Portrait of an Enzyme, a Complete Structural Analysis of a Multimodular β-N-Acetylglucosaminidase from Clostridium perfringens
2009; Elsevier BV; Volume: 284; Issue: 15 Linguagem: Inglês
10.1074/jbc.m808954200
ISSN1083-351X
AutoresE. Ficko-Blean, K. Gregg, Jarrett Adams, Jan‐Hendrik Hehemann, Mirjam Czjzek, Steven P. Smith, A.B. Boraston,
Tópico(s)Streptococcal Infections and Treatments
ResumoCommon features of the extracellular carbohydrate-active virulence factors involved in host-pathogen interactions are their large sizes and modular complexities. This has made them recalcitrant to structural analysis, and therefore our understanding of the significance of modularity in these important proteins is lagging. Clostridium perfringens is a prevalent human pathogen that harbors a wide array of large, extracellular carbohydrate-active enzymes and is an excellent and relevant model system to approach this problem. Here we describe the complete structure of C. perfringens GH84C (NagJ), a 1001-amino acid multimodular homolog of the C. perfringens μ-toxin, which was determined using a combination of small angle x-ray scattering and x-ray crystallography. The resulting structure reveals unprecedented insight into how catalysis, carbohydrate-specific adherence, and the formation of molecular complexes with other enzymes via an ultra-tight protein-protein interaction are spatially coordinated in an enzyme involved in a host-pathogen interaction. Common features of the extracellular carbohydrate-active virulence factors involved in host-pathogen interactions are their large sizes and modular complexities. This has made them recalcitrant to structural analysis, and therefore our understanding of the significance of modularity in these important proteins is lagging. Clostridium perfringens is a prevalent human pathogen that harbors a wide array of large, extracellular carbohydrate-active enzymes and is an excellent and relevant model system to approach this problem. Here we describe the complete structure of C. perfringens GH84C (NagJ), a 1001-amino acid multimodular homolog of the C. perfringens μ-toxin, which was determined using a combination of small angle x-ray scattering and x-ray crystallography. The resulting structure reveals unprecedented insight into how catalysis, carbohydrate-specific adherence, and the formation of molecular complexes with other enzymes via an ultra-tight protein-protein interaction are spatially coordinated in an enzyme involved in a host-pathogen interaction. Microbial and viral invaders of the human body often exploit host glycans to aid in adherence and then must contend with the protective and structural sugar layers to enable invasion and further spread of the infection. Some of the more spectacular bacterial infections, such as the severe myonecrotic infections caused by Streptococcus pyogenes and Clostridium perfringens, involve extensive tissue destruction (1Bryant A.E. Bayer C.R. Chen R.Y. Guth P.H. Wallace R.J. Stevens D.L. J. Infect. Dis. 2005; 192: 1014-1022Crossref PubMed Scopus (84) Google Scholar, 2Hynes W. Front. Biosci. 2004; 9: 3399-3433Crossref PubMed Scopus (42) Google Scholar, 3Smedley 3rd, J.G. Fisher D.J. Sayeed S. Chakrabarti G. McClane B.A. Rev. Physiol. Biochem. Pharmacol. 2004; 152: 183-204Crossref PubMed Scopus (161) Google Scholar, 4Stevens D.L. Bryant A.E. Clin. Infect. Dis. 2002; 35: S93-S100Crossref PubMed Scopus (97) Google Scholar). The tissue destruction and bacterial spread appears to be aided by a variety of carbohydrate-active enzymes, which break down the polysaccharides of the extracellular matrix or potentiate the activity of other cytolytic toxins (5Canard B. Garnier T. Saint-Joanis B. Cole S.T. Mol. Gen. Genet. 1994; 243: 215-224Crossref PubMed Scopus (67) Google Scholar, 6Flores-Diaz M. Alape-Giron A. Clark G. Catimel B. Hirabayashi Y. Nice E. Gutierrez J.M. Titball R. Thelestam M. J. Biol. Chem. 2005; 280: 26680-26689Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 7Johnson S. Gerding D.N. Rood J.I. McClane B.A. Songer J.G. Titball R.W. The Clostridia Molecular Biology and Pathogenesis. Harcourt Brace & Company, London1997: 117-140Google Scholar, 8Sheldon W.L. Macauley M.S. Taylor E.J. Robinson C.E. Charnock S.J. Davies G.J. Vocadlo D.J. Black G.W. Biochem. J. 2006; 399: 241-247Crossref PubMed Scopus (32) Google Scholar). A large number of other bacterial pathogens also feature carbohydrate-active enzymes as important virulence factors that figure in a variety of roles related to degrading and modifying host glycans (see for example Refs. 9Figura N. J. Clin. Gastroenterol. 