Identification of structural determinants for inhibition strength and specificity of wheat xylanase inhibitors TAXI-IA and TAXI-IIA Annick Pollet 1 , Stefaan Sansen 2 , Gert Raedschelders 3 , Kurt Gebruers 1 , Anja Rabijns 2 , Jan A. Delcour 1 and Christophe M. Courtin 1 1 Laboratory of Food Chemistry and Biochemistry and Leuven Food Science and Nutrition Research Centre (LFoRCe), Katholieke Universiteit Leuven, Belgium 2 Laboratory for Biocrystallography, Katholieke Universiteit Leuven, Belgium 3 Laboratory of Gene Technology, Katholieke Universiteit Leuven, Belgium Keywords Bacillus subtilis; inhibition; Triticum aestivum; X-ray structure; xylanase Correspondence C. M. Courtin, Laboratory of Food Chemistry and Biochemistry and Leuven Food Science and Nutrition Research Centre (LFoRCe), Katholieke Universiteit Leuven, Kasteelpark Arenberg 20 - bus 2463, B-3001 Leuven, Belgium Fax: +32 16 321997 Tel: +32 16 321917 E-mail: christophe.courtin@biw.kuleuven.be Note The atomic coordinates and structure factors of BSXÆTAXI-IA (PDB code 2B42) and BSXÆrTAXI-IIA (PDB code 3HD8) are deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ, USA (http://www.rcsb.org) (Received 12 March 2009, revised 15 May 2009, accepted 20 May 2009) doi:10.1111/j.1742-4658.2009.07105.x Triticum aestivum xylanase inhibitor (TAXI)-type inhibitors are active against microbial xylanases from glycoside hydrolase family 11, but the inhibition strength and the specificity towards different xylanases differ between TAXI isoforms. Mutational and biochemical analyses of TAXI-I, TAXI-IIA and Bacillus subtilis xylanase A showed that inhibition strength and specificity depend on the identity of only a few key residues of inhibitor and xylanase [Fierens K et al. (2005) FEBS J 272, 5872–5882; Raedschelders G et al. (2005) Biochem Biophys Res Commun 335, 512–522; Sørensen JF & Sibbesen O (2006) Protein Eng Des Sel 19, 205–210; Bourgois TM et al. (2007) J Biotechnol 130, 95–105]. Crystallographic anal- ysis of the structures of TAXI-IA and TAXI-IIA in complex with glycoside hydrolase family 11 B. subtilis xylanase A now provides a substantial explanation for these observations and a detailed insight into the structural determinants for inhibition strength and specificity. Structures of the xylan- ase–inhibitor complexes show that inhibition is established by loop interac- tions with active-site residues and substrate-mimicking contacts in the binding subsites. The interaction of residues Leu292 of TAXI-IA and Pro294 of TAXI-IIA with the )2 glycon subsite of the xylanase is shown to be critical for both inhibition strength and specificity. Also, detailed analysis of the interaction interfaces of the complexes illustrates that the inhibition strength of TAXI is related to the presence of an aspartate or asparagine residue adjacent to the acid ⁄ base catalyst of the xylanase, and therefore to the pH optimum of the xylanase. The lower the pH optimum of the xylanase, the stronger will be the interaction between enzyme and inhibitor, and the stronger the resulting inhibition. Structured digital abstract l MINT-7101869: BSX (uniprotkb:P18429) and TAXI-IA (uniprotkb:Q8H0K8) bind (MI:0407) by x-ray crystallography ( MI:0114) l MINT-7101880: BSX (uniprotkb:P18429) and TAXI-IIA (uniprotkb:Q53IQ4) bind (MI:0407) by x-ray crystallography ( MI:0114) Abbreviations ANX, Aspergillus niger xylanase A; ANXÆTAXI-IA, TAXI-IA in complex with ANX; BSX, Bacillus subtilis xylanase A; BSXÆrTAXI-IIA, recombinant TAXI-IIA in complex with BSX; BSXÆTAXI-IA, TAXI-IA in complex with BSX; GH, glycoside hydrolase family; PDB, protein data bank; TAXI, Triticum aestivum xylanase inhibitor. 3916 FEBS Journal 276 (2009) 3916–3927 ª 2009 The Authors Journal compilation ª 2009 FEBS Introduction Endo-b-1,4-d-xylanases (xylanases, E.C. 3.2.1.8) hy- drolyse b-1,4-linkages between the d-xylosyl residues of arabinoxylans in cereal grain cell walls, releasing (arabino)xylo-oligosaccharides of different lengths [5]. Based on sequence similarities and hydrophobic cluster analysis, most xylanases are classified in glycoside hydrolase families (GH) 10 and 11, with only a min- ority categorized in GH5, 7, 8 and 43 (http://www. cazy.org) [6]. GH11 xylanases have a molecular mass of approximately 20 kDa and display a b-jelly roll structure in which the substrate-binding groove is formed by the concave face of the inner b-sheet. The structure has been likened to a right hand, with a two-b-strand ‘thumb’ forming a lid over the active site. The active site is thus located in the ‘palm’ with two conserved glutamate residues located on either side of the extended open cleft [7,8]. Despite their high structural and sequence similari- ties, the pH optima of GH11 xylanases vary consider- ably from