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Structural and Functional Relationships in the Virulence-associated Cathepsin L Proteases of the Parasitic Liver Fluke, Fasciola hepatica

2007; Elsevier BV; Volume: 283; Issue: 15 Linguagem: Inglês

10.1074/jbc.m708521200

ISSN

1083-351X

Autores

Colin M. Stack, Conor R. Caffrey, Sheila Donnelly, Amritha Seshaadri, Jonathan Lowther, José F. Tort, Peter R. Collins, Mark W. Robinson, Weibo Xu, James H. McKerrow, Charles S. Craik, S. Geiger, Rachel Marion‐Letellier, Linda S. Brinen, John P. Dalton,

Tópico(s)

Parasite Biology and Host Interactions

Resumo

The helminth parasite Fasciola hepatica secretes cysteine proteases to facilitate tissue invasion, migration, and development within the mammalian host. The major proteases cathepsin L1 (FheCL1) and cathepsin L2 (FheCL2) were recombinantly produced and biochemically characterized. By using site-directed mutagenesis, we show that residues at position 67 and 205, which lie within the S2 pocket of the active site, are critical in determining the substrate and inhibitor specificity. FheCL1 exhibits a broader specificity and a higher substrate turnover rate compared with FheCL2. However, FheCL2 can efficiently cleave substrates with a Pro in the P2 position and degrade collagen within the triple helices at physiological pH, an activity that among cysteine proteases has only been reported for human cathepsin K. The 1.4-Å three-dimensional structure of the FheCL1 was determined by x-ray crystallography, and the three-dimensional structure of FheCL2 was constructed via homology-based modeling. Analysis and comparison of these structures and our biochemical data with those of human cathepsins L and K provided an interpretation of the substrate-recognition mechanisms of these major parasite proteases. Furthermore, our studies suggest that a configuration involving residue 67 and the “gatekeeper” residues 157 and 158 situated at the entrance of the active site pocket create a topology that endows FheCL2 with its unusual collagenolytic activity. The emergence of a specialized collagenolytic function in Fasciola likely contributes to the success of this tissue-invasive parasite. The helminth parasite Fasciola hepatica secretes cysteine proteases to facilitate tissue invasion, migration, and development within the mammalian host. The major proteases cathepsin L1 (FheCL1) and cathepsin L2 (FheCL2) were recombinantly produced and biochemically characterized. By using site-directed mutagenesis, we show that residues at position 67 and 205, which lie within the S2 pocket of the active site, are critical in determining the substrate and inhibitor specificity. FheCL1 exhibits a broader specificity and a higher substrate turnover rate compared with FheCL2. However, FheCL2 can efficiently cleave substrates with a Pro in the P2 position and degrade collagen within the triple helices at physiological pH, an activity that among cysteine proteases has only been reported for human cathepsin K. The 1.4-Å three-dimensional structure of the FheCL1 was determined by x-ray crystallography, and the three-dimensional structure of FheCL2 was constructed via homology-based modeling. Analysis and comparison of these structures and our biochemical data with those of human cathepsins L and K provided an interpretation of the substrate-recognition mechanisms of these major parasite proteases. Furthermore, our studies suggest that a configuration involving residue 67 and the “gatekeeper” residues 157 and 158 situated at the entrance of the active site pocket create a topology that endows FheCL2 with its unusual collagenolytic activity. The emergence of a specialized collagenolytic function in Fasciola likely contributes to the success of this tissue-invasive