional structural information for each of its component proteins, namely C5b, C6, C7, C8, and C9. membrane attack complex factor I-like module follistatin domain factor I adaptive Poisson-Boltzmann solver root mean square deviation heteronuclear single quantum coherence nuclear Overhauser effect. Classical, alternative, and lectin pathways of complement activation converge at a step in which C5 is cleaved to release activated C5b. Immediately following C5b formation, C6 and C7 bind sequentially; the C5b6 complex is soluble and relatively stable (3.Goldlust M.B. Shin H.S. Hammer C.H. Mayer M.M. J. Immunol. 1974; 113: 998-1007PubMed Google Scholar), but soluble C5b67 has a brief half-life and is proposed to attach rapidly to target membrane surfaces (4.Götze O. Müller-Eberhard H.J. J. Exp. Med. 1970; 132: 898-915Crossref PubMed Scopus (93) Google Scholar, 5.Hammer C.H. Nicholson A. Mayer M.M. Proc. Natl. Acad. Sci. U.S.A. 1975; 72: 5076-5080Crossref PubMed Scopus (42) Google Scholar). Subsequently, C8 binds to the nascent complex, inserting into the target membrane and causing disruptive rearrangements of the lipid bilayer. Finally the mature MAC, C5b6789n, forms by recruitment of between 10 and 16 copies of C9 that insert in the membrane to form the pore. Notably, once C5b is generated, MAC assembly requires no additional enzymatic triggers; this implies that individual components encompass highly specific, complementary binding sites that become exposed during MAC formation. Complement proteins C6, C7, C8 (α and β subunits), and C9 comprise the “MAC family” (Fig. 1a) (6.DiScipio R.G. Chakravarti D.N. Muller-Eberhard H.J. Fey G.H. J. Biol. Chem. 1988; 263: 549-560Abstract Full Text PDF PubMed Google Scholar). Family members share, in addition to a large central membrane attack complex perforin domain (7.Ponting C.P. Curr. Biol. 1999; 9: R911-R913Abstract Full Text Full Text PDF PubMed Google Scholar, 8.Husler T. Lockert D.H. Sims P.J. Biochemistry. 1996; 35: 3263-3269Crossref PubMed Scopus (12) Google Scholar, 9.Peitsch M.C. Amiguet P. Guy R. Brunner J. Maizel Jr., J.V. Tschopp J. Mol. Immunol. 1990; 27: 589-602Crossref PubMed Scopus (62) Google Scholar), several tandemly arranged, cysteine-rich modules of less than 80 amino acid residues each. These smaller modules include thrombospondin type I (10.Higgins J.M. Wiedemann H. Timpl R. Reid K.B. J. Immunol. 1995; 155: 5777-5785PubMed Google Scholar), low density lipoprotein receptor class A (11.Varret M. Rabés J.P. Thiart R. Kotze M.J. Baron H. Cenarro A. Descamps O. Ebhardt M. Hondelijn J.C. Kostner G.M. Miyake Y. Pocovi M. Schmidt H. Schuster H. Stuhrmann M. Yamamura T. Junien C. Béroud C. Boileau C. Nucleic Acids Res. 1998; 26: 248-252Crossref PubMed Scopus (86) Google Scholar) and modules similar in sequence to epidermal growth factor (Fig. 1a). C6 and C7 each contain an additional four modules at their carboxyl termini: two ∼60-residue complement control protein modules (12.Norman D.G. Barlow P.N. Baron M. Day A.J. Sim R.B. Campbell I.D. J. Mol. Biol. 1991; 219: 717-725Crossref PubMed Scopus (207) Google Scholar, 13.Soares D.C. Gerloff D.L. Syme N.R. Coulson A.F.W. Parkinson J. Barlow P.N. Prot. Eng. Des. Sel. 2005; 18: 379-388Crossref PubMed Scopus (43) Google Scholar), followed by two cysteine-rich modules composed of ∼75 residues each; these are the factor I-like modules (FIMs) (also known as factor I membrane attack complex domains (14.DiScipio R.G. Hugli T.E. J. Biol. Chem. 1989; 264: 16197-16206Abstract Full Text PDF PubMed Google Scholar, 15.Chamberlain D. Ullman C.G. Perkins S.J. Biochemistry. 1998; 37: 13918-13929Crossref PubMed Scopus (30) Google Scholar)), so named because of their apparent relatedness to an amino-terminal domain of complement factor I (fI) (Fig. 1b). Latent C5 was shown, in vitro, to bind reversibly to both C6 and C7 prior to activation. These interactions are distinct from and precede irreversible binding of C6 and subsequently C7 to C5b (18.Thai C.T. Ogata R.T. J. Immunol. 