acidic values (as low as 2) to alkaline values (as high as 11). The pH-dependent enzymatic catalysis by GH11 xylanases has been well studied. It has been demonstrated that the pH optima of the xylanases are correlated with the nature of the residue adjacent to the acid ⁄ base catalyst. In xylanases that function opti- mally under acidic conditions (pH < 5), this residue is aspartic acid, whereas it is asparagine in those that function optimally under more alkaline conditions (pH ‡ 5) [9–11]. Plants have evolved different classes of proteina- ceous inhibitors with the ability to counteract xylanases secreted by phytopathogens. To date, three distinct types of proteinaceous xylanase inhibitors have been isolated from wheat: Triticum aestivum xylanase inhibitor (TAXI) [12], xylanase-inhibiting protein [13] and thaumatin-like xylanase inhibitor [14]. These clas- ses of inhibitors show remarkable structural variety leading to different modes and specificities of inhibi- tion. TAXI-type inhibitors inhibit bacterial and fungal xylanases belonging to GH11 [15]. They are inhibitors with a high isoelectric point and occur in two mole- cular forms: an intact form with a molecular mass of approximately 40 kDa; and a processed form, consist- ing of two polypeptides of approximately 10 and 30 kDa, held together by one disulfide bond [15,16]. High-resolution 2D electrophoresis in combination with MS ⁄ MS analysis has identified large families of isoforms of TAXI-type inhibitors in wheat grain [17]. The amino acid sequences of TAXI-I and TAXI-II iso- forms share a high degree of identity (UniProt acces- sion nos: Q8H0K8, Q53IQ2, Q53IQ4 and Q53IQ3), but both types of inhibitors show different inhibition strengths and xylanase-inhibitor specificities. TAXI-I proteins show activity against a broad range of GH11 xylanases (Table 1) such as Bacillus subtilis xylanase A (BSX) and Aspergillus niger xylanase A (ANX), the latter being inhibited to a greater extent than the for- mer [18,19]. TAXI-II proteins have a very high inhibi- tion capacity against BSX, but are distinguished by the lack of activity against some other xylanases, such as ANX [2,18,19]. Two TAXI-I genes (encoding TAXI- IA [20] and TAXI-IB [2]) and two TAXI-II genes (encoding TAXI-IIA and TAXI-IIB) [2] were cloned and recombinantly expressed in Pichia pastoris. The 3D structure of TAXI-IA has been thoroughly characterized [21]. TAXI-IA consists of a two-b- barrel domain divided by an extended open cleft and displays structural homology with the pepsin-like family of aspartic proteases. The structure of TAXI- IA in complex with ANX (ANXÆTAXI-IA) revealed a direct interaction of the inhibitor with the active- site region of the enzyme and further substrate-mim- icking contacts with binding subsites filling the whole substrate-docking region [21]. The His374 TAXI-IA imidazole ring is located directly between the two catalytic glutamate residues of ANX and makes additional interactions with Asp37 ANX , Arg115 ANX and Tyr81 ANX . In the )1 glycon subsite, contacts are made between Phe375 TAXI-IA and Thr376 TAXI-IA and the xylanase, while, in subsite )2, Leu292 TAXI-IA mimics perfectly the position of a xylose bound in this subsite. On the aglycon side, TAXI-IA interferes with subsites +1 and +2 and prevents access to the aglycon end through steric hindrance. Mutational studies identified amino acids in the active site and in the thumb region of BSX and of TAXI-type inhibitors that are crucial for xylanase-inhibitor interaction [1–4]. Structural information on TAXI isoforms other than TAXI-IA is not available. Crystallographic analysis of TAXI-II, and of its interaction with xylanases, in par- ticular, could provide an explanation for its divergent inhibition specificity and verify the previously described hypothesis that its specificity depends on the identity of only a few residues [2,4]. The structures of TAXI-IA and recombinant TAXI-IIA described here, in complex with GH11 BSX (BSXÆTAXI-IA and BSXÆrTAXI-IIA, respectively), allowed identification of the structural determinants for the different TAXI- type xylanase inhibition strengths and specificities. Fine-tuned criteria could be deduced for the evaluation of TAXI-type inhibition specificity, with a predictive A. Pollet et al. Inhibition specificity of TAXI-type inhibitors FEBS Journal 276 (2009) 3916–3927 ª 2009 The Authors Journal compilation ª 2009 FEBS 3917 power on both the inhibitor as well as the enzyme side. Results Interaction interface of the BSXÆTAXI-IA complex For the description of TAXI-IA in the complex, the sec- ondary structure elements are denoted as described previ- ously [21]. In the BSXÆTAXI-IA complex, five TAXI-IA loop regions (L NiNj ,L HdCk ,L HfCs ,L HhCy and L CzCterm ) are