parasite. Clan CA papain-like cysteine peptidases, such as cathepsins B and L (1Rawlings N.D. Morton F.R. Barrett A.J. Nucleic Acids Res. 2006; 34: D270-D272Crossref PubMed Scopus (464) Google Scholar), are ubiquitous in helminth (worm) parasites of human and veterinary importance. These peptidases are involved in a variety of pathogen-specific functions, including penetration and migration through host tissues, catabolism of host proteins to peptides and amino acids, and modulation or suppression of host immune defenses by cleaving immunoglobulin or altering the activity of immune effector cells (2Tort J. Brindley P.J. Knox D. Wolfe K.H. Dalton J.P. Adv. Parasitol. 1999; 43: 161-266Crossref PubMed Google Scholar, 3Sajid M. Mckerrow J.H. Mol. Biochem. Parasitol. 2002; 120: 1-21Crossref PubMed Scopus (665) Google Scholar, 4Dalton J.P. Caffrey C.R. Sajid M. Stack C. Donnelly S. Loukas A. Don T. McKerrow J. Halton D.W. Brindley P.J. Maule A.G. Marks N.J. Parasitic Flatworms: Molecular Biology, Biochemistry, Immunology, and Physiology. CAB International, Wallingford, Oxon, UK2006: 1-36Google Scholar). The central role of Clan CA proteases in the survival of helminth parasites has positioned them as lead targets for the development of new chemotherapies and vaccines (5Wasilewski M.M. Lim K.C. Philips J. McKerrow J.H. Mol. Biochem. Parasitol. 1996; 81: 179-189Crossref PubMed Scopus (124) Google Scholar, 6Dalton J.P. O'Neill S.M. Stack C. Collins P. Walsh A. Sekiya M. Doyle S. Mulcahy G. Hoyle D. Khaznadji E. Moire N. Brennan G. Mousley A. Kreshchenko N. Maule A. Donnelly S. Int. Parasitol. 2003; 33: 1173-1181Crossref PubMed Scopus (224) Google Scholar, 7Abdulla M.H. Lim K.C. Sajid M. McKerrow J.H. Caffrey C.R. Plos Med. 2007; 4: e14Crossref PubMed Scopus (219) Google Scholar).Fasciola hepatica is a helminth parasite that causes liver fluke disease (fasciolosis) in cattle and sheep worldwide. It is most prevalent in Europe with infection rates increasing because of the emergence of drug-resistant parasites and possibly as a result of climate change (8Mitchell G.B. Maris L. Bonniwell M.A. Vet. Rec. 1998; 143: 399PubMed Google Scholar, 9Borgsteed F.H. Moll L. Vellema P. Gaasenbeek C.P. Vet. Rec. 2005; 156: 350-351Crossref PubMed Scopus (33) Google Scholar). Human fasciolosis has recently emerged as a major zoonosis in rural areas of South America (particularly Bolivia, Peru, and Equador), Egypt, and Iran where organized farm management practices are poor. It is estimated that worldwide over 2.4 million people are infected with F. hepatica and about 180 million are at risk of infection (10Mas-Coma S. Bargues M.D. Valero M.A. Int. J. Parasitol. 2005; 35: 1255-1278Crossref PubMed Scopus (642) Google Scholar, 11MacManus D.P. Dalton J.D. Parasitology. 2006; 133: S43-S61Crossref PubMed Scopus (160) Google Scholar).Secretion of cysteine proteases is associated with the virulence of F. hepatica and its capacity to infect a wide range of mammalian hosts (4Dalton J.P. Caffrey C.R. Sajid M. Stack C. Donnelly S. Loukas A. Don T. McKerrow J. Halton D.W. Brindley P.J. Maule A.G. Marks N.J. Parasitic Flatworms: Molecular Biology, Biochemistry, Immunology, and Physiology. CAB International, Wallingford, Oxon, UK2006: 1-36Google Scholar, 6Dalton J.P. O'Neill S.M. Stack C. Collins P. Walsh A. Sekiya M. Doyle S. Mulcahy G. Hoyle D. Khaznadji E. Moire N. Brennan G. Mousley A. Kreshchenko N. Maule A. Donnelly S. Int. Parasitol. 