Determination of thioxylo-oligosaccharide binding to family 11 xylanases using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry and X-ray crystallography Janne Janis1, Johanna Hakanpaa1, Nina Hakulinen1, Farid M Ibatullin2, Antuan Hoxha3, ă ăă Peter J Derrick3, Juha Rouvinen1 and Pirjo Vainiotalo1 Department of Chemistry, University of Joensuu, Finland Biophysics Division, Petersburg Nuclear Physics Institute, Gatchina, Russia Department of Chemistry, Mass Spectrometry Institute, University of Warwick, Coventry, UK Keywords Fourier transform ion cyclotron resonance (FT-ICR); noncovalent binding; thioxylooligosaccharides; X-ray crystallography; xylanases Correspondence P Vainiotalo, Department of Chemistry, University of Joensuu, PO Box 111, FI-80101 Joensuu, Finland Fax: +358 13 2513360 Tel: +358 13 2513362 E-mail: pirjo.vainiotalo@joensuu.fi (Received January 2005, revised March 2005, accepted 10 March 2005) doi:10.1111/j.1742-4658.2005.04659.x Noncovalent binding of thioxylo-oligosaccharide inhibitors, methyl 4-thioa-xylobioside (S-Xyl2-Me), methyl 4,4II-dithio-a-xylotrioside (S-Xyl3-Me), methyl 4,4II,4III-trithio-a-xylotetroside (S-Xyl4-Me), and methyl 4,4II, 4III,4IV-tetrathio-a-xylopentoside (S-Xyl5-Me), to three family 11 endo-1,4b-xylanases from Trichoderma reesei (TRX I and TRX II) and Chaetomium thermophilum (CTX) was characterized using electrospray ionization Fourier transform ion cyclotron resonance (FT-ICR) MS and X-ray crystallography Ultra-high mass-resolving power and mass accuracy inherent to FT-ICR allowed mass measurements for noncovalent complexes to within |DM|average of p.p.m The binding constants determined by MS titration experiments were in the range 104)103 M)1, decreasing in the series of S-Xyl5-Me ‡ S-Xyl4-Me > S-Xyl3-Me In contrast, S-Xyl2-Me did not bind to any xylanase at the initial concentration of 5–200 lm, indicating increasing affinity with increasing number of xylopyranosyl units, with a minimum requirement of three The crystal structures of CTX– inhibitor complexes gave interesting insights into the binding Surprisingly, none of the inhibitors occupied any of the aglycone subsites of the active site The binding to only the glycone subsites is nonproductive for catalysis, and yet this has also been observed for other family 11 xylanases in complex with b-d-xylotetraose [Wakarchuk WW, Campbell RL, Sung WL, Davoodi J & Makoto Y (1994) Protein Sci 3, 465–475, and Sabini E, Wilson KS, Danielsen S, Schulein M & Davies GJ (2001) Acta Crystallogr ă D57, 13441347] Therefore, the role of the aglycone subsites remains controversial despite their obvious contribution to catalysis Glycoside hydrolases [1] are ubiquitous enzymes involved in biochemical degradation of cellulose and hemicellulose, the main constituents of plant cell walls They cleave the glycosidic linkages between pyranose or furanose rings of disaccharides, oligosaccharides and polysaccharides Glycoside hydrolases can be classified on the basis of their substrate specificity, mechanism of action, or amino-acid sequence [2–5] To Abbreviations CTX, catalytic domain of Chaetomium thermophilum xylanase; ESI, electrospray ionization; FT-ICR, Fourier transform ion cyclotron resonance; GlcNAc, N-acetyl-D-glucosamine; Hex, hexose (Man ⁄ Gal); S-Xyl2-Me, methyl 4-thio-a-xylobioside; S-Xyl3-Me, methyl 4,4II-dithioa-xylotrioside; S-Xyl4-Me, methyl 4,4II,4III-trithio-a-xylotetroside; S-Xyl5-Me, methyl 4,4II,4III,4IV-tetrathio-a-xylopentoside; TRX I, Trichoderma reesei xylanase I; TRX II, Trichoderma reesei xylanase II; Xyl2, b-D-xylobiose; Xyl3, b-D-xylotriose; Xyl4, b-D-xylotetraose; Xyl5, b-D-xylopentaose; Xyl6, b-D-xylohexaose FEBS Journal 272 (2005) 2317–2333 ª 2005 FEBS 2317 Thioxylo-oligosaccharide binding to xylanases date, the sequence-based classification of glycoside hydrolases comprises more than 90 families, further categorized into 14 clans displaying the same structural folds and catalytic machinery [5,6] Xylan is the most abundant hemicellulose component in plant cell walls, mainly constituted of anhydro b-1,4-d-xylopyranose backbone Natural xylan usually contains various substituents, such as 4-O-methyla-1,2-d-glucuronic acid, a-1,2-d-glucuronic acid, a-1,3d-arabinofuranosyl, and 2-O ⁄ 3-O-acetyl, depending on the botanical origin Xylan accounts for 7–10% dry weight of softwoods, 15–30% of hardwoods and up to 30% of annual graminaceous plants [7,8] Endo-1,4-b-xylanases (EC 3.2.1.8) are O-glycoside hydrolases that catalyze a random hydrolysis of internal b-1,4-glycosidic linkages of d-xylan by a doubledisplacement mechanism, with a net retention of the anomeric configuration [8,9] The reaction proceeds through a covalent intermediate with oxocarbenium ion-like transition states, utilizing two conserved catalytic glutamate residues, a nucleophile and an acid ⁄ base catalyst [10,11] Xylanases have been generally classified in the glycoside hydrolase families 10 and 11, but recently xylanases associated with the families 5, 7, and 43 have also been reported [8,12–14] Figure shows the proposed reaction scheme for a family 11 xylanase Xylanases have a number of industrially important applications [15–21], such as roles in animal J Janis et al ă feeding [16,17], pulp processing [18,19] and baking [20,21] In addition, their potential use in the biomass conversion to liquid fuel (i.e bioethanol) has gained considerable interest [15] X-ray crystallography [22] has been extensively used to dissect catalytic mechanisms for glycoside hydrolases, particularly through the use of specific covalent or noncovalent inhibitors [11,12,23] Elegant experimental approaches providing snapshots along an enzymatic reaction co-ordinate have been presented, in which the crystal structures for each of the enzyme– substrate (Michaelis), covalent intermediate and product complexes have been determined and further kinetically analyzed [24,25] Both fluoro [24–32] and epoxyalkyl [33–37] glycosides have been successfully used to identify catalytic residues and gather information on the reaction mechanisms, such as the itinerary of the sugar ring conformations along the catalytic pathway [24,25,29,30] or the inversion of the roles