responsible for an extensive network of interactions, resulting in a total buried accessible surface area of 1248 A ˚ 2 (Fig. 1A). The TAXI-IA loop L CzCterm pro- trudes between the thumb and the fingers of the xylanase, inducing a displacement of the thumb-like loop compared with an uncomplexed BSX structure [protein data bank (PDB) code 2Z79] [22] (Fig. 1B). The shortest active site cleft-spanning distance of 5.6 A ˚ (Pro116 BSX C c to Trp9 BSX N e1 ) is lengthened to 8.8 A ˚ upon forma- tion of a complex with TAXI-IA. The opening of the substrate-binding cleft is accompanied by side-chain re-arrangements at the basis of the thumb. Re-orienta- tion of the Thr110 BSX side-chain, rotated 102° around the v 1 -torsion angle, results in the loss of a hydrogen bond with the side-chain of Gln127 BSX , which subsequently is involved in a close interaction with the main-chain carbonyl oxygen of Phe375 TAXI-IA . A further cascade of conformational changes upon association of TAXI-IA and BSX is observed in the aglycon-binding sites of the xylanase, determined by Tyr174 BSX (subsites +1 and +2) and Tyr88 BSX (subsite +3) [23]. Driven by the presence of TAXI-IA, the aromatic side-chain of Tyr174 BSX is pushed back to be re-oriented parallel to the xylanase surface, stabilized in its newly acquired posi- tion by the Asn63 BSX side-chain that underwent a similar conformational change. Asn63 BSX in turn pushes Tyr88 BSX outwards, from pointing into the substrate- binding cleft towards the solvent. As a result of the new orientation of Tyr174 BSX , the side-chain of Gln175 BSX is no longer stabilized and becomes solvent exposed. The enlarged total buried accessible surface area in the BSXÆ TAXI-IA complex compared with the ANXÆTAXI-IA complex (1248 A ˚ 2 versus 992 A ˚ 2 , [21]) can mainly be ascribed to the conformational changes in the BSX aglycon subsites as they lead to a better fit with the inhibitor. Interaction interface of the BSXÆrTAXI-IIA complex TAXI-IA and rTAXI-IIA have a highly similar basic architecture (Fig. 2). Much as for TAXI-IA, the rTAXI-IIA molecule has an overall two-b-barrel domain topology with a six-stranded antiparallel b-sheet that forms the backbone. For reasons of uniformity, the nomenclature denoting the TAXI-IA secondary structure [21] is used in the description of the rTAXI-IIA structure. Compared with the native TAXI-IA sequence, rTAXI-IIA possesses two extra amino acids at the N-terminus, which is reflected in the numbering. Also, it has six additional amino acids at the C-terminus. rTAXI-IIA loop regions L NiNj ,L HdCk ,L HfCs , L HhCy and L CzCterm are involved in an extensive network of interactions with BSX residues in the active-site cleft and the thumb region (Fig. 1C). Binding of rTAXI-IIA results in the burial of an accessible surface area, of 1203 A ˚ 2 , at the interface. rTAXI-IIA binding induces a partial opening of the Table 1. Summary of literature data on TAXI-I and TAXI-II activities towards different glycoside hydrolase family 11 xylanases. Xylanase Accession number pH optimum Inhibition by References TAXI-I TAXI-II Aspergillus niger XylA a P55329 3.0 +++ e n.i. e [19,37] Penicillium funiculosum XynB b Q8J0K5 2.5–4.5 +++ n.i. [24] Penicillium purpurogenum XynB a Q96W72 3.5 +++ + [19,38] Botrytis cinerea XynCB1 c B3VSG7 4.5 +++ n.i. [25] Hypocrea jecorina Xyn1 a P36218 4.5 +++ + [18,19] P. funiculosum XynC d Q9HFH0 5.0 +++ +++ [18,19,26] Trichoderma viride xylanase a Q9UVF9 5.0 +++ +++ [18,19] H. jecorina Xyn2 a P36217 6.0 ++ +++ [1,18,19] Bacillus subtilis XynA a P18429 6.0 ++ +++ [1,2,18] a Inhibition activities were determined by measuring residual xylanase activities using a colorimetric Xylazyme AX method with wheat arabin- oxylan, at 30 °C and pH 5.0, as described by Gebruers et al. [15]. b–d Inhibition activities were determined by measuring residual xylanase activities using a dinitrosalicylic acid reducing group assay with wheat arabinoxylan, at 42 °C and pH 4.2 (b) [24], at 30 °C and pH 4.5 (c) [25], or at 30 °C and pH 5.5 (d) [26]. e +++, very strong inhibition; ++, intermediate inhibition; +, weak inhibition; n.i., not inhibited. Inhibition specificity of TAXI-type inhibitors A. Pollet et al. 3918 FEBS Journal 276 (2009) 3916–3927 ª 2009 The Authors Journal compilation ª 2009 FEBS BSX hand, with a net lengthening of 3.1 A ˚ of the distance between Pro116 BSX C c at the tip of the thumb and Trp9 BSX N e1 at the fingers, much as for the BSXÆTAXI-IA complex (Fig. 1D). Several re-arrangements take place at the base of the thumb, with the establishment of a close hydrogen bond between main-chain Phe377 rTAXI-IIA oxygen and Gln127 BSX N e1 as the main driving force. The posi- tion of Tyr174 BSX in the aglycon subsites (subsites +1 and +2), however, is