2004; 173: 4547-4552Crossref PubMed Scopus (39) Google Scholar). It is hypothesized that the C56 and C57 preactivation complexes ensure that C6 and C7 are maintained proximal to C5 in the plasma. This may be significant because activated C5b is labile (19.Cooper N.R. Müller-Eberhard H.J. J. Exp. Med. 1970; 132: 775-793Crossref PubMed Scopus (113) Google Scholar, 20.DiScipio R.G. Smith C.A. Muller-Eberhard H.J. Hugli T.E. J. Biol. Chem. 1983; 258: 10629-10636Abstract Full Text PDF PubMed Google Scholar), hence swift assembly of C5b67 is advantageous. Within this preactivation complex, critical interactions occur between the carboxyl-terminal C345C domain of C5, C5-C345C (21.Thai C.T. Ogata R.T. J. Immunol. 2003; 171: 6565-6573Crossref PubMed Scopus (23) Google Scholar), and the carboxyl-terminal FIM pair of both C6 and C7 (22.DiScipio R.G. Linton S.M. Rushmere N.K. J. Biol. Chem. 1999; 274: 31811-31818Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 23.Thai C.T. Ogata R.T. J. Immunol. 2005; 174: 6227-6232Crossref PubMed Scopus (23) Google Scholar). The involvement of these domains in MAC formation was demonstrated using recombinant proteins, where either C7-FIMs or C5-C345C inhibited the binding of C7 to C5b6 and inhibited complement-mediated erythrocyte lysis (23.Thai C.T. Ogata R.T. J. Immunol. 2005; 174: 6227-6232Crossref PubMed Scopus (23) Google Scholar). The FIMs of C6, however, although shown to promote MAC assembly, do not appear to be essential for MAC formation (22.DiScipio R.G. Linton S.M. Rushmere N.K. J. Biol. Chem. 1999; 274: 31811-31818Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). C7-FIMs have a stronger affinity than C6-FIMs for C5-C345C, suggesting that C7-FIMs may displace C6-FIMs during MAC assembly (23.Thai C.T. Ogata R.T. J. Immunol. 2005; 174: 6227-6232Crossref PubMed Scopus (23) Google Scholar). Thus, interactions between C5- C345C and FIMs are key to the early assembly of MAC, and their structural basis is an important target of investigations. The structure of the C5-C345C domain is well established (24.Bramham J. Thai C.T. Soares D.C. Uhrín D. Ogata R.T. Barlow P.N. J. Biol. Chem. 2005; 280: 10636-10645Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 25.Fredslund F. Laursen N.S. Roversi P. Jenner L. Oliveira C.L. Pedersen J.S. Nunn M.A. Lea S.M. Discipio R. Sottrup-Jensen L. Andersen G.R. Nat. Immunol. 2008; 9: 753-760Crossref PubMed Scopus (105) Google Scholar); however, there has been no three-dimensional structural information available for any of the FIMs or for any other domains within C6 or C7. The closely related FIM within fI has been postulated to resemble a follistatin domain (26.Ullman C.G. Perkins S.J. Biochem. J. 1997; 326: 939-941Crossref PubMed Scopus (21) Google Scholar). Intriguingly, however, disulfide mapping of human C6 isolated from plasma appeared to exclude that possibility (27.Lengweiler S. Schaller J. DiScipio R.G. Rickli E.E. Biochim. Biophys. Acta. 1997; 1342: 13-18Crossref PubMed Scopus (4) Google Scholar). The three-dimensional arrangement of the neighboring FIMs, and the extent of interactions between them, has also been a mystery. We previously described a protein construct comprising the carboxyl-terminal pair of FIMs from human C7 (18.Thai C.T. Ogata R.T. J. Immunol. 2004; 173: 4547-4552Crossref PubMed Scopus (39) Google Scholar), which folds homogeneously and binds to C5 in surface plasmon resonance assays. Here we report the solution structure of this consecutive pair of FIMs. This new structure reveals that, despite previous evidence to the contrary, each FIM adopts a follistatin-like fold, and the two FIMs are intimately associated to form a homodimer-like, pseudosymmetrical carboxyl terminus of C7. This work, therefore, serendipitously provides the first published structure of a follistatin-domain pair in the absence of ligand and suggests that conformational changes within FIM pairs accompany ligand binding. Novel structures of the FIMs from both C6 and fI have been modeled based upon our NMR-derived solution structure of the C7-FIMs. C7-FIMs, encompassing residues Asn693 to Gln843 of C7 with an amino-terminal His6 tag, was cloned previously (18.Thai C.T. Ogata R.T. J. Immunol. 2004; 173: 4547-4552Crossref PubMed Scopus (39) Google Scholar), and the recombinant protein was expressed in the Origami B strain of Escherichia coli as previously described (18.Thai C.T. Ogata R.T. J. Immunol. 