of the catalytic glutamates [34] Substrate derivatives with a fluorine atom at the 2-O-position of the xylose or glucose moiety (e.g 2-deoxy-2-fluoroglycosides) slow down the formation of the intermediates by inductively destabilizing the oxocarbenium transition states and eliminating an important hydrogen bond to the 2-Oposition [24] Epoxyalkyl glycosides bind to the enzymes by forming a covalent bond to the putative nucleophile [34] Fig Proposed reaction scheme for a retaining family 11 xylanase, with b-D-xylopentaose as a model substrate Putative glycone (from )3 to )1) and aglycone (+1 and +2) subsites at the enzyme active site have been numbered as described in [77] The reaction proceeds by a nucleophilic attack of the catalytic glutamate on the anomeric carbon of the xylopyranoside ring (at the )1 subsite) to produce a covalent glycosyl– enzyme intermediate via an oxocarbenium ion-like transition state At this point, the first product (b-D-xylobiose) is released The intermediate is then hydrolyzed via a second transition state to give the second product (b-D-xylotriose) and the free enzyme The proposed conformations for the xylopyranose ring in the )1 subsite have been indicated 2318 FEBS Journal 272 (2005) 2317–2333 ª 2005 FEBS J Janis et al ¨ In recent years, many noncovalent glycosidase inhibitors, e.g glyco-, xylo-, manno-, and galacto-configured aza [38,39], imino [40–42] tetrahydropyridoazole ([23] and references therein), and hydroximolactam [43] sugar derivatives have been introduced Imino-sugar inhibitors, for instance, are potential transition state mimics by virtue of the protonated nitrogen atom that highly resembles the oxocarbenium ion-like transitionstate structure [12] However, only a handful of the above inhibitors possess considerable aglycone (i.e the subsites that bind the ‘aglycone’ leaving group portion of the substrate; Fig 1) specificity However, interactions of the substrate with the aglycone subsites play an important role in transition-state stabilization along the catalytic pathway [28] Attention has been drawn to thio-oligosaccharides (Fig 2) as being promising noncovalent inhibitor candidates for structural biology studies among glycoside hydrolases [44–50] Such oligosaccharides, in which two or more carbohydrate residues are incorporated via S-glycosidic linkage(s), should conserve the global geometry of the natural substrate while being hydrolytically inert [44] In fact, changes only occur at the glycosidic bond The length ˚ and angle for the S-glycosidic bond are 1.83 A and 97°, whereas the respective values for O-glycosides are ˚ ˚ 1.41 A and 117°, resulting in a difference of % 0.35 A between the adjacent sugar rings [49] During the past 15 years, electrospray ionization (ESI) MS [51] has become an increasingly important analytical technique for the study of protein structure and function Of particular interest is the use of ESIMS in the studies of noncovalent interactions [52–54], as valuable parameters such as stoichiometry and binding constants can be determined A Fourier transform ion cyclotron resonance (FT-ICR) MS [55,56] has extended the possibilities of MS in protein analysis because of its inherently ultra-high mass-resolving power and mass accuracy For instance, protein–ligand [57], protein–carbohydrate [58,59], protein–peptide Fig Structures of thioxylo-oligosaccharide inhibitors From top to bottom: S-Xyl5-Me, S-Xyl4-Me, S-Xyl3-Me, and S-Xyl2-Me FEBS Journal 272 (2005) 2317–2333 ª 2005 FEBS Thioxylo-oligosaccharide binding to xylanases [60], and protein–RNA [61] interactions have been analyzed by ESI FT-ICR MS, providing valuable thermodynamic and kinetic data The filamentous fungus Trichoderma reesei is an efficient xylanase producer, expressing at least four xylanases, of which TRX I and TRX II are the most characterized [62,63] CTX is a thermostable xylanase expressed from Chaetomium thermophilum, another filamentous fungus [64] TRX I, TRX II and CTX, associated with family 11 of glycoside hydrolases, are folded into a single domain (b-jelly roll) structure comprising two parallel b-sheets and a single a-helix TRX II and CTX have been previously studied using ESI FT-ICR [64–66] The complex structures of TRX II with covalently attached epoxyalkyl xylosides have been obtained using X-ray crystallography [34– 37] In this paper, we report the characterization of the noncovalent binding of thioxylo-oligosaccharide inhibitors to TRX I, TRX II and CTX using high-field ESI FT-ICR MS and X-ray crystallography Results and Discussion ESI FT-ICR analyses Figure presents typical 9.4 T ESI FT-ICR mass spectra of TRX I, TRX II and CTX in 10 mm ammonium acetate buffer (pH 6.8) The resolving power of % 150 000 (defined as m ⁄ DmFWHM, where m is the ion mass and DmFWHM is the peak full width at half-maximum) allowed isotopic distributions, a consequence of the contributions of heavier isotopes (primarily 13C and 15N), to be well resolved (Fig 3B, inset) Each peak represents an unresolved superimposition of several isotopic compositions of the same nominal mass (actually differing by a few mDa), except the first peak, which represents the monoisotopic mass (i.e all hydrogens are 1H, all carbons are 12C, all nitrogens are 14N, etc.) However, the monoisotopic peak is often undetectable for proteins > 10 kDa, except for isotope-depleted proteins [67] Hence, the molecular masses reported hereafter refer to the masses calculated from the most abundant isotopic peaks (exp.) or the sequence-derived, most abundant elemental composition of a protein (theor.) The charge state for the species at a given mass-to-charge (m ⁄ z) ratio can be readily assigned, as the spacing between the adjacent isotopic peaks corresponds to a reciprocal of the charge, i.e z)1 for the species [M + zH]z+, which then allows the accurate mass to be unequivocally determined All proteins exhibited narrow charge state distributions of mainly four charge states, from 7+ to 10+, 2319 Thioxylo-oligosaccharide binding to xylanases Fig 9.4 T ESI FT-ICR mass spectra of lM TRX I (A), 10 lM TRX II (B), and 10 lM CTX (C) in 10 mM ammonium acetate buffer (pH 6.8) Numbers (n+) denote charge states (B) The inset