different from that in the BSXÆTAXI-IA complex. In the BSXÆ rTAXI-IIA com- plex Tyr174 BSX is highly stabilized through a hydro- phobic stacking interaction with Pro375 rTAXI-IIA and is therefore found in a different conformation than the uncomplexed xylanase structure and the BSXÆ TAXI-IA complex. When looking at the residues contributing to the interface area, again the behav- iour of Tyr174 BSX is most aberrant. Whereas com- AB CD Fig. 1. (A,C) Overall structure of the BSXÆ- TAXI-IA (PDB 2B42) (A) and BSXÆrTAXI-IIA (PDB 3HD8) (C) complexes. His374 ⁄ 376 on the C-terminal loop L CzCterm is shown in sticks (red) and is located directly between the two active-site glutamic acids (Glu78 and Glu172) of the xylanase and Asn35 (yel- low sticks). TAXI-IA is displayed in orange, rTAXI-IIA is shown in green and BSX is shown in blue. (B,D) Cascade of conforma- tional changes upon association of TAXI-IA (B) and rTAXI-IIA (D) with BSX in the agly- con-binding sites of the xylanase, deter- mined by Tyr174 (subsites +1 and +2) and Tyr88 (subsite +3). The structure in yellow is the uncomplexed xylanase taken from PDB 2Z79 [22]; and the xylanase repre- sented in blue is taken from the BSXÆTAXI- IA and BSXÆrTAXI-IIA complex structures. Catalytic residues are displayed in red. Fig. 2. Superimposition of TAXI-IA (orange) (PDB 2B42) on TAXI-IIA (green) (PDB 3HD8). Despite local discrepancies, primarily confined to loop regions, both TAXI structures display a highly similar archi- tecture. A. Pollet et al. Inhibition specificity of TAXI-type inhibitors FEBS Journal 276 (2009) 3916–3927 ª 2009 The Authors Journal compilation ª 2009 FEBS 3919 plexation with TAXI-IA results in the burial of 65 A ˚ 2 of the Tyr174 BSX solvent-accessible surface, upon rTAXI-IIA binding Tyr174 BSX is much better stabilized by interaction with Pro375 rTAXI-IIA , burying 100 A ˚ 2 . For Tyr88 BSX , the re-orientation and stabiliza- tion in its new position are identical to what was observed for BSXÆTAXI-IA. Another striking difference with the BSXÆTAXI-IA complex is the nature and con- tribution to the total contact area of Pro294 rTAXI-IIA , compared with that of Leu292 TAXI-IA (6.8% and 10.1%, respectively). Structural basis for the inhibition of BSX by TAXI-IA and rTAXI-IIA BSXÆTAXI-IA In the BSXÆTAXI-IA structure the imidazole side-chain of His374 TAXI-IA is located directly between the two catalytic glutamate residues of BSX (Fig. 3A). In this position, the N e2 atom of the imidazole side-chain is highly stabilized through hydrogen-bonded contacts with Glu172 BSX O e2 (2.9 A ˚ ), Glu172 BSX O e1 (3.0 A ˚ ) and Tyr80 BSX O f (2.8 A ˚ ), while the more positive N d1 atom is involved in a weak electrostatic interaction with the negatively charged Glu78 BSX O e2 over a distance of 3.7 A ˚ and in a water-bridged contact with the Pro116 BSX main-chain O. Moreover, the main-chain His374 TAXI-IA N is tightly bonded to Asn35 BSX N d2 (2.6 A ˚ ) and the main-chain Phe375 TAXI-IA O is hydro- gen-bonded to Gln127 BSX N e2 (2.7 A ˚ ). To assess the interactions of TAXI-IA with the glycon-binding subsites of BSX, the superimposition of the BSXÆTAXI-IA complex with the structure of a catalytically inactive B. subtilis xylanase mutant complexed with xylotriose (PDB code 2QZ3) [22] was inspected (Fig. 3A*). The His374 TAXI-IA N e2 atom nearly coincides with the xylose C1 atom in subsite )1, and, in subsite )2, five Leu292 TAXI-IA atoms (N, C a , C b ,C c and C d1 ) get close to the atomic positions of C5, O5, C1, C2 and O2 of the xylose in subsite )2. In this way, Leu292 TAXI-IA accomplishes an efficient bur- ial of the hydrophobic surface of Trp9 BSX , constituting subsite )2, resulting in a tight binding through a sig- nificant hydrophobic effect. Furthermore, as a conse- quence of the conformational changes of Tyr174 BSX and Tyr88 BSX in the aglycon subsites of BSX, induced upon binding of TAXI-IA, additional interactions can be observed. Contacts between Gln187 TAXI-IA main- chain O and Tyr174 BSX O f (2.9 A ˚ ), Gln187 TAXI-IA main-chain O and Asn63 BSX N d2 (3.2 A ˚ ), and AA* BB* CC* Fig. 3. A detailed view of the interactions in the xylanase active site for the BSXÆTAXI-IA (PDB 2B42) (A), the BSXÆrTAXI-IIA (PDB 3HD8) (B) and the ANXÆTAXI-IA (PDB 1T6G) [21] (C) complexes. In A*, B* and C* an identical situation is shown as in A, B and C, respectively, with a substrate molecule bound in the active site of the xylanase (taken from the superimposition with the structure of PDB 2QZ3 for BSX and PDB 2QZ2 for ANX) [22] to illustrate the sub- strate mimicry in the )2 glycon subsite. TAXI-IA is displayed in orange, rTAXI-IIA in green, BSX in blue and ANX in grey. Grey labels indicate the amino acids involved in the interactions; black labels denote the