2003; 33: 1173-1181Crossref PubMed Scopus (224) Google Scholar, 12Collins P.R. Stack C.M. O'Neill S.M. Doyle S. Ryan T. Brennan G.P. Mousley A. Stewart M. Maule A.G. Dalton J.P. Donnelly S. J. Biol. Chem. 2004; 279: 17038-17046Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 13Irving J.A. Spithill T.W. Pike R.N. Whisstock J.C. Smooker P.M. J. Mol. Evol. 2003; 57: 1-15Crossref PubMed Scopus (60) Google Scholar, 14Beckham S.A. Law R.H. Smooker P.M. Quinsey N.S. Caffrey C.R. McKerrow J.H. Pike R.N. Spithill T.W. Biol. Chem. 2006; 387: 1053-1061Crossref PubMed Scopus (33) Google Scholar). Cathepsin L1 (FheCL1) and cathepsin L2 (FheCL2) are the two major peptidases secreted by the infective larvae that traverse the host intestinal wall, by the migratory stages that penetrate the liver tissues, and by the mature adult parasites that reside in the bile ducts and feed on host blood, which they ingest through the punctured bile duct wall (4Dalton J.P. Caffrey C.R. Sajid M. Stack C. Donnelly S. Loukas A. Don T. McKerrow J. Halton D.W. Brindley P.J. Maule A.G. Marks N.J. Parasitic Flatworms: Molecular Biology, Biochemistry, Immunology, and Physiology. CAB International, Wallingford, Oxon, UK2006: 1-36Google Scholar, 6Dalton J.P. O'Neill S.M. Stack C. Collins P. Walsh A. Sekiya M. Doyle S. Mulcahy G. Hoyle D. Khaznadji E. Moire N. Brennan G. Mousley A. Kreshchenko N. Maule A. Donnelly S. Int. Parasitol. 2003; 33: 1173-1181Crossref PubMed Scopus (224) Google Scholar, 15Hanna R.E. Trudgett A.G. Parasite Immunol. 1983; 5: 409-425Crossref PubMed Scopus (47) Google Scholar). Experiments using purified native enzymes demonstrated that FheCL1 and FheCL2 efficiently degrade host hemoglobin, immunoglobulin, and interstitial matrix proteins such as fibronectin, laminin, and native collagen (6Dalton J.P. O'Neill S.M. Stack C. Collins P. Walsh A. Sekiya M. Doyle S. Mulcahy G. Hoyle D. Khaznadji E. Moire N. Brennan G. Mousley A. Kreshchenko N. Maule A. Donnelly S. Int. Parasitol. 2003; 33: 1173-1181Crossref PubMed Scopus (224) Google Scholar, 16Berasain P. Goni F. McGonigle S. Dowd A. Dalton J.P. Frangione B. Carmona C. J. Parasitol. 1997; 83: 1-5Crossref PubMed Scopus (120) Google Scholar, 17Berasain P. Carmona C. Frangione B. Dalton J.P. Goni F. Exp. Parasitol. 2000; 94: 99-110Crossref PubMed Scopus (114) Google Scholar). Although FheCL1 and FheCL2 exhibited similar substrate specificities, FheCL2 showed a greater affinity for peptides containing Pro residues in the P2 position (18Dowd A.J. Smith A.M. McGonigle S. Dalton J.P. Eur. J. Biochem. 1994; 223: 91-98Crossref PubMed Scopus (99) Google Scholar, 19Dowd A.J. McGonigle S. Dalton J.P. Eur. J. Biochem. 1995; 232: 241-260Crossref PubMed Scopus (43) Google Scholar, 20Dowd A.J. Tort J. Roche L. Ryan T. Dalton J.P. Mol. Biochem. Parasitol. 1997; 88: 163-174Crossref PubMed Scopus (54) Google Scholar). We proposed that by producing proteases with overlapping specificity the parasite could digest these host macromolecules more efficiently, and therefore more effectively penetrate host organs (6Dalton J.P. O'Neill S.M. Stack C. Collins P. Walsh A. Sekiya M. Doyle S. Mulcahy G. Hoyle D. Khaznadji E. Moire N. Brennan G. Mousley A. Kreshchenko N. Maule A. Donnelly S. Int. Parasitol. 2003; 33: 1173-1181Crossref PubMed Scopus (224) Google Scholar, 16Berasain P. Goni F. McGonigle S. Dowd A. Dalton J.P. Frangione B. Carmona C. J. Parasitol. 1997; 83: 1-5Crossref PubMed Scopus (120) Google Scholar).The F. hepatica cathepsin Ls belong to a lineage that eventually gave rise to the mammalian cathepsin Ls from which the mammalian cathepsin Ks diverged (2Tort J. Brindley P.J. Knox D. Wolfe K.H. Dalton J.P. Adv. Parasitol. 