shows the isotopic distribution for the charge state 9+, with white spheres representing the theoretical abundance distribution Glycosylated protein forms (TRX IHex, TRX IIGlcNAc and CTXGlcNAc) at each charge state have been denoted by # (see text for details) at m ⁄ z 2000–3500 We have previously shown that in these conditions, TRX II exists in a single conformation which represents the native protein structure [65,66] On the basis of the mass spectra presented in Fig 3, all proteins had a variable degree of modification The first peaks at each charge state in the mass spectrum of TRX I (Fig 3A) represent the native protein (19 046.920 ± 0.011 Da exp., 19 046.939 Da theor.), and the second peaks are due to a mass increment of % 162 Da, consistent with the post-translationally attached hexose (Hex, +162.053 Da), the form (TRX IHex) comprising less than 5% The first peaks in the mass spectrum of TRX II (Fig 3B) represent the native protein (20 824.823 ± 0.008 Da exp., 20 824.850 Da theor.) with the N-terminal glutamine 2320 J Janis et al ă existing in its cyclized pyrrolidonecarboxylic acid ()17.027 Da) form The second peaks correspond to a mass increment of % 203 Da, consistent with the previously observed N-glycosylation by a single N-acetyld-glucosamine (GlcNAc, +203.076 Da; TRX IIGlcNAc) [65,66], comprising % 30% of the protein content Exactly the same modifications as in TRX II were determined in CTX (Fig 3C), although the glycosylated protein form (CTXGlcNAc) was the major form (21 680.114 ± 0.021 Da exp., 21 680.289 Da theor.) comprising % 90% of the total protein content The observed modifications of TRX II and CTX agree with our earlier reports [64–66] The mass data are summarized in Table Close examination of the peaks at m ⁄ z 2900–3300 in the ESI FT-ICR spectrum of TRX II (Fig 3B) also revealed the existence of noncovalent protein dimers The peak at m ⁄ z % 3200 represents [(TRX II)2 + 13H]13+ (41 649.665 ± 0.029 Da exp., 41 649.700 Da theor.), whereas the peak at m ⁄ z % 2975 is a composite, in which [TRX II + 7H]7+ and [(TRX II)2 + 14H]14+ are overlapping each other (Fig 4) This results in a superimposition of the two isotopic distributions, one with the peak spacing of % 0.143 (z ¼ 7) representing the monomer, and the other with % 0.071 (z ¼ 14) representing the dimer Also, a heterodimer, TRX II–TRX IIGlcNAc, was observed at m ⁄ z % 2990 (14+) and % 3220 (13+) We have previously reported noncovalent dimerization of TRX II upon heatinduced conformational change [63] Also, TRX I had minor peaks in the mass spectra representing noncovalent dimer (Fig 3A), but CTX did not dimerize under any solution conditions, regardless of the close structural homology with TRX II Although the only significant difference between TRX II and CTX was the extent of N-glycosylation, this would not explain the absence of the dimeric form of CTX, as the heterodimer was present in the case of TRX II Observation of noncovalent protein–inhibitor complexes The formation of noncovalent protein–inhibitor complexes was readily observed by mixing appropriate aliquots of xylanase and thio-oligosaccharide solutions before direct analysis by MS Figure represents the ESI FT-ICR mass spectra of 10 lm TRX II with 50 lm methyl 4,4II,4III,4IV-tetrathio-a-xylopentoside (S-Xyl5-Me), 50 lm methyl 4,4II,4III-trithio-a-xylotetroside (S-Xyl4-Me), and 50 lm methyl 4,4II-dithioa-xylotrioside (S-Xyl3-Me) in 10 mm ammonium acetate buffer (pH 6.8) Comparison with the spectrum in Fig 3B reveals the presence of the noncovalent : FEBS Journal 272 (2005) 2317–2333 ª 2005 FEBS J Janis et al ă Thioxylo-oligosaccharide binding to xylanases Table The most abundant isotopic masses for TRX I, TRX II and CTX xylanases and their noncovalent thioxylo-oligosaccharide inhibitor complexes The data were obtained using 9.4 T ESI FT-ICR MS n.b., No binding (the peaks at the expected m ⁄ z for any charge states of the CTX–S-Xyl2-Me complex were not detected with [S-Xyl2-Me]initial ¼ 5–200 lM) Protein (modifications) Inhibitor Mexp (Da)a TRX I (none) None S-Xyl2-Me S-Xyl3-Me S-Xyl4-Me S-Xyl5-Me None S-Xyl2-Me S-Xyl3-Me S-Xyl4-Me S-Xyl5-Me None S-Xyl2-Me S-Xyl3-Me S-Xyl4-Me S-Xyl5-Me 19046.920 n.b 19508.080 19656.081 19804.091 20824.823 n.b 21285.981 21433.993 21582.033 21680.114 n.b 22141.411 22289.412 22437.463 TRX II (PCA) CTX (PCA, GlcNAc) Mtheor (Da)b ± 0.011 ± ± ± ± 0.032 0.012 0.033 0.008 ± ± ± ± 0.037 0.022 0.034 0.021 ± 0.057 ± 0.063 ± 0.063 DM (p.p.m.)c 19046.939 19359.026 19508.048 19656.067 19804.087 20824.850 21136.942 21285.964 21433.983 21582.002 21680.189 21993.279 22141.298 22289.317 22437.336 ) 1.0 + + + ) 1.6 0.7 0.2 1.3 + + + ) 0.8 0.4 1.4 3.5 + 5.1 + 4.3 + 5.7 a Mean ± SD measured over the charge state distributions b Calculated from the elemental composition of a protein and an inhibitor, including observed post-translational modifications c Calculated from DM (p.p.m.) ¼ [(Mexp–Mtheor) ⁄ Mexp] · 106 Fig Expansion of the 9.4 T ESI FT-ICR mass spectrum of 10 lM TRX II in 10 mM ammonium acetate (pH 6.8) at m ⁄ z 2920–3060 showing the group of peaks representing monomers (charge state 7+) and noncovalent dimers (charge state 14+) of TRX II and its glycosylated form (TRX IIGlcNAc) From left to right: [TRX II + 7H]7+ ⁄ [(TRX II)2 + 14H]14+ (the inset shows the superimposed isotopic distributions with the peak spacing of Dm ⁄ z ¼ 0.143 for the monomer and Dm ⁄ z ¼ 0.071 for the dimer), [TRX II + TRX IIGlcNAc + 14H]14+ (the inset shows isotopic distribution with Dm ⁄ z ¼ 0.071 for the dimer), and [TRX IIGlcNAc + 7H]7+ (no inset) protein–inhibitor complexes on the basis of the new peaks at the expected m ⁄ z values The accurate mass data for the complexes are summarized in Table However, no complexes could be detected for methyl 4-thio-a-xylobioside (S-Xyl2-Me), at the initial concentration of 5–200 lm, suggesting no interaction or the dissociation constant being a high millimolar FEBS Journal 272 (2005) 2317–2333 ª 2005 FEBS concentration, unreachable with our mass spectrometer Similar results for the thio-oligosaccharide binding were observed for TRX I and CTX (Table 1) The glycosylation in TRX II did not have any influence on the binding