atom names as they are used throughout the description of these structures. Inhibition specificity of TAXI-type inhibitors A. Pollet et al. 3920 FEBS Journal 276 (2009) 3916–3927 ª 2009 The Authors Journal compilation ª 2009 FEBS Gln190 TAXI-IA N e2 and Tyr88 BSX O f (2.9 A ˚ ), further stabilize the complex by induced fit and physically block the binding of substrate in the aglycon subsites (Fig. 4A). Interactions between the thumb region of BSX and TAXI-IA are established through Asp320 TAXI-IA O d2 and Asp121 BSX O d2 (3.4 A ˚ ), and Glu354 TAXI-IA O e1 and Arg122 BSX N f2 (2.8 A ˚ ). Asp11 BSX O d2 , located in the outer finger region, inter- acts with Arg371 TAXI-IA N e (3.7 A ˚ ). BSXÆrTAXI-IIA Although the interactions between the inhibitor key residues His376 rTAXI-IIA and Phe377 rTAXI-IIA and the xylanase active site are very similar to those of the BSXÆTAXI-IA structure, rTAXI-IIA induces a slightly larger distortion of the active-site architecture, reflected in somewhat longer intermolecular distances. The His376 rTAXI-IIA imidazole side-chain is hydrogen- bonded with its N e2 atom to the acid ⁄ base catalyst Glu172 BSX O e2 (2.9 A ˚ ) and Glu172 BSX O e1 (2.9 A ˚ ), while the positive N d1 atom points towards the nega- tively charged nucleophile Glu78 BSX O e2 over a dis- tance of at least 5.2 A ˚ , forming a weak electrostatic interaction (Fig. 3B). Other interactions are nearly invariable with respect to the BSXÆTAXI-IA model: a water-bridged contact between His376 rTAXI-IIA N d and Pro116 BSX main-chain O, a hydrogen bond between main-chain His376 rTAXI-IIA N and Asn35 BSX O d2 (2.9 A ˚ ), and an interaction between main-chain Phe377 rTAXI-IIA O hydrogen-bonded to Gln127 BSX N e2 (3.1 A ˚ ). In the BSXÆrTAXI-IIA complex, however, Tyr80 BSX is no longer involved in a contact with His376 rTAXI-IIA . The superimposition with the structure of the catalytically inactive B. subtilis xylanase mutant complexed with xylotriose (PDB code 2QZ3) [22] revealed some differ- ences (Fig. 3B*). Whereas Leu292 TAXI-IA coincides with the xylose moiety bound in the )2 subsite, in the case of rTAXI-IIA, Pro294 rTAXI-IIA is responsible for the substrate mimicry in this subsite. The envelope confor- mation of Pro294 rTAXI-IIA (with N, C a ,C c and C d copla- nar, and C b located above this plane) superimposes perfectly on the )2 xylose unit in chair conformation (C1 up, and C2, C3, C5 and O coplanar). This very stable Pro294 rTAXI-IIA conformation maximizes the burial of the Trp9 BSX side-chain accessible surface. In the aglycon subsites, further inhibitor–enzyme interactions both contribute to complex stabilization and reinforce the occlusion of the substrate-binding positions. Contacts are established between Gln189 rTAXI-IIA main-chain O and Asn63 BSX N d2 (3.1 A ˚ ), and between Gln192 rTAXI-IIA N e2 and Tyr88 BSX O f (2.3 A ˚ ) (Fig. 4B). Also, several interactions are made between Arg122 BSX in the thumb region and rTAXI-IIA, in particular with Asp322 rTAXI-IIA O d2 (4.1 A ˚ ), Glu356 rTAXI-IIA O e1 (2.8 A ˚ ) and Lys317 rTAXI-IIA main-chain O (2.8 A ˚ ). Finally, inter- action is observed between Arg373 rTAXI-IIA N e and Asp11 BSX O d2 (3.7 A ˚ ), similarly to the BSXÆTAXI-IA complex. Discussion The strength and specificity of inhibition of TAXI-I- and TAXI-II-type inhibitors differ strongly (Table 1). Analysis of the structures of the BSXÆTAXI-IA and BSXÆrTAXI-IIA complexes presented here, and of the ANXÆTAXI-IA complex described previously (Fig. 3C,C*) [21], basically reveal the same inhibition mechanism. First, His374 ⁄ 376 completely blocks the active site through intense contacts with the xylanase A B Fig. 4. A detailed view of the interactions in the aglycon-binding sites of the xylanase for the BSXÆTAXI-IA (PDB 2B42) (A) and the BSXÆrTAXI-IIA (PDB 3HD8) (B) complexes. TAXI-IA is displayed in orange, rTAXI-IIA in green and BSX in blue. Grey labels indicate the amino acids involved in the interactions; black labels denote the atom names as they are used throughout the description of these structures. A. Pollet et al. Inhibition specificity of TAXI-type inhibitors FEBS Journal 276 (2009) 3916–3927 ª 2009 The Authors Journal compilation ª 2009 FEBS 3921 active site amino acids. Second, parallel to the sub- strate–enzyme interactions involved in the reaction mechanism of xylanases, the glycon subsites are firmly occupied by strong hydrophobic interactions, perfectly mimicking the natural substrate. Finally, further contacts between TAXI-type inhibitors and xylanase residues constituting the aglycon subsites, prevent the access to the aglycon end through steric hindrance, thus filling the whole substrate-docking region. The above-described interactions of TAXI-type inhibitors with the active