1997; 25: S149-S163Crossref PubMed Scopus (35) Google Scholar, 10Galen J.E. Ketley J.M. Fasano A. Richardson S.H. Wasserman S.S. Kaper J.B. Infect. Immun. 1992; 60: 406-415Crossref PubMed Google Scholar, 11Shelburne S.A. Davenport M.T. Keith D.B. Musser J.M. Trends Microbiol. 2008; 16: 318-325Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). A common feature of these microbial carbohydrate-active enzymes involved in processing eukaryotic glycans is their structural complexity; such proteins are frequently very large (over 1000 amino acids) and can comprise numerous modules and domains (12Abbott D.W. Eirin-Lopez J.M. Boraston A.B. Mol. Biol. Evol. 2008; 25: 155-167Crossref PubMed Scopus (52) Google Scholar, 13Adams J.J. Gregg K. Bayer E.A. Boraston A.B. Smith S.P. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 12194-12199Crossref PubMed Scopus (33) Google Scholar, 14Chitayat S. Gregg K. Adams J.J. Ficko-Blean E. Bayer E.A. Boraston A.B. Smith S.P. J. Mol. Biol. 2008; 375: 20-28Crossref PubMed Scopus (15) Google Scholar, 15Ficko-Blean E. Boraston A.B. J. Biol. Chem. 2006; 281: 37748-37757Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 16Thobhani S. Ember B. Siriwardena A. Boons G.J. J. Am. Chem. Soc. 2003; 125: 7154-7155Crossref PubMed Scopus (63) Google Scholar). C. perfringens, a myonecrotic ("flesh-eating") bacterial pathogen and a leading cause of food-borne gastrointestinal illness, deploys numerous protein exo-toxins that work together to increase the pathogen's virulence (3Smedley 3rd, J.G. Fisher D.J. Sayeed S. Chakrabarti G. McClane B.A. Rev. Physiol. Biochem. Pharmacol. 2004; 152: 183-204Crossref PubMed Scopus (161) Google Scholar, 17Rood J.I. Cole S.T. Microbiol. Rev. 1991; 55: 621-648Crossref PubMed Google Scholar). Among its exo-toxin proteins are a considerable battery of large extracellular carbohydrate-active enzymes (supplemental Fig. S1), including the hyaluronidase μ-toxin, which destroys the polysaccharide hyaluronan, and the large sialidases (NanJ and NanI), which remove terminal sialic acid sugars and enhance the lethal cytolytic phospholipase activity of the α-toxin (5Canard B. Garnier T. Saint-Joanis B. Cole S.T. Mol. Gen. Genet. 1994; 243: 215-224Crossref PubMed Scopus (67) Google Scholar, 6Flores-Diaz M. Alape-Giron A. Clark G. Catimel B. Hirabayashi Y. Nice E. Gutierrez J.M. Titball R. Thelestam M. J. Biol. Chem. 2005; 280: 26680-26689Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 18Boraston A.B. Ficko-Blean E. Healey M. Biochemistry. 2007; 46: 11352-11360Crossref PubMed Scopus (77) Google Scholar). The individual modules of such carbohydrate-active enzymes perform a variety of functions, which are most commonly catalysis, protein-carbohydrate interactions, or protein-protein interactions, and these contribute to the overall function and efficiency of the protein. However, because of their large size and flexibility, properties that make them recalcitrant to structural analyses, and their multifunctional natures, such large proteins have required a reductionist approach to their study whereby individual modules are heterologously produced for thorough structural and functional analysis. This has provided considerable insight into the individual functions of the modules. However, how the functions of the modules are spatially coordinated to cooperatively influence the overall activity of the full-length enzymes, knowledge that would have considerable biological relevance, is largely unknown as the complete structures of large carbohydrate-active bacterial virulence factors, for the most part, resist determination. The considerable number of large and multimodular carbohydrate-active enzymes produced by C. perfringens makes this organism an excellent and relevant model system for the study of complex carbohydrate-active enzymes involved in bacterial pathogenesis. GH84C (NagJ) from C. perfringens is a homolog of the μ-toxin that comprises 1001 amino acids and four distinct modules: an N-terminal catalytic module followed by a family 32 carbohydrate-binding module (CBM32), 5The abbreviations used are: CBM32, family 32 carbohydrate-binding module; Coh, cohesin; FN3, fibronectin type III; Doc, dockerin; SAXS, small-angle x-ray scattering; FIVAR, found in various architectural regions module. a cohesin module (Coh), and a C-terminal fibronectin type III module (FN3) (Fig. 1). The structural and biochemical properties of the isolated catalytic module, CBM32, and Coh have been described separately (13Adams J.J. Gregg K. Bayer E.A. Boraston A.B. Smith S.P. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 12194-12199Crossref PubMed Scopus (33) Google Scholar, 14Chitayat S. Gregg K. Adams J.J. Ficko-Blean E. Bayer E.A. Boraston A.B. Smith S.P. J. Mol. Biol. 2008; 375: 20-28Crossref PubMed Scopus (15) Google Scholar, 15Ficko-Blean E. Boraston A.B. J. Biol. Chem. 2006; 281: 37748-37757Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 19Rao F.V. Dorfmueller H.C. Villa F. Allwood M. Eggleston I.M. van Aalten D.M. EMBO J. 2006; 25: 1569-1578Crossref PubMed Scopus (167) Google Scholar). These informative studies have revealed that the catalytic module is similar to human O-GlcNAcase and functions as an exo-β-d-N-acetylglucosaminidase, whereas the CBM32 preferentially recognizes the non-reducing terminus of N-acetyllactosamine (β-d-galactosyl-1,4-β-d-N-acetylglucosamine) bearing carbohydrate receptors (15Ficko-Blean E. Boraston A.B. J. Biol. Chem. 2006; 281: 37748-37757Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 19Rao F.V. Dorfmueller H.C. Villa F. Allwood M. Eggleston I.M. van Aalten D.M. EMBO J. 2006; 25: 1569-1578Crossref PubMed Scopus (167) Google Scholar). The Coh module is perhaps the most unique component, because it functions to recognize and bind ultra-tightly to dockerin modules (Doc), such as that present at the C terminus of the μ-toxin, and plays a role in forming higher order complexes with other large C. perfringens exo-toxins, all of which are thought to contribute to the virulence of this bacterium (13Adams J.J. Gregg K. Bayer E.A. Boraston A.B. Smith S.P. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 12194-12199Crossref PubMed Scopus (33) Google Scholar). However, the overall three-dimensional structure and therefore spatial distribution of the cognate modules of GH84C remain unknown, as do the structure and function of the FN3 module. This information would provide considerable insight into the structural coordination of modular functions in this protein and provide a powerful model for understanding modularity in the carbohydrate-active enzymes of C. perfringens as well as carbohydrate-active virulence factors in other bacterial pathogens, which also frequently display modular complexity. Intact GH84C proved intractable to heterologous production and in vitro analysis. However, a less conventional approach that combined x-ray crystallography and small-angle x-ray scattering (SAXS) was applied in a "dissect-and-build" approach to generate a solution structure model of the complete enzyme. The reconstructed architecture of GH84C in complex with a Doc-containing fragment of the μ-toxin reveals unprecedented insight into how the recognition of substrate by the catalytic module, interaction of a CBM with a carbohydrate receptor, and recruitment of other carbohydrate-active enzymes through a high affinity protein-protein interaction are spatially coordinated in an enzyme involved in a host-pathogen interaction. Furthermore, the methodology employed here should be broadly applicable to a range of large and flexible multimodular proteins. Materials-Unless otherwise stated, chemicals, carbohydrates, glycoproteins, and polysaccharides were purchased from Sigma. Cloning, Protein Production, and Purification-The DNA fragments encoding the modules and combinations thereof from GH84C were amplified by PCR from C. perfringens genomic DNA (ATCC 13124) and, unless previously cloned, cloned into pET 28a(+) using described methods and the primers listed in supplemental Tables S1 and S2 (15Ficko-Blean E. Boraston A.B. J. Biol. Chem. 2006; 281: 37748-37757Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 20Ficko-Blean E. Boraston A.B. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2005; 61: 834-836Crossref PubMed Scopus (19) Google Scholar, 21Ficko-Blean E. Stubbs K.A. Nemirovsky O. Vocadlo D.J. Boraston A.B. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 6560-6565Crossref PubMed Scopus (71) Google Scholar). With the exception of Coh-FN3 and FIVAR-Doc, all constructs encoded polypeptides comprising a hexa-histidine tag fused to the desired module(s) by an N-terminal thrombin protease cleavage site. Coh-FN3 and FIVAR-Doc had C-terminal, non-cleavable hexa-histidine tags. GH84C catalytic module and FIVAR-Doc were produced, purified by immobilized metal affinity chromatography, buffer-exchanged, and concentrated using previously described methods (13Adams J.J. Gregg K. Bayer E.A. Boraston A.B. Smith S.P. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 12194-12199Crossref PubMed Scopus (33) Google Scholar, 20Ficko-Blean E. Boraston A.B. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2005; 61: 834-836Crossref PubMed Scopus (19) Google Scholar, 22Chitayat S. Adams J.J. Furness H.S. Bayer E.A. Smith S.P. J. Mol. Biol. 2008; 381: 1202-1212Crossref PubMed Scopus (13) Google Scholar). This process was followed by further purification by size exclusion chromatography using a Sephacryl S-200 size exclusion column (Amersham Biosciences). The CBM32-Coh·FIVAR-Doc complex was prepared by mixing the two polypeptides with the FIVAR-Doc in ∼1.5-fold molar excess. The complex was purified using a Sephacryl S-200 size exclusion column. Protein concentrations were determined using UV absorbance at 280 nm and calculated extinction coefficients (23Gasteiger E. Gattiker A. Hoogland C. Ivanyi I. Appel R.D. Bairoch A. Nucleic Acids Res. 2003; 31: 3784-3788Crossref PubMed Scopus (3480) Google Scholar). X-ray Crystallography-Crystallization and data collection procedures have been described previously for GH84C catalytic module (20Ficko-Blean E. Boraston A.B. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2005; 61: 834-836Crossref PubMed Scopus (19) Google Scholar). The hexahistidine tag was removed from the GH84C-CBM32 construct by incubation with thrombin protease for 16 h. The mixture was separated on a Sephacryl S-200 size exclusion column in 20 mm Tris-HCl, pH 8.0. All protein constructs were concentrated to 10–25 mg/ml for crystallization trials. Crystallization was performed using the hanging drop vapor diffusion method. GH84C-CBM32 was crystallized at 18 °C in 0.1 m Tris-HCl, pH 7.5, 0.1 m MgCl2, 15% (w/v) polyethylene glycol 4000. These crystals were cryoprotected in the crystallization solution supplemented with 20% ethylene glycol (v/v). The Coh-FN3 modular pair was crystallized in 0.1 m sodium acetate, pH 3.5, 4% (w/v) polyethylene glycol 8000. These crystals were cryoprotected in 20% (w/v) polyethylene glycol 400 with mother liquor. Diffraction data for GH84C-CBM32 was collected with a Rigaku R-AXIS IV++ area detector coupled to an MM-002 x-ray generator with Osmic "blue" optics and an Oxford Cryostream 700 at 113 K. Diffraction data for Coh-FN3 were collected at 100 K on beamline X6A at the National Synchrotron Light Source (Brookhaven National Laboratories). All data were processed with Crystal Clear/d*trek (24Pflugrath J.W. Acta Crystallogr. D Biol. Crystallogr. 1999; 55: 1718-1725Crossref PubMed Scopus (1417) Google Scholar). Data collection statistics are given in Table 1.TABLE 1X-ray crystal diffraction data collection and refinement statisticsGH84C-CBMGH84C catalyticCoh-FN3Data collectionSpace groupP21I212121C2Cell dimensionsa, b, c (Å)46.85, 69.54, 136.78130.39, 150.05, 155.43148.25, 48.64, 36.90α, β, γ (°)90.00, 95.78, 90.0090.00, 90.00, 90.0090.00, 96.19, 90.00Resolution (Å)20.00-3.30 (3.39-3.30)aValues in parentheses are for the highest resolution shells.20.00-2.10 (2.15-2.10)20.00-1.80 (1.86-1.80)Rmerge0.114 (0.287)0.103 (0.397)0.030 (0.170)I/σI4.4 (2.2)12.4 (4.8)30.0 (8.0)Completeness (%)92.8 (94.7)99.9 (100.0)95.7 (77.9)Redundancy2.5 (2.5)16.1 (15.7)7.1 (6.1)RefinementNo. reflections31,4971,384,042166,316Unique reflectionsbAll F > 2σF.12,42085,96523,338Rwork/Rfree0.325/0.3700.198/0.2550.224/0.294No. atomsProtein5,6774,644 (A); 4,632 (B)1750Ligands3 (Ca)4 (Ca); 3 (Na); 25 (CAC)4 (ACT)Water281,195318B-factors (Å2)Protein52.829.3 (A); 32.1 (B)28.3Ligands71.8 (Ca)44.3 (Ca); 22.0 (Na); 71.7 (CAC)30.2 (ACT)Water27.534.338.7r.m.s.d.Bond lengths (Å)0.0060.0100.018Bond angles (°)0.9341.3891.732Ramachandran statistics (%)Favored84.190.491.6Allowed15.59.78.4Disallowed0.50.00.0a Values in parentheses are for the highest resolution shells.b All F > 2σF. Open table in a new tab The structure of the GH84C catalytic module was solved by molecular replacement using PHASER (25Zwart P.H. Afonine P.V. Grosse-Kunstleve R.W. Hung L.W. Ioerger T.R. McCoy A.J. McKee E. Moriarty N.W. Read R.J. Sacchettini J.C. Sauter N.K. Storoni L.C. Terwilliger T.C. Adams P.D. Methods Mol. Biol. 2008; 426: 419-435Crossref PubMed Scopus (437) Google Scholar) to find the positions of two GH84C molecules in the asymmetric unit. The preliminary coordinates of the family 84 glycoside hydrolase from Bacteroides thetaiotaomicron, kindly provided by Dr. Gideon Davies prior to deposition (PDB accession code 2J47) (26Dennis R.J. Taylor E.J. Macauley M.S. Stubbs K.A. Turkenburg J.P. Hart S.J. Black G.N. Vocadlo D.J. Davies G.J. Nat. Struct. Mol. Biol. 2006; 13: 365-371Crossref PubMed Scopus (165) Google Scholar), was used as a search model. Three iterations of manual model correction using COOT (27Emsley P. Cowtan K. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23389) Google Scholar), refinement with REFMAC (28Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13870) Google Scholar), and solvent flattening with 2-fold non-crystallographic symmetry averaging using DM (29Cowtan K.D. Zhang K.Y. Prog. Biophys. Mol. Biol. 1999; 72: 245-270Crossref PubMed Scopus (242) Google Scholar) were required to obtain a model of roughly 60% completeness with partially built side chains. Phases from this process were then sufficient for ARP/ wARP (30Morris R.J. Perrakis A. Lamzin V.S. Acta Crystallogr. D Biol. Crystallogr. 2002; 58: 968-975Crossref PubMed Scopus (221) Google Scholar) to build a virtually complete model with docked side chains, which was then manually completed using COOT and refinement with REFMAC. Waters were added using the ARP/wARP option in REFMAC. The GH84C-CBM32 modular pair structure was solved by molecular replacement by first running MOLREP (31Vagin A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4153) Google Scholar) using the GH84C catalytic module as a search model to find the single molecule in the asymmetric unit. MOLREP was run a second time to place the CBM32 model (PDB accession 2J7M) in the asymmetric unit. Successive rounds of model correction were done using COOT and refinement with REFMAC. A limited number of waters was added manually. Due to substantial disorder in portions of this structure rather than use a bulk B-factor, we opted to refine individual B-factors. The structure of the Coh-FN3 modular pair was determined by molecular replacement using PHASER and the coordinates of the isolated Coh module (PDB accession 2O4E) as a search model to find the single molecule of Coh-FN3 in the asymmetric unit. Although the molecular replacement solution contained only ∼50% of the asymmetric unit contents, the initial phases from restrained refinement with REFMAC were of sufficient quality for ARP/wARP to build a complete model, including the FN3 module, with docked side chains. Model correction was done manually using COOT and refinement with REFMAC. Waters were added using the ARP/wARP option in REFMAC. In all cases, 5% of the reflections were flagged as "free" to monitor refinement procedures and judge model quality (32Brunger A.T. Nature. 1992; 355: 472-475Crossref PubMed Scopus (3864) Google Scholar). Model validation was performed with SFCHECK (33Vaguine A.A. Richelle J. Wodak S.J. Acta Crystallogr. D Biol. Crystallogr. 1999; 55: 191-205Crossref PubMed Scopus (859) Google Scholar) and PROCHECK (34Roman A. Laskowski M.W.M. Moss D.S. Thornton J.M. J. App. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). All model statistics are shown in Table 1. The coordinates and structure factors for the GH84C catalytic module, GH84C-CBM32, and Coh-FN3 have been deposited with the pdb codes of 2v5c, 2v5d, and 2w1n, respectively. SAXS-SAXS data were collected at the X33 beamline of the European Molecular Biology Laboratory (Deutsches Electronen Synchrotron, Hamburg) using an MAR345 image plate detector or a Pilatus 500K detector. A 4.2 mg/ml solution of bovine serum albumin was measured as a reference and for calibration. The scattering patterns were measured with an exposure time of 2 min at 288 K. The wavelength was 1.5 Å. The sample-to-detector distance was set at 2.4 m, leading to scattering vectors q (defined as q = 4π /λ sinθ, where 2θ is the scattering angle) ranging from 0.06 Å–1 to 0.5 Å–1. The concentration of the protein samples ranged from 0.92 mg/ml to 13.48 mg/ml, depending on the protein, and each protein was measured at three to five concentrations. Background scattering was measured after each protein sample using the buffer solution and subsequently subtracted from the protein scattering patterns after proper normalization and correction from detector response. The radii of gyration (Rg) were derived from the Guinier approximation: I(q) = I(0) exp(–q2R2g/3), where I(q) is the scattered intensity and I(0) is the forward scattered intensity (35Guinier A.F. Fournet F. Small Angle Scattering of X-rays. Wiley Interscience, New York1955Google Scholar). The radius of gyration and I(0) are inferred from the slope and the intercept, respectively, of the linear fit of ln[I(q)] versus q2 in the q-range q·Rg < 1.12. At low angles, the scattered intensities were very well approximated by the Guinier law, and revealed some repulsive interparticle interactions at high concentrations. All scattering curves were indicative of monomeric states of the molecules in solution. The distance distribution function P(r) was calculated on the merged curve by the Fourier inversion of the scattering intensity I(q) using GNOM (36Svergun D. J. Appl. Crystallogr. 1992; 25: 495-503Crossref Scopus (2971) Google Scholar) and GIFT (37Bergmann A. Fritz G. Glatter O. J. Appl. Crystallogr. 2000; 33: 1212-1216Crossref Scopus (213) Google Scholar). The low resolution shapes of the protein constructs were determined ab initio from the scattering curve using the program GASBOR (38Svergun D.I. Petoukhov M.V. Koch M.H. Biophys. J. 2001; 80: 2946-2953Abstract Full Text Full Text PDF PubMed Scopus (1141) Google Scholar). Several independent fits were run with no symmetry restriction, and the stability of the solution was checked. These solutions were subsequently compared with the program DAMAVER (39Volkov V.V. Svergon D.I. J. Appl. Crystallogr. 2003; 36: 860-864Crossref Scopus (1618) Google Scholar), which computes the normalized spatial discrepancy value for the various obtained shapes (40Putnam C.D. Hammel M. Hura G.L. Tainer J.A. Q. Rev. Biophys. 2007; 40: 191-285Crossref PubMed Scopus (891) Google Scholar). In all cases, calculations led to highly similar forms with normalized spatial discrepancy values ranging between 0.8 and 1.3. The atomic crystallographic structures of the individual modules were positioned in the envelopes using PyMOL. For each structural model obtained the theoretical SAXS profile, the Rg, and the corresponding fit to the experimental data were calculated using the program CRYSOL (41Petoukhov M.V. Eady N.A. Brown K.A. Svergun D.I. Biophys. J. 2002; 83: 3113-3125Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). The goodness of fit for all atomic models, as well as the low resolution models, with the experimental data were determined using the discrepancy χ, defined according to Konarev et al. (42Konarev P.V. Volkov V.V. Sokolova A.V. Koch M.H.J. Svergun D.I. J. Appl. Crystallogr. 2003; 36: 1277-1282Crossref Scopus (2359) Google Scholar). SAXS data are summarized in Table 2, and experimental SAXS curves and their fits are shown in supplemental Figs. S2 and S3.TABLE 2Structural parameters of GH84C constructs obtained by SAXSConstruct (module boundariesaConstruct boundaries are shown relative to the amino acid numbering in the intact GH84C protein.)Molecular massbValues are calculated including the N- or C-terminal six-histidine tags.Number of amino acidsbValues are calculated including the N- or C-terminal six-histidine tags.RgDmaxAb initio modelingdx (GASB)Normalized spatial discrepancykDaÅÅGH84C-CBM32-Coh (31-909)10090237 ± 1116 ± 31.01.3GH84C-CBM32 (31-767)82.376035 ± 6103 ± 62.31.3CBM32-Coh (625-909)32.430726 ± 385 ± 31.70.9CBM32-Coh·FIVAR-Doc (625-909/NAcNA, not applicable.)47.944731 ± 395 ± 31.41.2Coh-FN3 (765-1001)27.024637 ± 6118 ± 6NDdND, not determined.NDBovine serum albumin66.358231 ± 190 ± 2NDNDa Construct boundaries are shown relative to the amino acid numbering in the intact GH84C protein.b Values are calculated including the N- or C-terminal six-histidine tags.c NA, not applicable.d ND, not determined. Open table in a new tab Structure of the GH84C Catalytic Module-To determine the overall architecture of the enzyme we dissected GH84C into a series of modular combinations. These constructs were successfully overproduced in Escherichia coli and purified in sufficient quantities for structural analysis by x-ray crystallography and SAXS (see "Experimental Procedures" for details). The catalytic module construct of GH84C comprising the residues 41–624 of the full protein sequence was crystallized, and its structure was determined by x-ray crystal diffraction to 2.1 Å (Table 1 and Fig. 2A). This fragment of GH84C comprises three domains that are not distinguishable through amino acid-based sequence comparisons. The N-terminal domain (amino acids 41–177) is a mixed β-sheet structure consisting of six β-strands sandwiched between three α-helices; two helices on one face of the β-sheet and one helix on the other. A central domain (amino acids 178–470) adopts a (β/α)8 barrel lacking the 7th helix, whereas the C-terminal domain (amino acids 471–624) is an elongated five-helix bundle (Fig. 2A). This is identical to the structure of a GH84C fragment from C. perfringens strain 13 reported by Rao et al. to 2.25 Å, and thus the additional structural and functional properties of this catalytic region have been discussed in detail previously (19Rao F.V. Dorfmueller H.C. Villa F. Allwood M. Eggleston I.M. van Aalten D.M. EMBO J. 2006; 25: 1569-1578Crossref PubMed Scopus (167) Google Scholar). Positioning the Carbohydrate-binding Module-A GH84C fragment comprising the catalytic module and the adjacent CBM32, herein called GH84C-CBM32, was also crystallized, and its structure was determined by x-ray crystallography to 3.3-Å resolution (Table 1 and Fig. 2B). Although these data were measured to comparatively low resolution the availability of the high resolution structures of the isolated catalytic module and CBM32, the latter previously determined at resolutions as high as 1.4 Å (15Ficko-Blean E. Boraston A.B. J. Biol. Chem. 2006; 281: 37748-37757Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), made the placement of the modules by molecular replacement relatively facile and accurate. Despite the lack of excessively high mosaicity (<1) or anisotropy in the data, the refined R-factors for this structure remained relatively high at over 30%. This structure displayed substantial disorder in the N-terminal domain and portions of the (α/β)8 barrel. Indeed, the Wilson B-factor for this data were 62 Å2, which was roughly in keeping with the average refined B-factor of 52 Å2 for the protein; however, the B-factors for the N-terminal domain and portions of the (α/β)8 barrel approached 65–75 Å2 indicating their relative disorder. Thus, we primarily attributed the high R-factors to this disorder, which made it difficult to accurately model portions of the structure. This structure is important, however, not for the accurate appointment of individual atoms but for its disclosure of the relative placement of the protein's domains. The GH84C-CBM32 structure reveals the positioning of the CBM32 at the extremity of the rigid α-helical linker domain with the long axis of the CBM32 at roughly right angles to the axis of the α-helical bundle (Fig. 2B). The α-helical bundle and the CBM32 are separated by a short linker, which was clearly visible in electron density maps thus allowing unambiguous assignment of the CBM32 to the correct catalytic module in the crystal lattice. The C terminus of the CBM32 is positioned approximately at the tip of the α-helical bundle where, in the intact enzyme, the Coh module would immediately begin (Fig. 2B). The solution conformation of the GH84C-CBM32 modular pair was also analyzed by SAXS (Table 2). The ab initio calculation of SAXS molecular envelopes for GH84C-CBM32 consistently yielded extended forms comprising a large globular region and a smaller region (Fig. 2C). An atomic mod
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