as the same ratio of protein–inhibitor complex ⁄ free protein was obtained at each charge state for both the glycosylated and nonglycosylated protein forms The same was observed for CTX Therefore, the subsequent analyses for the calculation of binding constants were made on the basis of the major protein forms only (i.e nonglycosylated protein form representing TRX II and glycosylated protein form representing CTX) In addition to equimolar complexes, : and : protein–inhibitor complexes were typically present with higher initial inhibitor concentrations (Fig 5) Complexes with higher stoichiometric compositions are probably due to nonspecific aggregation on the electrospray process [59] On the basis of the crystal structures, TRX I, TRX II and CTX have each only a single binding site Therefore, the subsequent molecules apparently bind to the protein surface by weak electrostatic forces, e.g hydrogen bonds Determination of binding constants MS titration experiments [68] were performed to determine binding constants for protein–inhibitor complexes as explained in Experimental Procedures The mass spectra were first background subtracted Assuming that the observed protein ion intensities reflect the true 2321 Thioxylo-oligosaccharide binding to xylanases J Janis et al ă determination of binding constants because of their low abundance and the fact that no peaks representing dimers were actually observed at ligand concentrations higher than 20 lm The MS titration curves for TRX I, TRX II and CTX are presented in Fig On the basis of the data presented in Fig 6, even at the highest ligand concentrations the proteins were still far from saturation, indicating relatively low binding constants The nonspecific binding was also observed in the titration experiments; the relatively important two and three ligand binding indicated that the nonspecific binding constants were of the same order of magnitude For simplicity, we will consider here only the case of a protein with a single specific binding site As will be explained in more detail in the next few paragraphs, this situation corresponds well to TRX I, TRX II and CTX xylanases In such cases, the binding constant, Ka, can be expressed as Ka ẳ ẵPL ẵPẵL 1ị in which [PL] is the concentration of the protein–ligand complex, and [P] and [L] are the concentrations of the free protein and free ligand (i.e inhibitor), respectively [68] Expressed in terms of r, defined as the number of ligands bound to one protein molecule, Eqn (1) can be written as: r¼ Fig 9.4 T ESI FT-ICR mass spectra of 10 lM TRX II with 50 lM S-Xyl5-Me (A), 50 lM S-Xyl4-Me (B) and 50 lM S-Xyl3-Me (C) in 10 mM ammonium acetate buffer (pH 6.8) Noncovalent protein– inhibitor complexes are indicated (A) The insets show the isotopic distributions for [TRX II + S-Xyl5-Me + 9H]9+ and [TRX II + (S-Xyl5Me)2 + 9H]9+ Only the 9+ charge states have been denoted for clarity protein concentrations, one can readily calculate the free and bound protein concentrations [68] Consequently, the free ligand concentration can also be calculated Previously it has been shown that different charge states can represent different binding patterns, reflecting different conformations [60] The intensities used for these calculations were therefore summed over the charge state distributions Moreover, in FT-ICR the signal intensity (i.e the induced image current) increases linearly with the charge state [55] Hence, the data were charge-normalized by dividing the intensity of each signal by the respective z This should reduce any bias introduced by a possible shift of the charge state distribution caused by ligand binding The dimeric protein forms were disregarded in the 2322 na Ka ẵL ỵ Ka ẵL 2ị in which na ẳ r [L] plotted vs r is called the Scatchard plot and is a straight line with a slope of –Ka In the case of nonspecific binding (in our case, PL2 and PL3 complexes), the number of ligands bound to one protein molecule is proportional to the concentration of free ligand Therefore, another term has to be implemented in Eqn (2) giving: r¼ na Ka ẵL ỵ Knsp ẵLịa ỵ Ka ẵL 3ị in which Knsp is a binding constant for the nonspecific protein–ligand complexes, with negative or positive co-operativity (the coefficient a) MS measurements readily provided the values of r as follows: P IPL ỵ 2IPL2 ỵ 3IPL3 ị 4ị rẵLinitial ; ẵPinitial ị ẳ P IP ỵ IPL ỵ IPL2 ỵ IPL3 Þ in which IP and IPLn are the intensities of a free protein and different protein–ligand complexes summed over the charge states and the isotopic distributions The free ligand concentration was then: FEBS Journal 272 (2005) 2317–2333 ª 2005 FEBS J Janis et al ă Thioxylo-oligosaccharide binding to xylanases Fig MS titration curves for TRX I (5 lM), TRX II (10 lM) and CTX (10 lM) with S-Xyl5-Me, S-Xyl4-Me and S-Xyl3-Me (initial concentrations of 0–100 lM) [PLn] is the concentration for 1:n protein–inhibitor complex with n ¼ 1–3, calculated from the 9.4 T ESI FT-ICR intensity data [L] is the free inhibitor concentration ẵL ẳ ẵLinitial rẵPinitial ð5Þ To obtain values for Ka and Knsp, nonlinear curve fittings based on the Levenberg–Marquardt algorithm were performed using Microcal origin 6.1 (Origin Laboratory Corp., Northampton, MA, USA) Briefly, starting from the given set of parameters, the sum of squared residuals of Eqn (3) from the experimental data points was minimized by performing a series of iterations Figure shows the binding isotherm, i.e r as a function of the free ligand concentration and the fit to Eqn (3) (solid line), obtained in the case of TRX I and S-Xyl5-Me On the basis of the Ka obtained, one can calculate the binding free energy (for specific binding) at a given temperature from the general expression DGbind ¼ –RTlnKa The Ka, Knsp, a values determined and DGbind values calculated are presented in Table All of the thioxylo-oligosaccharide complexes had specific binding constants (Ka) within the range 103)104 m)1, whereas the nonspecific binding constants (Knsp) were % 102)103 m)1 (Table 2) The a values were % in most cases, suggesting no significant co-operativity in the nonspecific binding For both FEBS Journal 272 (2005) 2317–2333 ª 2005 FEBS Fig Binding isotherm for