1999; 43: 161-266Crossref PubMed Google Scholar). Mammalian cathepsin L is ubiquitously expressed in tissues and performs a housekeeping function in protein turnover, but it also plays a part in more specialized functions such as antigen processing and presentation, hormone and protease activation, and extracellular matrix turnover (21Ishidoh K. Kominami E. Biol. Chem. 1998; 379: 131-135PubMed Google Scholar). Cathepsin K, on the other hand, exhibits a more restricted expression profile being predominantly found in osteoclasts but also in multinucleated giant cells, macrophages, and lung epithelial cells (22Drake F.H. Dodds R.A. James I.E. Connor J.R. Debouck C. Richardson S. Lee-Rykaczewski E. Coleman L. Rieman D. Barthlow R. Hastings G. Gowen M. J. Biol. Chem. 1996; 271: 12511-12516Abstract Full Text Full Text PDF PubMed Scopus (617) Google Scholar, 23Buhling F. Reisenauer A. Gerber A. Kruger S. Weber E. Brömme D. Roessner A. Ansorge S. Welte T. Rocken C. J. Pathol. 2001; 195: 375-382Crossref PubMed Scopus (83) Google Scholar). A specific role for cathepsin K in bone resorption by osteoclasts has been related to the ability of the protease to cleave the covalently linked triple helices of native collagen, a unique property among the mammalian papain-like cysteine proteases (24Atley L.M. Mort J.S. Lalumiere M. Eyre D.R. Bone (Elmsford). 2000; 26: 241-247Crossref PubMed Scopus (84) Google Scholar). This unusual property was attributed to the presence of a tyrosine residue at position 67 within the S2 subsite of cathepsin K that interacts with proline in the P2 of substrates, including the Gly-Pro-Xaa repeat sequence (where Xaa is mainly proline or 4-trans-l-hydroxyproline) found in collagen. A parallel therefore exists between mammalian cathepsin K and the F. hepatica FheCL2 as the latter can also cleave substrates with a P2 proline and possesses a tyrosine residue at the corresponding position 67.To understand the role of the major secreted cathepsin L proteases of F. hepatica in the virulence of the parasite and its adaptation to various hosts, it is important to elucidate their biochemical properties and relate these to structure and function. Therefore, in this study, we have characterized the substrate specificity of active recombinant forms of FheCL1 and FheCL2. These properties were further explored by preparing variants of FheCL1 in which specific substitutions were made within the S2 subsite of the active site (positions 67 and 205) to simulate those residues present in human cathepsins L and K. In addition, the 1.4-Å three-dimensional structure of a variant FheCL1 zymogen, in which the active site Cys was replaced by a Gly (FheproCL1Gly25), has been determined by x-ray crystallography. For FheCL2, the three-dimensional structure has been constructed via homology-based modeling. Analysis and comparison of these major parasite proteases with the human cathepsins L and K provide a structural interpretation of the substrate-recognition mechanisms.EXPERIMENTAL PROCEDURESMaterials—Z-Phe-Arg-NHMec, 6The abbreviations used are:Z-Phe-Arg-NHMecbenzyloxycarbonyl-l-phenylalanyl-l-arginine-4-methylcourmarinyl-7-amideDTTdithiothreitolE-64trans-epoxysuccinyl-l-leucylamido(4-guanidino)butanePS-SCLpositional scanning synthetic combinatorial libraryZ-Phe-Ala-CHN2benzyloxycarbonyl-l-phenylalanyl-l-alanine-diazomethyl ketoneTostosylPBSphosphate-buffered salineBoct-butoxycarbonylPDBProtein Data Bank. Z-Leu-Arg-NHMec, Z-Pro-Arg-NHMec, Z-Val-Pro-Arg-NHMec, Z-Gly-Pro-Arg-NHMec, Z-Ala-Gly-Pro-Arg-NHMec, Z-Phe-Arg-NHMec, Z-Gly-Pro-Lys-NHMec, and Z-Phe-Ala-CHN2 were obtained from Bachem (St. Helens, UK). Z-Leu-Arg-NHMec was purchased from Peptide Institute Inc. (Japan). E-64, DTT, and EDTA were obtained from Sigma. Cathepsin K inhibitor II was purchased from BD Biosciences. Prestained molecular weight markers and the AvrII and SnaBI restriction enzymes were obtained from New England Biolabs. Primers were obtained from Sigma-Genosys. The pPIC9K vector and Pichia pastoris strain GS115 were obtained from Invitrogen. Nickel-nitrilotriacetic acid-agarose and columns were obtained from Qiagen (Crawley, UK). Collagen, calf skin, was purchased