2004; 173: 4547-4552Crossref PubMed Scopus (39) Google Scholar, 28.Phelan M.M. Thai C.T. Herbert A.P. Bella J. Uhrin D. Ogata R.T. Barlow P.N. Bramham J. Biomol NMR Assign. 2009; 3: 49-52Crossref PubMed Scopus (3) Google Scholar). The His6-tagged protein was captured using a Co2+ affinity column, the His6 tag was then cleaved with thrombin before noncleaved material, and the His6 tag was removed via a re-pass down the Co2+ affinity column. The cleaved protein was purified by reverse phase high performance liquid chromatography (Supelco Discovery BIO Wide Pore C8 column; Supelco Inc., PA), eluting with an acetonitrile gradient in the presence of 0.1% (v/v) trifluoroacetic acid, before lyophilization and resuspension into NMR buffer. Characterization of the purified protein was carried out using polyacrylamide gel electrophoresis and electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry. NMR sample conditions were optimized by analysis of two-dimensional 15N,1H HSQC spectra at a range of protein concentrations (0.1–1.0 mm), salt concentrations (0–150 mm), buffers (Arg-Glu (29.Golovanov A.P. Hautbergue G.M. Wilson S.A. Lian L.Y. J. Am. Chem. Soc. 2004; 126: 8933-8939Crossref PubMed Scopus (318) Google Scholar) and phosphate), temperatures (10–60 °C), and pH values (3.0–7.0). Final optimal sample conditions were a protein concentration of 300 μm in 20 mm potassium phosphate, pH 6.5, 25 °C, with 10% v/v 2H2O. A standard suite of NMR experiments was implemented to obtain nearly complete resonance assignment (28.Phelan M.M. Thai C.T. Herbert A.P. Bella J. Uhrin D. Ogata R.T. Barlow P.N. Bramham J. Biomol NMR Assign. 2009; 3: 49-52Crossref PubMed Scopus (3) Google Scholar). The spectra were processed using the Azara suite of programs (provided by Wayne Boucher and the Department of Biochemistry, University of Cambridge, Cambridge, UK), and resonance assignment was carried out using the Analysis package (30.Vranken W.F. Boucher W. Stevens T.J. Fogh R.H. Pajon A. Llinas M. Ulrich E.L. Markley J.L. Ionides J. Laue E. D Proteins. 2005; 59: 687-696Crossref PubMed Scopus (2324) Google Scholar). Assignment of the NOE spectroscopy data was carried out using a combination of 10% manual peak assignment and 90% automated assignment within the structure calculation software CYANA 2.1 (31.Herrmann T. Guntert P. Wuthrich K. J. Mol. Biol. 2002; 319: 209-227Crossref PubMed Scopus (1329) Google Scholar, 32.Guntert P. Methods Mol. Biol. 2004; 278: 353-378Crossref PubMed Scopus (1170) Google Scholar). All of the proline residues were defined as trans on the basis of chemical shifts and NOEs between Hα(Pron−1) and Hδ(Pron). All 18 cysteines were inferred to be disulfide-bonded on the basis of accurate mass spectrometry. Therefore, although no specific disulfide linkages were initially incorporated, all of the cysteine residues were defined as in the oxidized state in the structure calculations. The incidence of hydrogen bonds between backbone amides and carboxyl groups was determined on the basis of amide exchange retardation as observed in 15N,1H HSQC spectra collected 1 h after the protein was transferred to 99.9% (v/v) deuterated buffer. The hydrogen bond acceptor of the protected amide was then inferred from the corresponding network of NOEs. The CYANA calculation comprised a seven-cycle routine using combined, automated, NOE assignment, and structure determination. The upper limit distance constraints generated were transferred into the crystallography and NMR system (33.Bünger 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. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16967) Google Scholar, 34.Brunger A.T. Nat. Protoc. 2007; 2: 2728-2733Crossref PubMed Scopus (1131) Google Scholar) using the program Format Converter within the CCPN suite (30.Vranken W.F. Boucher W. Stevens T.J. Fogh R.H. Pajon A. Llinas M. Ulrich E.L. Markley J.L. Ionides J. Laue E. D Proteins. 2005; 59: 687-696Crossref PubMed Scopus (2324) Google Scholar) to include ambiguous disulfide restraints and to perform a structure refinement using explicit water. Structure calculations proceeded iteratively with inclusion of hydrogen bonds, ambiguous disulfide bridges, dihedral restraints generated using TALOS (35.Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2738) Google Scholar), and, finally, unambiguous disulfide bridges. An ensemble of 25 structures was calculated based on the converged set from 100 structures calculated. In addition a further set of structures were calculated without disulfide bonds Cys773–Cys782 and Cys776–Cys789 explicitly constrained; this yielded 23 converged structures (from 100) of differing disulfide linkages in this region, although as expected, the overall structure was not affected. All 48 converged structures were deposited in the Protein Data Bank (36.Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. Nucleic Acids Res. 2000; 28: 235-242Crossref PubMed Scopus (27551) Google Scholar) under code 2WCY. NMR relaxation data were assessed from 15N T1 and T2 (37.Kay L.E. Nicholson L.K. Delaglio F. Bax A. Torchia D.A. J. Magn. Reson. 1992; 97: 359-375Google Scholar, 38.Grzesiek S. Bax A. J. Am. Chem. Soc. 1993; 115: 12593-12594Crossref Scopus (1015) Google Scholar) and the 1H,15N HSQC heteronuclear NOE experiment (38.Grzesiek S. Bax A. J. Am. Chem. Soc. 1993; 115: 12593-12594Crossref Scopus (1015) Google Scholar). The quality of the data and structures were analyzed using Whatif (39.Vriend G. J. Mol. Graph. 1990; 8: 52-56Crossref PubMed Scopus (3374) Google Scholar), and the Ramachandran statistics were checked using PROCHECK (40.Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar, 41.Morris A.L. MacArthur M.W. Hutchinson E.G. Thornton J.M. Proteins. 1992; 12: 345-364Crossref PubMed Scopus (1407) Google Scholar). For analysis of the structures calculated, intermodular interactions including hydrophobic interactions, hydrogen bonds, and salt bridges were assessed using PIC (42.Tina K.G. Bhadra R. Srinivasan N. Nucleic Acids Res. 2007; 35: W473-W476Crossref PubMed Scopus (726) Google Scholar) and MolSurfer (43.Gabdoulline R.R. Wade R.C. Walther D. Nucleic Acids Res. 2003; 31: 3349-3351Crossref PubMed Scopus (48) Google Scholar, 44.Gabdoulline R.R. Wade R.C. Walther D. Trends Biochem. Sci. 1999; 24: 285-287Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). The incidence of secondary structure elements was identified using STRIDE (45.Frishman D. Argos P. Proteins Struct. Funct. Genet. 1995; 23: 566-579Crossref PubMed Scopus (2045) Google Scholar). Surface potentials were determined using the Adaptive Poisson-Boltzmann Solver (APBS) (46.Baker N.A. Sept D. Joseph S. Holst M.J. McCammon J.A. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 10037-10041Crossref PubMed Scopus (5877) Google Scholar) within PyMOL (DeLano Scientific, San Carlos, CA), GRASP-2 (47.Nicholls A. Sharp K.A. Honig B. Proteins Struct. Funct. Genet. 1991; 11: 281-296Crossref PubMed Scopus (5316) Google Scholar, 48.Petrey D. Honig B. Method Enzymol. 2003; 374: 492-509Crossref PubMed Scopus (198) Google Scholar), and the MOLCAD module (49.Heiden W. Moeckel G. Brickmann J. J. Comput. Mol. Des. 1993; 7: 503-514Crossref PubMed Scopus (175) Google Scholar) of SYBYL v6.9 (Tripos Associates, St. Louis, MO). The buried surface area at the intermodular junctions was calculated using GETAREA 1.0 (50.Fraczkiewicz R. Braun W. J. Comput. Chem. 1998; 19: 319-333Crossref Scopus (874) Google Scholar), being computed as: (SA Modulei + SA Modulej) − SA Bimoduleij. Intermodular angles were determined using the same protocol as previously described (51.Barlow P.N. Steinkasserer A. Norman D.G. Kieffer B. Wiles A.P. Sim R.B. Campbell I.D. J. Mol. Biol. 1993; 232: 268-284Crossref PubMed Scopus (185) Google Scholar, 52.Soares D.C. Barlow P.N. Morikis D. Lambris J.D. Structural Biology of the Complement System. CRC Press, Inc., Boca Raton, FL2005: 19-62Google Scholar). Superimpositions of structures were performed using MultiProt (53.Shatsky M. Nussinov R. Wolfson H.J. Proteins Struct. Funct. Bioinformat. 