lM TRX I with S-Xyl5-Me ([L]initial ¼ 0–100 lM) at 293 K For details of the calculations of r and [L], see text and Eqns (1–5) TRX I and TRX II, a decreasing trend in the specific binding constants was observed as follows: S-Xyl5-Me > S-Xyl4-Me > S-Xyl3-Me > S-Xyl2-Me > > For CTX, a similar trend, S-Xyl5-Me % S-Xyl4-Me S> Xyl3-Me > S-Xyl2-Me, was observed In fact, none > > 2323 Thioxylo-oligosaccharide binding to xylanases J Janis et al ă Table Thermodynamic parameters for thioxylo-oligosaccharide inhibitor binding to TRX I, TRX II, and CTX xylanases The data were obtained in 10 mM ammonium acetate (pH 6.8) at 293 K using 9.4 T ESI FT-ICR MS n.b., No binding (the peaks at the expected m ⁄ z for any charge state of CTX–S-Xyl2-Me complex were not detected within [S-Xyl2-Me]initial ¼ 5–200 lM) Protein Inhibitor Ka · 10)3 (M)1) TRX I S-Xyl2-Me S-Xyl3-Me S-Xyl4-Me S-Xyl5-Me S-Xyl2-Me S-Xyl3-Me S-Xyl4-Me S-Xyl5-Me S-Xyl2-Me S-Xyl3-Me S-Xyl4-Me S-Xyl5-Me n.b 1.4 4.5 11.0 n.b 2.5 7.0 9.1 n.b 2.2 12.0 10.9 TRX II CTX DGbind (kJỈmol)1) Knsp · 10)3 (M)1)a a ± 0.2 ± 0.5 ± 0.7 )17.7 )20.5 )22.7 n.d.b 0.84 0.55 1.0 1.1 1.0 ± 0.5 ± 0.9 ± 0.2 )19.1 )21.6 )22.2 0.54 0.71 1.6 1.0 1.0 1.0 ± 0.3 ± 1.1 ± 0.9 )18.8 )22.9 )22.7 0.64 0.92 0.92 1.0 1.0 1.1 a The fitting procedure did not provide error for Knsp probably because of several minima reached on replicate runs b Too low to be accurately determined of the xylanases had detectable affinity for S-Xyl2-Me, at the initial concentration of 5–200 lm This may be because the Ka values for the S-Xyl2 complexes were in the range 1–100 m)1, which is undetectable with our instrument These observations clearly show that the ligand binding is highly influenced by the number of xylopyranosyl units, with a minimum requirement of three Protein crystallography The final model of CTX contained 191 residues (on the basis of the ESI FT-ICR data, CTX contained 196 amino-acid residues with an N-terminal pyrrolidonecarboxylic acid and glycosylation by a single GlcNAc; neither the last five C-terminal amino acids nor the carbohydrate residue were visible in the crystal structures of CTX or CTX–S-Xyl5-Me complex) in both of the two molecules (A and B) of the asymmetric unit, 450 water molecules, three sulfate ions and two inhibitor molecules (S-Xyl5-Me) partly attached to the active site (Fig 8) In the electrondensity maps, three xylopyranose rings of the inhibitor molecules could be observed in the active site of both molecules (Fig 9) The xylopyranose rings, all adopting a normal 4C1 ground-state conformation, were observed only in the )1, )2 and )3 subsites (for the nomenclature, see [69]), missing the point of catalysis which occurs between subsites )1 and +1 Additional densities were observed in the glycone 2324 Fig Cartoon representation (A) and surface representation (B) of the crystal structure of CTX with S-Xyl5-Me The observed part of the inhibitor (three xylopyranose rings) is shown at the active site Carbon atoms of the inhibitor are coloured in purple, oxygen atoms in red, and sulfur atoms in orange The figure was created with PYMOL [86] ends of the inhibitor chains in both molecules, but no more xylopyranose rings could be unambiguously fitted into those electron densities In the active site of molecule B, two of the rings, and 2, were packed between two tryptophan residues (Trp19 and Trp80), with sugar ring located just outside the active-site, forming hydrogen bonds only with water molecules The hydrogen bonds formed between CTX and S-Xyl5-Me are listed in Table and schematically represented for molecule B in Fig 9C Similar results were obtained for other inhibitors (data not presented), except for S-Xyl2-Me When crystals were soaked in the solution containing S-Xyl2Me and further in 2-methyl-2,4-pentanediol before measurement at 120 K, no electron density caused by the inhibitor was detected However, some residual density was observed, consistent with 2-methyl-2,4FEBS Journal 272 (2005) 2317–2333 ª 2005 FEBS J Janis et al ă Thioxylo-oligosaccharide binding to xylanases Table Hydrogen bonds formed between CTX xylanase and S-Xyl5-Me inhibitor Inhibitor atoma ⁄ side chain hydroxy group Residue ⁄ water ˚ Distance (A) A1 ⁄ 1-Ob A1 ⁄ 1-O A1 ⁄ 2-O A1 ⁄ 3-O A2 ⁄ 2-O A2 ⁄ 2-O A2 ⁄ 3-O A2 ⁄ 3-O A3 ⁄ 2-O A3 ⁄ 2-O A3 ⁄ 3-O B1 ⁄ 1-O B1 ⁄ 1-O B1 ⁄ 3-O B2 ⁄ 2-O B2 ⁄ 2-O B2 ⁄ 3-O B2 ⁄ 3-O B3 ⁄ 2-O B3 ⁄ 3-O Glu178 OE2 HOH440 HOH438 HOH441 Tyr78 OH Tyr172 OH Tyr172 OH HOH1 HOH450 HOH357 HOH450 Glu178 OE1 HOH192 HOH436 Tyr78 OH Tyr172 OH Tyr172 OH HOH6 HOH145 HOH372 3.08 2.60 2.55 2.52 2.86 3.00 2.70 2.53 2.58 2.66 2.86 2.94 2.61 2.68 2.74 3.09 2.73 2.58 3.01 2.91 a A and B refer to the corresponding molecules in the asymmetric unit, and numbers refer to the xylopyranose rings at the corresponding glycone subsites, )1, )2 and )3 b Side-chain methoxy group at 1-O-position Fig Final 2Fo-Fc electron density map of the active site of CTX in molecule A (top) and molecule B (middle) Contour level is 1.0 r Water molecules are depicted as red spheres The figure was created with PYMOL [86] Schematic representation of the interactions of CTX with S-Xyl5-Me inhibitor (in molecule B) is shown at the bottom pentanediol bound to the active site On the other hand the electron density for S-Xyl2-Me was actually detected in subsites )1 and )2 when the measurements were performed at room temperature This suggests that the cryo-protectant replaces the bound inhibitor before the cryogenic measurement, which is consistent with the observations of low binding affinity by MS The overall conformations of molecules A and B ˚ were quite alike (rmsd ¼ 0.85 A for 1495 atoms) However, both molecules in the asymmetric unit contained several residues with signs of multiple FEBS Journal 272 (2005) 2317–2333 ª 2005 FEBS conformations Most of these residues were located on the surface of the enzyme, and only residues TyrB74 and GluB178 were fitted into two conformations in the final model, as these conformations are relevant to ligand binding In addition, molecule B contained a loop region (residues 162–167) that was slightly disordered In the disordered region, the main chain of the protein was intact, but clear signs of