from Calbiochem. Precast 4-20% gradient SDS-polyacrylamide gels were purchased from Gradipore (Australia).Expression and Purification of Recombinant Cathepsin L Zymogens in Yeast—F. hepatica procathepsin L1 (FheCL1) and procathepsin L2 (FheCL2) were amplified by PCR from the pAAH5 Saccharomyces cerevisiae expression vector into which the full-length cDNA had been cloned previously in our laboratory (12Collins P.R. Stack C.M. O'Neill S.M. Doyle S. Ryan T. Brennan G.P. Mousley A. Stewart M. Maule A.G. Dalton J.P. Donnelly S. J. Biol. Chem. 2004; 279: 17038-17046Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 25Roche L. Dowd A.J. Tort J. McGonigle S. MacSweeney A. Curley G.P. Ryan T. Dalton J.P. Eur. J. Biochem. 1997; 232: 241-246Google Scholar). FheCL1 variants (FheCL1 L67Y and FheCL1 L205A) were synthesized and incorporated an SnaBI restriction site at the 5′ end of the gene and an AvrII restriction site and His6 tag sequence at the 3′ end (Geneart, Regensburg, Germany). The 980-bp fragments were ligated into pCR-Script cloning vector (Stratagene), which were transformed into competent Escherichia coli for amplification. Inserts were digested from plasmid preparations with AvrII and SnaBI and inserted in-frame with the yeast α-factor at the AvrII/SnaBI site of P. pastoris expression vector pPIC9K (Invitrogen). Plasmids were linearized with SacI and then transformed into chemically competent GS115 cells (Invitrogen) as described previously (12Collins P.R. Stack C.M. O'Neill S.M. Doyle S. Ryan T. Brennan G.P. Mousley A. Stewart M. Maule A.G. Dalton J.P. Donnelly S. J. Biol. Chem. 2004; 279: 17038-17046Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). All inserts were sequenced to ensure congruence with original cDNAs.P. pastoris yeast transformants were cultured in 500 ml of buffered glycerol complex medium broth, buffered to pH 8.0, in 5-liter baffled flasks at 30 °C until an A600 of 2-6 was reached (12Collins P.R. Stack C.M. O'Neill S.M. Doyle S. Ryan T. Brennan G.P. Mousley A. Stewart M. Maule A.G. Dalton J.P. Donnelly S. J. Biol. Chem. 2004; 279: 17038-17046Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Cells were harvested by centrifugation at 2000 × g for 5 min, and protein expression was induced by resuspending in 100 ml of buffered minimal methanol medium broth, buffered at pH 6.0 containing 1% methanol (20Dowd A.J. Tort J. Roche L. Ryan T. Dalton J.P. Mol. Biochem. Parasitol. 1997; 88: 163-174Crossref PubMed Scopus (54) Google Scholar). Recombinant proteins were purified from yeast medium by affinity chromatography using nickel-nitrilotriacetic acid-agarose (Qiagen) (12Collins P.R. Stack C.M. O'Neill S.M. Doyle S. Ryan T. Brennan G.P. Mousley A. Stewart M. Maule A.G. Dalton J.P. Donnelly S. J. Biol. Chem. 2004; 279: 17038-17046Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 26Stack C.M. Donnelly S. Lowther J. Xu W. Collins P.R. Brinen L.S. Dalton J.P. J. Biol. Chem. 2007; 282: 16532-16543Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Purified recombinant zymogens were dialyzed against phosphate-buffered saline (PBS) and stored at -20 °C. The 37-kDa zymogens were autocatalytically activated and processed to 24.5-kDa mature enzymes by incubation for 2 h at 37 °C in 0.1 m sodium citrate buffer, pH 5.0, containing 2 mm DTT and 2.5 mm EDTA. The mixture was then dialyzed against PBS, pH 7.3. The proportion of functionally active recombinant protein in these preparations was determined by titration against E-64.P1-P4 Specificity Using a Positional Scanning Synthetic Combinatorial Library—The substrate specificities of FheCL1, FheCL1 L67Y, and FheCL1 L205A and FheCL2 were determined using a complete diverse positional scanning synthetic combinatorial library (PS-SCL) (27Choe Y. Leonetti