site and surrounding regions of the xylanase are in agreement with previously reported results of mutational studies of BSX by Sørensen & Sibbesen [3] and Bourgois et al. [4], which are summa- rized in Table 2. Modification of Glu127 BSX in the )1 glycon subsite, involved in a hydrogen-bonding inter- action with Phe375 TAXI-IA and Phe377 rTAXI-IIA , and of Asp11 BSX , which interacts with Arg371 TAXI-IA and Arg373 rTAXI-IIA , resulted in TAXI insensitivity [3,4]. Xylanase mutants, where thumb-region residues Arg122 BSX and Asp121 BSX , that interact with several residues of TAXI-IA and rTAXI-IIA, were replaced, were less sensitive to TAXI-type inhibitors [3]. The fact that BSX mutants which had decreased inhibitor sensi- tivities also had decreased enzyme activities [3,4], confirms that TAXI binding is accomplished by sub- strate mimicry in the active site of the xylanase. The seemingly minimal disparities between TAXI-IA and rTAXI-IIA, and between the enzyme–inhibitor complexes, suggest that the inhibition strength and specificity of TAXI-IA ⁄ TAXI-IIA reside in the subtle difference of only a few amino acid residues. In this study, in-depth analysis of the enzyme–inhibitor com- plexes allowed identification of two structural features that determine the xylanase–TAXI interaction. First, based on the structural analysis provided here, the stronger inhibition of ANX than of BSX by TAXI-I, as reported by Gebruers et al. and Fierens et al. [1,19] (Table 1), can be explained as follows. Figure 3A,C shows that the orientation of the His374 side-chain differs between the ANXÆ TAXI-IA and BSXÆTAXI-IA complexes. In contrast to the conformational change observed in TAXI-IA for this His374 TAXI-IA upon complexation with ANX [21], in the BSXÆTAXI-IA complex the side-chain has an orientation identical to that in the uncomplexed struc- ture. The basis for the (re)orientation of the imidazole side-chain is found in the mechanism of action of both xylanases. In ANX (or more general: ‘acidic’ xylanases), the side-chain of Asp37 ANX has the lowest pK a value of the residues involved in the catalytic action, and hence is negatively charged at the pH optimum [9]. This negative charge is the driving force for the conformational perturbation of His374 TAXI-IA upon complexation with the inhibitor. Re-orientation of the histidine allows charge complementarity between the positively charged N d1 atom of the imid- azole side-chain and the negatively charged Asp37 ANX [21]. As a consequence, in the ANXÆTAXI-IA com- plex, the main electrostatic interaction is with the acid ⁄ base catalyst, which induces a pH dependency of the inhibition profile. Moreover, the induced fit of TAXI-IA upon complexation with ANX results in a strong salt bridge between the more positively charged N d atom of the imidazole side-chain of His374 TAXI-IA and the negatively charged Asp37 ANX O d2 that will substantially contribute to an increased affinity of the inhibitor for the enzyme and complex stabilization. By contrast, in BSX (or ‘alkaline’ xylan- ases), the pH optimum is not influenced by the aspar- agine residue adjacent to the acid ⁄ base catalyst and, in the complex, the main electrostatic interaction is with the catalytic nucleophile that remains deproto- nated throughout a broad pH range. Hence, no con- formational changes are needed for TAXI-IA to reach charge compatibility and the pH dependency of the inhibition will be less pronounced. Furthermore, the rather long-distance salt bridge thus formed in the BSXÆTAXI-IA complex will not contribute sub- stantially to the affinity and stability of the complex. This could be the basis for the weaker inhibition by TAXI-I of BSX than of ANX. So, one could argue Table 2. Inhibition of BSX mutants by a mixture of TAXI-type inhibi- tors, as reported by Sørensen & Sibbesen [3] (A) and by recombi- nant TAXI-I and TAXI-II, as reported by Bourgois et al. [4] (B). A Xylanase Inhibition (IC 50 ) a TAXI BSX 3.8 D11Y ⁄ F ⁄ K >> 100 D121K 39.6 R122F 12.4 B Xylanase Inhibition (IC 50 ) a TAXI-I TAXI-II BSX 2.2 2.1 W9Y 6.7 >> 100 N35D 0.5 0.5 Q127K >> 100 >> 100 a The IC 50 value is defined as the half-maximal inhibitory concen- tration under the conditions of the assay [3] [4]. Inhibition specificity of TAXI-type inhibitors A. Pollet et al. 3922 FEBS Journal 276 (2009) 3916–3927 ª 2009 The Authors Journal compilation ª 2009 FEBS that the lower the pH optimum of the xylanase (i.e. the lower the pK a value of the aspartate residue adja- cent to the acid ⁄ base catalyst), the more pronounced the induced fit will be, and the stronger the resulting salt-bridge. Thus, the inhibition strength of TAXI-IA seems to depend on the pH optimum of the inhibited xylanase. Earlier results from biochemical testing of