peptide flipping and several side-chain conformations could be seen, and fitting of the residues to the electron density was challenging The main differences were the two conformations of the catalytic glutamate, Glu178, in molecule B but not in molecule A and the disordered loop region in molecule B that was unambiguous in molecule A Glu178 has one unambiguous conformation in molecule A, where it points towards the other catalytic glutamate, Glu87 This conformation is also present in molecule B together with another conformation, in which Glu178 is bent away from the inhibitor molecule Movement of Glu178 forces the tyrosine residue, Tyr74, to adopt another conformation in molecule B The single position of Tyr74 in molecule A is an intermediate of the two conformations found in molecule B 2325 Thioxylo-oligosaccharide binding to xylanases A few differences were observed when comparing the CTX–S-Xyl5-Me complex with the native structure of CTX The space group and cell dimensions in the complex structure were the same as for the native protein, and the native structure also contained two molecules in the asymmetric unit In molecule B, the catalytic glutamate of the native protein adopted a single conformation, the one described as bent away from the inhibitor in the complex structure An arginine residue, Arg105, on the surface of the native protein was in a more extended conformation in the native protein compared with the CTX–S-Xyl5-Me complex in both A and B molecules This is probably due to the packing of the molecules, as Arg105 bends away to make space for the inhibitor molecule of the adjacent enzyme molecule Evaluation of inhibitor binding and implications for catalysis Ultra-high mass-resolving power and high mass accuracy inherent to FT-ICR, as demonstrated here, allowed unequivocal identification of the different protein forms as well as noncovalent protein complexes The binding constants for thioxylo-oligosaccharides were determined using ‘classical’ titration experiments Such analyses are feasible using ESI-MS intensity data to represent the thermodynamic equilibrium of free protein and protein–ligand complexes in solution [68] However, nonspecific protein–carbohydrate complexes, which can be even energetically preferred in the gas phase [70], often arise during the electrospray process A large amount of nonspecific binding complicates the analysis because of its indefinable manner Here, we distinguished between these two types of binding from the crystal structures, given that only a single binding site exists in each xylanase, capable of occupying only one inhibitor molecule An equimolar titration, recently described for the determination of protein– carbohydrate association constants [59], might have been a better approach as it diminishes the extent of nonspecific binding Unfortunately, there are no other reports on the binding of xylo-oligosaccharides or thioxylo-oligosaccharides to family 11 xylanases for which the binding constants had been determined However, some comparison can be on the basis of xylo-oligosaccharidebinding thermodynamics reported for isolated carbohydrate-binding domains from Clostridum thermocellum Xyn10B (X6b domain, family 10 [71]) and Pseudomonas cellulosa Xyn10C (CBM15 domain, family 15 [72]) xylanases, which display a similar b-jelly roll fold, characteristic of family 11 xylanases 2326 J Janis et al ¨ The affinity for xylo-oligosaccharide binding determined using isothermal titration calorimetry of both domains decreased in the series b-d-xylohexaose (Xyl6) > b-d-xylopentaose (Xyl5) > b-d-xylotetraose (Xyl4) > b-d-xylotriose (Xyl3), with no detectable affinity for b-d-xylobiose (Xyl2), which is consistent with our results However, absolute affinity values for X6b were % 10-fold higher than the values for CBM15 and the values reported here A similar trend was seen in the Michaelis constants (KM) for Penicillium simplicissium xylanase from family 10 vs the length of the oligosaccharide (KM ¼ 1.4, 3.1, 5.1 and 7.9 mm for Xyl6, Xyl5, Xyl4, and Xyl3, respectively), with no hydrolysis occurring in the case of Xyl2 [73] On the basis of the results, it remains controversial why such a correlation between the number of xylopyranose rings and the binding constants was observed for the thio-oligosaccharides given that none of the sugar rings occupied any of the aglycone subsites The electron densities for three sugar rings were observed only in the )1, )2 and )3 subsites Yet, the same has previously been observed with catalytically incompetent Bacillus circulans E172C [74] and Bacillus agaradhaerens E94A [75] mutant xylanases in complex with Xyl4 In the case of B circulans xylanase, only two carbohydrate residues were unequivocally fitted into the electron densities The authors suggested that either the enzyme had a small amount of residual activity (i.e being able to hydrolyze Xyl4), which sounds questionable from the mechanistic point of view (E172C mutation) and in view of the KM and kcat values reported in the same paper, or that the enzyme requires a larger substrate for tight binding In CTX, the main contribution to the binding in the glycone subsites together with multiple hydrogen bonds (Table 3) is apparently the hydrophobic interactions of the proximal sugar ring (A2, B2) with the conserved active-site tryptophan residue (Trp19) (Fig 8C) There were no clear electron densities for the sugar rings A4 ⁄ B4 and A5 ⁄ B5 (some partial disordered density was, in fact, visible for the sugar ring B4 outside of the active site) However, the third sugar ring (A3 ⁄ B3) makes hydrogen bonds only to the adjacent water molecules, with no direct contacts to the protein (Fig 9C), the same as with B agaradhaerens E94A xylanase [75] Therefore, the major contributions to the binding in the glycone side must come from the interactions within the subsites )1 and )2 However, the thermodynamic data suggest that, although the importance of the glycone subsites in substrate binding is more evident, some affinity must remain in the aglycone subsites to explain the results In fact, the results could be easily interpreted in view of the proposed model for FEBS Journal 272 (2005) 2317–2333 