F. Greenbaum D.C. Lecaille F. Bogyo M. Brömme D. Ellman J.A. Craik C.S. J. Biol. Chem. 2006; 281: 12824-12832Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar). Screens were performed at 25 °C in 0.1 m sodium acetate, 0.1 m NaCl, 0.01 m DTT, 0.001 m EDTA, 0.01% Brij-35, 1% Me2SO (from the substrates), pH 5.5. Aliquots of 25 nmol in 1 μl from each of 20 sub-libraries of the P1, P2, P3, and P4 libraries were added to the wells of a 96-well Microfluor-1 U-bottom plate (Dynex Technologies). The final concentration of each compound of the 8000 compounds per well was 31.25 nm in a 100-μl final reaction volume. The assays were initiated by addition of preactivated enzyme, and the reaction was monitored with a SpectraMax Gemini fluorescence spectrometer (Molecular Devices) with excitation at 380 nm, emission at 460 nm, and cutoff at 435 nm. Screens were performed in duplicate and triplicate for wild type and mutated enzymes, respectively.Enzyme Assays and Kinetics with Fluorogenic Peptide Substrates—Initial rates of hydrolysis of the fluorogenic dipeptide substrates were measured by monitoring the release of the fluorogenic leaving group, NHMec, at an excitation wavelength of 380 nm and an emission wavelength of 460 nm using a Bio-Tek KC4 microfluorometer. kcat and Km values were determined using nonlinear regression analysis. Initial rates were obtained at 37 °C over a range of substrate concentrations spanning Km values (0.2-200 μm) and at fixed enzyme concentrations (0.5-5 nm). Assays were performed in PBS, pH 7.3, and 100 mm sodium acetate buffer, pH 5.5, each containing 2.5 mm DTT and 2.5 mm EDTA.Rate constants for the inactivation of enzyme by Z-Phe-Ala-CHN2 and cathepsin K inhibitor II were determined from progress curves in the presence of substrate (28Morrison J.F. Walsh C.T. Adv. Enzymol. 1988; 61: 201-301PubMed Google Scholar, 29Tian W.X. Tsou C.L. Biochemistry. 1982; 21: 1028-1032Crossref PubMed Scopus (301) Google Scholar). When substrate and inhibitor bind to enzyme in rapid equilibrium and the substrate concentration does not change significantly during the course of the assay, the concentration of product, [P], at time t after the start of the reaction is given by Equation 1, [P]=V0kobs(1-exp(-kobst))+A0(Eq. 1) where v0 is the initial rate of reaction; kobs is the rate of inactivation, and A0 is the background fluorescence. kobs is related to the inhibitor concentration by Equation 2, kobs=kinact[I][I]+Ki1+[S]Km(Eq. 2) When [I] << Ki plots of kobs versus [I] were linear with slope equal to an apparent second-order rate constant kobs/[I]. This value was then corrected for substrate concentration and the Michaelis constant to determine a true second-order rate constant kinact/Ki.The initial rate v0 is related to inhibitor concentration by Equation 3, V0=Vmax[S]Km1+[I]Ki(app)+[S](Eq. 3) Because the inactivation was carried out with [S] = Km, Equation 3 reduces to Equation 4, V0=Vmax2+[I]Ki(app)(Eq. 4) An apparent inhibition constant Ki(app) for the formation of the initial reversible enzyme-inhibitor complex prior to inactivation was determined by plotting v0 against [I] and fitting to Equation 4.Collagen Digestion—Calf skin collagen type 1 was solubilized in 0.2 m acetic acid at a concentration of 2 mg/ml and dialyzed for 2 days against 0.1 m sodium acetate, pH 4.0, 0.1 m sodium acetate, pH 5.5, or PBS, pH 7.3. Reactions contained 10 μg of dialyzed collagen type 1, 1 mm DTT, and 2 mm EDTA and 5.47 μm activated peptidase in a final volume of 100 μl of one of the above buffers. Reactions were performed at 28 °C for 3 and 20 h or at 37 °C for 30 min. All reactions were stopped by the addition of 10 μm E-64. Collagen digests were analyzed by 4-20% gradient SDS-PAGE under reducing conditions and stained with