TAXI-IA and TAXI-IA His374 mutants, described by Fierens et al. [1], are in accordance with this conclu- sion. The lower the pH optimum of the tested xylan- ase, the more the binding affinity was deleteriously affected by His374 replacement. Binding affinity reduction ranged from a fivefold decrease with BSX to a total lack of interaction with ANX. Moreover, replacement of Asn35 BSX with the corresponding Asp37 ANX resulted in a BSX mutant with increased TAXI-I sensitivity [4] (Table 2), validating the above- described theory. Second, TAXI-II type inhibitors, unlike TAXI-I type inhibitors, do not inhibit ANX. Inhibition of BSX by TAXI-II, by contrast, is stronger than inhibition of this xylanase by TAXI-I [2]. As outlined earlier, comparison of the BSXÆTAXI-IA and the BSXÆrTAXI-IIA struc- tures shows that the active-site blocking by His374 ⁄ His376 is relatively well conserved in both complexes. Furthermore, the extra amino acids at the C-terminus of rTAXI-IIA do not directly intervene in xylanase binding, despite the crucial role of the loop L CzCterm in the inhibition interaction. Therefore, to find determinants of the TAXI-IA ⁄ TAXI-IIA specificity, a more detailed analysis was performed. The results of this analysis showed discrepancies in the interactions at the )2 (Trp9) BSX-binding subsite. Pro294 rTAXI-IIA –as a result of the ring structure – shares more equivalent positions with the xylose )2 sugar ring atoms compared with the Leu292 TAXI-IA side-chain atoms and hence accomplishes a mimicry with a higher degree of likeness to the substrate than TAXI-IA, which is also reflected in a slightly better burial of Trp9 BSX by Pro294 rTAXI-IIA than by the more voluminous Leu292 TAXI-IA . This explains the stronger inhibition of BSX by TAXI-IIA than by TAXI-I. Also, ANX has a tyrosine instead of a tryptophan in binding site )2. Pro294 rTAXI-IIA is not able to accomplish the same substrate mimicry at the )2 subsite of ANX. These views are in line with previously reported results of affinity tests that were performed by Raedschelders et al. [2] using engineered rTAXI-IIA and by Bourgois et al. [4] using engineered BSX. Chang- ing Pro294 rTAXI-IIA into leucine, to generate the Leu294 ⁄ His376 combination present in TAXI-IA, resulted in the ability of rTAXI-IIA to inhibit ANX, while inhibition activity towards BSX fell back to a moderate level. A BSX mutant, where Trp9 BSX was exchanged for Tyr10 ANX , was no longer inhibited by rTAXI-IIA and displayed a lower TAXI-I sensitivity (Table 3), illustrating the incompatibility between Pro294 rTAXI-IIA and Tyr10 ANX and the tighter binding between Pro294 rTAXI-IIA and Trp9 BSX than between Leu294 TAXI-IA and Trp9 BSX . This confirms the crucial role of Leu294 TAXI-IA and Pro294 rTAXI-IIA for inhibition specificity. In summary, the first interaction of the inhibitors with the xylanase active site can be identified as the interaction between the residue on position 374 or 376 of TAXI-IA or TAXI-IIA, respectively, and the xylan- ase amino acid located next to the acid ⁄ base catalyst. For the inhibitor, a histidine has been found in all sequences identified so far, with exception of the TAXI-IIB and TAXI-IV sequences (Uniprot accession nos Q53IQ3 and Q5TMB2, respectively) where a gluta- mine takes position 376. On the xylanase side, the aspartate or asparagine adjacent to the acid ⁄ base cata- Table 3. Data collection and refinement statistics of the structures of the BSXÆTAXI-IA and BSXÆrTAXI-IIA complexes. BSXÆTAXI-IA BSXÆrTAXI-IIA Data collection Space group C2 P2 1 Wavelength (A ˚ ) 0.934 0.811 Resolution limit (A ˚ ) a 2.5 (2.64–2.50) 2.38 (2.44–2.38) Cell parameters a = 107.89 A ˚ a = 77.35 A ˚ b = 95.33 A ˚ b = 60.30 A ˚ c = 66.31 A ˚ c = 134.19 A ˚ b = 122.4° b = 101.48° X-ray source ID14-EH1 ESRF BW7A DESY Total observations 51556 447271 Unique reflections a 20136 (2965) 44570 (955) Completeness of all data (%) a 98.0 (98.0) 97.5 (97.5) Mean I ⁄ r a 8.7 (3.2) 7.7 (3.8) R sym (%) a,b 5.8 (20.1) 7.6 (34.1) Refinement Resolution range (A ˚ ) 29.36–2.50 40.0–2.39 Number of reflections used 18166 41604 Reflections in R free set 1986 2427 R cryst ⁄ R free c 0.181 ⁄ 0.239 0.211 ⁄ 0.266 Number of atoms Protein 4047 8233 Solvent 65 230 Root mean square deviations d Bond lengths (A ˚ ) 0.013 0.027 Bond angles (°) 1.42 2.23 PDB entry 2B42 3HD8 a Values in parentheses are for the highest resolution shell. b R sym ¼ R h R j <IðhÞ> À IðhÞ j       =R h R j <IðhÞ>, where <I(h)> is the mean intensity of symmetry-equivalent reflections. c R cryst =R free ¼ R F o jjÀj F c jjj =R F o jj , where F o and F c are the observed and calculated struc- ture factors, respectively. d Root mean square deviations relate to the Engh and Huber parameters. A. Pollet et al. Inhibition