ê 2005 FEBS J Janis et al ă xylo-oligosaccharide binding to TRX II [63] in which, at least five putative subsites, from )2 to +3, could be modeled As there is no reported crystal structure in which xylo-oligosaccharides would completely traverse the active site for any family 11 xylanase, the experimental verification for the +1, +2 and +3 subsites remains controversial For family 11 xylanases, it has been demonstrated that catalysis is performed via a covalent reaction intermediate adopting an unusual 2,5B (boat) conformation [29,30,75], in constrast with the 4C1 (chair) conformation observed for other b-glycosidases The distortion of the xylopyranose ring from its ground-state conformation at the point of cleavage (i.e in the )1 subsite) plays an important role in the catalytic mechanism utilized by glycoside hydrolases As the 2,5B conformation also fulfills the stereochemical constraints for the oxocarbenium-ion-like transition states (sp2-hybridized C1 coplanar with C5, O1 and C2), catalysis in family 11 members probably takes a 4C1 fi 2H3 fi 2S0 fi 2,5B route [75] For family 10, a 4C1 fi 1S3 fi 4H3 fi 4C1 itinerary has been proposed instead [25] (C, H, S and B refer to chair, half-chair, skew-boat and boat conformations, respectively, and numbers denote the orientations of the respective ring atoms from the reference plane [87]) However, in our case all of the detected xylopyranosides adopted a low-energy 4C1 conformation, including the one occupying the )1 subsite In the crystal structures reported for barley b-d-glucan glucohydrolase in complex with 4,4III,4V-trithiocellohexaose [48] and B agaradhaerens endoglucanase Cel5A in complex with methyl 4,4II,4III,4IV-tetrathio-a-cellopentoside [49], all xylopyranose rings also adopted a 4C1 conformation In contrast, Fusarium oxysporum endoglucanase Cel7A in complex with the same inhibitor as with Cel5A revealed a considerable distortion towards the 1,4B (‘sofa’) conformation [45], displaying a preferred quasiaxial orientation for the leaving group In addition, an unusual 2S0 conformation has also been reported for Humicola insolens cellobiohydrolase Cel6A in complex with methyl cellobiosyl-4-thio-b-cellobioside [46,50] Distorted Michaelis complexes of retaining glycoside hydrolases are believed to reflect the incipient transitionstate conformations [25,50,75] However, a large variation among the different conformations obtained for Michaelis complexes of different glycoside hydrolases suggests that it may be difficult to assess whether the observed conformation is catalytically relevant [50] As there was no observable distortion from the 4C1 ground-state conformation, we looked for any geometric constraints arising from S-glycosidic bonds In fact, the crystal structure of CTX–S-Xyl5-Me superimposed with B agaradhaerens E94A xylanase in FEBS Journal 272 (2005) 2317–2333 ª 2005 FEBS Thioxylo-oligosaccharide binding to xylanases Fig 10 Superposition of the crystal structures of CTX in complex with S-Xyl5-Me (in purple) and B agaradhaerens xylanase in complex with Xyl4 (in blue) The PDB codes are 1XNK and 1H4H, respectively Superpositioning was performed with O [73], and the figure was created with PYMOL [86] complex with Xyl4 shows that there are significant differences in the locations of the xylopyranose rings at the active site (Fig 10) The xylopyranose rings in the )2 subsite are almost superimposable, whereas the other two observed rings for S-Xyl5-Me in the )1 and )3 subsites display considerable movement com˚ pared with Xyl4 This results in 5.80 A in molecule A ˚ in molecule B between the nucleophile and 3.78 A (Glu87 OE2) and the anomeric carbon (C1) This may partly explain the nonproductive binding of thioxylo-oligosaccharide inhibitors to CTX without distortion to the expected 2,5B conformation [75] Experimental procedures Protein expression and purification Native, DNA-encoded TRX I and TRX II xylanases (Swiss-Prot accession codes P36218 and P36217, respectively), originally obtained from Cultor Ltd, were produced in a T reesei Rut-C30 mutant strain and purified as previously reported [61] TRX II is now commercially available from Hampton Research (Hampton Research, Aliso Viejo, CA, USA) The catalytic domain of the recombinant CTX xylanase (GenBank accession code AJ508931) expressed from T reesei (hereafter referred to as CTX) was kindly provided by O Turunen (Helsinki University of Technology, Helsinki, Finland) CTX was initially purified using cation-exchange and hydrophobicinteraction chromatography and characterized by SDS ⁄ PAGE and ESI FT-ICR analyses as described previously in detail [64] Further purification of the protein solutions for MS analyses is described in Sample preparation, below 2327 Thioxylo-oligosaccharide binding to xylanases Thioxylo-oligosaccharides S-Xyl2-Me, S-Xyl3-Me, S-Xyl4-Me, and S-Xyl5-Me (Fig 2) were produced by stereoselective synthesis from S-glycosyl isothiourea precursors and characterized using elemental analysis and 1H ⁄ 13C NMR spectroscopy Experimental synthesis and purification protocols have been reported elsewhere in detail [76,77] Several precursors were also characterized using ESI FT-ICR MS [77] The stock solutions were prepared from thioxylo-oligosaccharides by accurately weighing and dissolving in ultra-pure water (Elga LabWater, High Wycombe, Bucks, UK) to a final concentration of 1–10 mm Sample preparation All chemicals, typically the highest HPLC grade, were purchased from Sigma-Aldrich (Gillingham, Dorset, UK) Protein stock solutions were first desalted over a PD-10 column (Amersham Biosciences Ltd, Amersham, Bucks, UK) previously equilibrated with 10 mm ammonium acetate (pH 6.8) Protein-containing fractions were concentrated using Millipore Ultrafree (5 kDa cut-off) centrifugal filter devices (Millipore, Bedford, MA, USA) by ultracentrifugation at °C Finally, protein concentrations were determined from A280 For TRX II, A280 ¼ corresponds to a concentration of 18.5 lm (0.37 mgỈmL)1) [66], equivalent to a molar absorption coefficient of 54 050 m)1Ỉcm)1 For TRX I and CTX, the molar absorption coefficients (46 940 and 62 870 m)1Ỉcm)1, respectively) were calculated using a GPMAW 6.01 [78] from the A280 contribution of Tyr, Trp, and Cys [79] The calculated coefficient for TRX II, 55 900 m)1Ỉcm)1, differs from the experimental value for less than 4% Mass spectrometry All MS measurements were performed using a Bruker BioAPEX II ESI FT-ICR instrument (Bruker Daltonics, Billerica, MA, USA), described previously in detail [66,80] Briefly, the instrument was equipped with an external ESI source (Analytica, Branford, CT, USA) having a dielectric Pyrex capillary with Pt ⁄ Ni-coated ends (VCapExit ¼ 80–120) and a radio frequency hexapole (operated at 5.2 MHz and 500 Vp-p) used for external ion accumulation Samples were directly infused into the ion source at a flow rate of 1.0 lLỈ min)1 Carbon dioxide (69 kPA at 200 °C) was used as a drying gas ESI-generated ions were accumulated for 1.0 s (D1) and transferred within 5.5 ms (P2) to an Infinity ICR cell [81], located inside a 9.4 T central-field passively shielded superconducting magnet (Magnex Scientific Ltd, Abingdon, Oxon, UK) Other ion source parameters were adjusted to the most appropriate values Ions were trapped by a ‘SideKick’ technique before frequency-sweep excitation (35–103 kHz, % 72 Vp-p) and broadband detection 2328 J Janis et al ă (36–96 kHz) All measurements were carried out with 512 k (524 288) data points, and 64 coadded time-domain transients were recorded The transients were subjected to fast Fourier transform, magnitude calculation, and one zero-fill Mass spectra were externally calibrated using an ES Tuning Mix (Hewlett Packard, Palo Alto, CA, USA) by a twoparameter frequency-to-m ⁄ z calibration function [82] Mass spectral acquisition and postprocessing were performed using Bruker xmass 5.0.1 ⁄ 6.0.1 software Titration experiments MS titration experiments were performed in order to determine binding constants for xylanase–thioxylo-oligosaccharide complexes Experiments were carried out with xylanases at a fixed concentration of 5.0 or 10.0 lm in 10 mm ammonium acetate buffer (pH 6.8), by varying the concentration of oligosaccharide Briefly, appropriate aliquots of protein and thioxylo-oligosaccharide solutions were mixed up to obtain initial oligosaccharide concentrations of 5–100 lm The samples were equilibrated at 20 °C for 10 before MS analyses To ascertain that the true chemical equilibrium had been reached within 10 min, xylanases (10 lm) were incubated with S-Xyl5-Me (50 lm) overnight and analyzed as previously The same relative abundances of the free and bound enzymes were observed with either 10 or the extended incubation periods within the error level of the experiments Some of the ion source parameters were carefully adjusted to minimize the unintended dissociation of the weak noncovalent complexes For quantitative analysis (i.e calculation of the binding constants), this has crucial importance as relative MS intensities should, as close as possible, reflect the true chemical equilibrium in solution [68] For instance, the capillary exit potential, VCapExit, was typically set to 80– 120, as values of 160 clearly induced dissociation (data not presented) The drying gas temperature adjusted between 25 and 300 °C did not have any effect on the relative free protein complex ratios (except the number of sodium adducts present), and therefore 200 °C was chosen as the optimized value for maintaining good desolvation conditions X-ray crystallography The catalytic domain of CTX was crystallized from solution containing 1.2 m ammonium sulfate as precipitant in 0.1 m Hepes buffer (pH 7.0) using a hanging-drop vapor diffusion method at room temperature The protein stock solution was diluted with 10 mm sodium citrate buffer (pH 5.0) to an approximate concentration of mgỈmL)1 Crystallization was initiated by mixing equal amounts of protein and crystallization solutions The crystals grew in the space group P21212 To obtain complex structures with thioxylo-oligosaccharides, the crystals were soaked for h in the crystallization solutions containing 20 mm inhibitor FEBS Journal 272 (2005) 23172333 ê 2005 FEBS J Janis et al ă Thioxylo-oligosaccharide binding to xylanases before rapid soaking in the crystallization solution containing 40% (v ⁄ v) 2-methyl-2,4-pentanediol, acting as a cryoprotectant Alternatively, cocrystallization was performed (data not presented) with all thioxylo-oligosaccharides, with similar results to the soaking experiments X-ray diffraction measurements were carried out using a Rigaku RU-200HB ˚ copper rotating anode source (wavelength 1.54 A) operated at 50 kV and 100 mA, equipped with Osmic Confocal optics (Osmic Inc., Auburn Hills, MI, USA) and an R-AXIS IIC imaging plate detector (Rigaku ⁄ MSC, Woodlands, TX, USA) The crystals diffracted to a resolution of ˚ 1.55 A at 120 K The data were processed with denzo and scaled with scalepack [83] The native structure of CTX [64] was used as a starting point in the model construction The initial model was refined with cns [84], and electrondensity maps were visualized with program o [85] The asymmetric unit contained two molecules, A and B Parameters of the measurement and refinement statistics for the CTX–S-Xyl5-Me complex are summarized in Table Atomic co-ordinates for the crystal structures of TRX I, TRX II, CTX, and CTX–S-Xyl5-Me complex can be found Table X-ray diffraction measurement and refinement statistics for the CTX–S-Xyl5-Me Complex Inhibitor Space group ˚ Unit cell dimensions (A) Unit cell angles (°) ˚ Resolution range (A) Completeness (%) Rsym (%) I ⁄ Ir Number of observations Number of unique reflections Temperature (K) Rfactor (%) Rfree (%) ˚ Rmsd bond lengths (A) Rmsd bond angles (°) Number of all atoms Number of protein atoms Number of waters Number of inhibitor atoms ˚ Baverage (A2) Protein main chain atoms Protein side chain atoms All protein atoms Inhibitor atoms Water atoms Other atoms All atoms PDB code Methyl 4,4II,4III,4IVtetrathio-a-D-xylopentaoside P21212 a ¼ 108.24, b ¼ 57.59, c ¼ 66.21 90, 90, 90 1.55–99.0 (1.55–1.61)a 92.8 (52.1) 6.6 (29.6) 1.7 (1.04) 166670 56502 120 19.7 22.4 0.0053 1.52 3533 3011 450 58 14.2 16.3 15.2 30.6 28.1 36.6 17.2 1XNK a Values in parentheses are for 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