Coomassie Brilliant Blue R-250.Production of Inactive Variant FheproCL1 Gly25—For the purpose of obtaining a high resolution three-dimensional structure of FheCL1, an inactive enzyme was produced by replacing the active site Cys residue at position 25 in the mature domain by a Gly (12Collins P.R. Stack C.M. O'Neill S.M. Doyle S. Ryan T. Brennan G.P. Mousley A. Stewart M. Maule A.G. Dalton J.P. Donnelly S. J. Biol. Chem. 2004; 279: 17038-17046Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 26Stack C.M. Donnelly S. Lowther J. Xu W. Collins P.R. Brinen L.S. Dalton J.P. J. Biol. Chem. 2007; 282: 16532-16543Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). This FheproCL1 Gly25 enzyme migrated as a single protein of 37 kDa on reducing 12% SDS-PAGE, which represents the full zymogen containing a prosegment and mature enzyme domain (data not shown).Data Collection, Structure Solution, and Crystallographic Refinement of FheproCL1 Gly25—Initial crystallization screening experiments were performed at the Hauptman-Woodward Institute high throughput crystallization laboratory. A total of 1536 conditions were tested using a nanoscale microbatch-under-oil method, resulting in several preliminary hits that suggested a route to diffraction-quality crystals (30Luft J.R. Collins R.J. Fehrman N.A. Lauricella A.M. Veatch C.K. DeTitta G.T. J. Struct. Biol. 2003; 142: 170-179Crossref PubMed Scopus (253) Google Scholar). Ultimately, high quality crystals were grown in-house via vapor diffusion in sitting drops. One μl of 10 mg/ml FheproCL1 Gly25 enzyme was mixed with 1 μl of the precipitating agent, 0.2 m sodium thiocyanate in 20% polyethylene glycol 3350, and allowed to equilibrate at 23 °C over a 100-μl reservoir of precipitating agent. Crystalline plates formed within 2 days; however, full-size growth to plates greater than 75 μm in thickness took nearly 2 months.Diffraction data were collected at the Advanced Light Source, beam line 8.3.1, using monochromatic (Si-111) radiation of 1.11588 Å (31MacDowell A.A. Celestre R.S. Howells M. McKinney W. Krupnick J. Cambie D. Domning E.E. Duarte R.M. Kelez N. Plate D.W. Cork C.W. Earnest T.N. Dickert J. Meigs G. Ralston C. Holton J.M. Alber T. Berger J.M. Agard D.A. Padmore H.A. J. Synchrotron. Radiat. 2004; 11: 447-455Crossref PubMed Scopus (81) Google Scholar). An ADSC Quantum 210 2 × 2 CCD array detector was used with low temperature conditions of 100 K at the crystal position. Crystals of the single mutant protein were flash-cooled in liquid nitrogen after being soaked for ∼1.5 min in a cryoprotectant solution of crystal growth solution plus 50% 2-methyl-2,4-pentane diol. High and low resolution datasets were collected from the same crystal. Data processing was completed with MOSFLM (32Leslie A.G. Acta Crystallogr. Sect. D Biol. Crystallogr. 2006; 62: 48-57Crossref PubMed Scopus (964) Google Scholar) and SCALA. The structure was solved via molecular replacement using the MOLREP program of the CCP4 suite (33Collaborative Computational Project, No. 4 (1994) Acta Crystallogr. Sect. D Biol. Crystallogr. 50, 760-763Google Scholar) with a polyserine search model derived from the 1.8 Å structure of 1CS8 (human procathepsin L). The topmost solution had an R-factor value of 0.535 and correlation coefficient of 0.288, each several σ levels above the next best solution, which had corresponding statistics of 0.604 and 0.083, respectively. One unique solution was found with one molecule in the asymmetric unit and a starting Rfactor of 0.526. The initial molecular replacement solution was improved using ARP/wARP as implemented in the CCP4 program suite (34Lamzin V.S. Perrakis A. Wilson K.S. Rossman F.M.G. Arnold E. Crystallography of Biological Macromolecules. Kluwer Academic Publishing, Dordrecht, Netherlands2001: 720-722Google Scholar) resulting in a model that was better than 85% complete. Iterative rounds of visualization and manual model building and refinement were completed with QUANTA (Accelrys, San Diego) and Refmac5 with anisotropic atomic displacement parameters (35Murshudov G.N. Vagin A. Dodson E. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13779) Google Scholar), respectively. Water molecules were added automatically using ARPwaters in CCP4 (36Perrakis A. Sixma T.K. Wilson K.S. Lamzin V.S. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 448-455Crossref PubMed Scopus (479) Google Scholar) and were manually verified. In the final stages of refinement, XPLEO (37van den Bedem H. Lotan I.J. Latombe C.L. Deacon A.M. Acta Crystallogr. Sect. D Biol. Crystallogr. 2005; 61: 2-13Crossref PubMed Scopus (52) Google Scholar) was used to improve the fit of two areas of ambiguous density in the structure. Final visualization and manual adjustments to the structure as well as final assessment of water molecules were completed with COOT (38Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (22810) Google Scholar). Crystallographic parameters and statistics are summarized in Table 1, and final atomic coordinates have been deposited with the Protein Data Bank, accession ID 2O6X (RCSB040763).TABLE 1Crystallographic parameters: data collection and refinement statistics Note the values given in parentheses under Data Collection are for highest resolution bin (1.48-1.40 Å).Data Collection Space groupP21212 Unit cell parametersa57.17 Åb105.78 Åc49.11 Å Wavelength1.1159 Å Temperature100 K Resolution1.4 Å Total no. of reflections333,582 (19,545) Total unique reflections59,595 (8465) Completeness99.7 (98.7%) Redundancy5.6 (2.3) Rmerge0.080 (0.549) Rp.i.m.0.029 (0.431) 〈I〉/〈σI〉14.6 (1.8)Refinement Resolution range (Å)52.93–1.40 Å No. of reflections56,141 Rfactor0.128 Rfree0.165 Free reflections5.05% Average B factor (Å2)Protein15.39Water34.09 Root mean square deviation from idealBond lengths0.020 ÅBond angles1.79° Ramachandran PlotResidues in most favored regions238 (90.5%)Residues in additional allowed regions25 (9.5%)Residues in generously allowed regions0 (0.0%)Residues in disallowed regions0 (0.0%) Open table in a new tab Homology-based Molecular Modeling—A model structure of the mature domain of FheCL2 was built using Modeler (release 8, version 1), a program for protein structure modeling (39Marti-Renom M.A. Stuart A. Fiser A. Sanchez R. Melo F. Sali A. Annu. Rev. Biophys. Biomol. Struct. 2000; 29: 291-325Crossref PubMed Scopus (2530) Google Scholar, 40Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10389) Google Scholar, 41Fiser A. Do R.K. Sali A. Protein Sci. 2000; 9: 1753-1773Crossref PubMed Scopus (1602) Google Scholar). The 1.8 Å structure of human procathepsin L (PDB code 1CS8), the 2.2 Å structure of human cathepsin K (PDB code 1ATK), and our 1.4 Å solved structure of FheproCL1 Gly25 were used as three-dimensional templates of related fold. Generated models were visualized and compared with COOT (37van den Bedem H. Lotan I.J. Latombe C.L. Deacon A.M. Acta Crystallogr. Sect. D Biol. Crystallogr. 2005; 61: 2-13Crossref PubMed Scopus (52) Google Scholar) and with PyMOL (42DeLano W.L. The PyMOL Molecular Graphics System, Version 1.0, Delano Scientific. San Carlos, CA2002Google Scholar).Sequence Analysis—F. hepatica cathepsin L protein sequences were aligned using Clustal X 1.81. Phylogenetic trees were generated from the alignment by the boot-strapped (1000-trial) neighbor-joining method using MEGA (43Kumar S. Tamura K. Jakobsen I.B. Nei M. Bioinformatics (Oxf.). 2001; 17: 1244-1245Crossref PubMed Scopus (4545) Google Scholar).RESULTSActive Site

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