2004; 56: 143-156Crossref PubMed Scopus (369) Google Scholar). The three-dimensional structure of C6-FIMs was modeled using, as a template, the structure of C7-FIMs closest to that of the mean from the ensemble generated in structure calculations. The single FIM in fI (fI-FIM) was modeled using the carboxyl-terminal FIM (C7-FIM2) from the closest-to-mean structure of C7-FIMs as the template. C6-FIMs and fI-FIM share 32 and 25% sequence identity with their templates, respectively. The optimal alignment between the targets and template sequences was achieved using an initial multiple sequence alignment of homologous protein sequences from a range of differing genera employing the program PROMALS3-D (54.Pei J. Tang M. Grishin N.V. Nucleic Acids Res. 2008; 36: W30-W34Crossref PubMed Scopus (120) Google Scholar). Related sequences for C6-FIMs were first identified via the [email protected] server (55.Combet C. Blanchet C. Geourjon C. Deléage G. Trends Biochem. Sci. 2000; 25: 147-150Abstract Full Text Full Text PDF PubMed Scopus (1438) Google Scholar) with a BLAST (56.Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (70747) Google Scholar) search against the UniProt data base (57.Apweiler R. Bairoch A. Wu C.H. Barker W.C. Boeckmann B. Ferro S. Gasteiger E. Huang H. Lopez R. Magrane M. Martin M.J. Natale D.A. O'Donovan C. Redaschi N. Yeh L.S. Nucleic Acids Res. 2004; 32: D115-119Crossref PubMed Google Scholar). Because the FIM from fI exhibits a lower degree of sequence similarity to its template sequence, in this case a remote hidden Markov model (58.Eddy S.R. Bioinformatics. 1998; 14: 755-763Crossref PubMed Scopus (4066) Google Scholar), search methodology was employed to identify related sequences prior to alignment (data not shown). The resulting target-template alignments were manually refined from the multiple sequence alignment to place gaps optimally guided by positioning of predicted and identified secondary structure elements. Twenty models were built for each protein using the program Modeler 9v6 (59.Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10563) Google Scholar), and the one with the lowest objective function score was selected as the representative model. The quality of the models was assessed using PROCHECK v3.5.4 (40.Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar, 41.Morris A.L. MacArthur M.W. Hutchinson E.G. Thornton J.M. Proteins. 1992; 12: 345-364Crossref PubMed Scopus (1407) Google Scholar) and ProQ (60.Wallner B. Elofsson A. Protein Sci. 2003; 12: 1073-1086Crossref PubMed Scopus (584) Google Scholar). The recombinant protein fragment C7-FIMs, encompassing residues Asn693 to Gln843 of human C7 with an amino-terminal His6 tag (and where Gln843 is the carboxyl-terminal residue of the complete C7 protein), was expressed as a soluble, folded protein in the Origami B strain of E. coli. The affinity tag was removed by thrombin, leaving four non-native residues (Gly-Ser-His-Met) at the new amino terminus. Protein expression levels in rich or isotopically enriched media were typically 4 mg liter−1, yielding sufficient high quality protein to enable a solution state structure determination of C7-FIMs by NMR spectroscopy. Protein purity and the presence of nine disulfide bonds were confirmed by mass spectrometry (28.Phelan M.M. Thai C.T. Herbert A.P. Bella J. Uhrin D. Ogata R.T. Barlow P.N. Bramham J. Biomol NMR Assign. 2009; 3: 49-52Crossref PubMed Scopus (3) Google Scholar). The 15N-, and 15N,13C-labeled samples of C7-FIMs yielded high quality NMR spectra, thus permitting assignment of 90% of 1H, 15N, and 13C nuclei (28.Phelan M.M. Thai C.T. Herbert A.P. Bella J. Uhrin D. Ogata R.T. Barlow P.N. Bramham J. Biomol NMR Assign. 2009; 3: 49-52Crossref PubMed Scopus (3) Google Scholar). The missing backbone assignments arise from the four non-native amino-terminal residues and three stretches of residues within FIM2 (Cys773–Gly774, Cys776–Trp779, and Asp783–Lys788). There was an absence of observable backbone resonances corresponding to these regions despite exploration of a variety of protein concentrations, ionic strengths, temperatures, pH values, and field strengths. All eight proline residues were judged to be trans on the basis of chemical shift differences δCβ − δCγ and NOEs between Hδ (Pron) and Hα (Pron−1). Initial NMR structures were determined using 2702 NOE-derived restraints. A total of 154 ϕ and ψ dihedral angle restraints were deduced using TALOS (35.Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2738) Google Scholar) and incorporated into subsequent calculations. Early structure calculations containing only NOE data confirmed 28 hydrogen bonds that had been inferred by deuterium exchange; these were incorporated into the next round of calculations. Seven of the nine disulfide bonds inferred from mass spectrometry were clearly identified by supporting NOEs (often Cβ-Cβ) and by the proximity of pairs of cysteines in these initially calculated structures (Cys702–Cys713, Cys715–Cys750, Cys721–Cys743, Cys728–Cys763, Cys791–C825, Cys797–Cys818, and Cys805–Cys838). These disulfide bonds were therefore included (61.Nilges M. J. Mol. Biol. 1995; 245: 645-660Crossref PubMed Scopus (322) Google Scholar) in subsequent refinement calculations. The remaining four cysteines (Cys773, Cys776, Cys782, and Cys789) lie in, or close to, an apparently flexible region of FIM2. Consequently, the complete disulfide bonding pattern of C7-FIMs could not be unambiguously determined from the NMR data; nor could it be uniquely determined by protease digest and mass spectrometry because of the lack of appropriate cleavage sites. In the final structure calculations, disulfide bonds Cys773–Cys782 and Cys776–Cys789 were inferred on the basis of the alignment of FIM2 with FIM1 and in agreement with the previously observed preference for such a linkage arrangement, termed a disulfide β-cross, in other protein structures (62.Harrison P.M. Sternberg M.J. J. Mol. Biol. 1996; 264: 603-623Crossref PubMed Scopus (112) Google Scholar). The ensemble of 25 structures with lowest NOE-derived energies converged well overall, as may be judged from backbone overlays and from the root mean square deviations (RMSDs) of the Cα coordinates and the heavy atoms (Fig. 2, a and b, and Table 1). The initial residues of FIM2 (Cys773, Gly774, Pro777, and Leu778), which lie adjacent to the intermodular linker, form a solvent-exposed loop that did not converge during structure calculations because of the low number of NOEs observed in this region; this is reflected in the high RMSDs of the relevant Cα coordinates (Fig. 2b). Relaxation experiments (T1, T2, and 1H,15N NOE) identified residues Ala767–Ala772, as undergoing motion faster than the overall molecular tumbling (Fig. 2b). These residues are located in the linker region connecting the two modules indicating a significant amount of flexibility here; this is reflected by the lack of convergence observed in this region, depicted in the overlay (Fig. 2a). The surface-exposed loop within the β-hairpin (see below) of FIM2, Asp783–Ser787, is also likely to be very flexible; again there is a dearth of detectable NMR signals for these residues, indicative of conformational movement on the intermediate timescale (milliseconds). However, because of the lack of observed resonances, this dynamic behavior could not be confirmed by NMR relaxation data. The relaxation data also identified two solvent-exposed residues within FIM1 and FIM2 that may be considered mobile: Asn754 within FIM1 and Arg792 within FIM2.TABLE 1Structural statistics for the lowest energy structuresNumber of lowest energy structures25Upper limit distance constraintsIntraresidue618Sequential770Intermediate range (2–4 residues)402Long range (>4)912Total2702Intermodular124FIM1 to linker11FIM2 to linker38Hydrogen bonds28Disulfides explicit9Root mean square deviations (Å)All heavy atoms1.53Backbone atoms Cα, N, COFIM10.74FIM20.94Both modules0.95Skew, twist, and tilt angles (°)28, 161, 100Ramachandran assessment (%)Most favored81.5Additionally allowed17.2Generously allowed1.1Disallowed0.2Coarse packing Whatif score−1.53Buried surface area of intermodular junction (Å2)1237.69" @default.
• W2078046435 created "2016-06-24" @default.
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• W2078046435 date "2009-07-01" @default.
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• W2078046435 title "Solution Structure of Factor I-like Modules from Complement C7 Reveals a Pair of Follistatin Domains in Compact Pseudosymmetric Arrangement" @default.
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• W2078046435 doi "https://doi.org/10.1074/jbc.m901993200" @default.
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