specificity of TAXI-type inhibitors FEBS Journal 276 (2009) 3916–3927 ª 2009 The Authors Journal compilation ª 2009 FEBS 3923 lyst determines the pH optimum. This enables us to state that, for the principal xylanase–TAXI interaction, the Asp ⁄ His combination results in a higher affinity than the Asn ⁄ His combination. The enzyme–inhibitor contact in the )2 xylanase- binding subsite can be brought back to the residue on positions 292 or 294 of TAXI-IA or TAXI-IIA, respectively, and the xylanase amino acid constituting the )2 subsite. Except for TAXI-IIA (Pro294), the TAXI residue on position 292 ⁄ 294 is a leucine. The nature of the amino acid constituting subsite )2 has been shown to be important for the pH optimum of the xylanase. For ‘acidic’ xylanases, glycon subsite )2 corresponds to a tyrosine, while a tryptophan is found for ‘alkaline’ xylanases [11]. Hence, for the second important xylanase–TAXI interaction, a higher affinity for the Trp ⁄ Pro combination than for the Trp ⁄ Leu combination is expected. This in turn leads to a much higher affinity than the Tyr ⁄ Pro combination. Although based on only two main interactions, these two criteria nicely rationalize the results of studies per- formed previously, where inhibition tests were carried out using different native xylanases (Table 1). In spite of the fact that inhibition tests were carried out by different authors under different conditions, for each single xylanase, inhibition by TAXI-I and TAXI-II was tested under the same conditions, allowing com- parison. Acidic xylanases, such as ANX, Penicil- lium funiculosum XynB, Penicillium purpurogenum XynB and Hypocrea jecorina Xyn1, have an aspartate residue adjacent to the acid ⁄ base catalyst, and the )2 glycon subsite is formed by a tyrosine. Therefore, the presently defined criteria for the TAXI-inhibition spec- ificity indicate a weak or absent inhibition by TAXI- IIA. When probing these xylanases for their sensitivity against TAXI-type inhibitors, they were indeed less sensitive towards TAXI-inhibition, because they are not, or are only weakly, affected by TAXI-II type inhibitors [18,19,24] (Table 1). For xylanase XynCB1 from Botrytis cinerea, also an acidic xylanase with a pH optimum of 4.5, one would expect a decreased sus- ceptibility for TAXI-II inhibition. Inhibition tests indeed confirm that XynCB1 is inhibited by TAXI-I and not by TAXI-II [25]. Surprisingly, this xylanase contrasts sharply with the other uninhibited xylanases, because, despite its low pH-optimum, the residue next to the acid ⁄ base catalyst is an asparagine, and a tryp- tophan residue constitutes the )2 glycon subsite. Struc- tural analysis of B. cinerea XynCB1 could produce interesting results because additional factors may be involved in the inhibition interaction between this xylanase and TAXI-type inhibitors. The basic xylanases P. funiculosum XynC, Tricho- derma viride xylanase, H. jecorina Xyn2 and BSX are inhibited by TAXI-II type inhibitors [2,18,19,26]. They have an asparagine residue next to the acid ⁄ base cata- lyst in combination with a tryptophan residue in the )2 glycon subsite. An exception is the P. funiculosum xylanase XynC, for which a pH optimum of 5 results from a combination of an aspartate and a tryptophan residue. Both our criteria on the strength and specific- ity of the inhibition, however, indicate an increased susceptibility of this xylanase for inhibition by TAXI, which is in line with the determined inhibition specific- ity [26] (Table 1). The elucidation of the molecular architecture of complexes of TAXI-IA and TAXI-IIA with xylanases considerably contributes to the understanding of TAXI-type xylanase inhibition. The structures hold key information on the features of TAXI that are indispensable for the inhibitory action. Combined with mutational and biochemical data from previous stud- ies, structural analysis of the xylanase–TAXI com- plexes provides an integrated view on the inhibition of xylanases by TAXI-type inhibitors. Materials and methods Production and purification of xylanases and xylanase inhibitors TAXI-I (i.e. a mixture of TAXI-IA and TAXI-IB) was purified from wheat whole meal (cv. Soissons) by cation- exchange chromatography and affinity chromatography [27]. The production (in P. pastoris), and purification, of recombinant TAXI-IIA (rTAXI-IIA) were carried out as described by Raedschelders et al. [2]. GH11 BSX was purified from the Grindamyl H640 enzyme preparation (Danisco, Brabrand, Denmark) by cation-exchange chroma- tography [27,28]. The BSXÆTAXI-I and BSXÆrTAXI-IIA complexes were prepared by incubation of TAXI-I or rTAXI-IIA with an excess amount of BSX and purified by cation-exchange chromatography, as described by Sansen et al. [28]. Crystallization of TAXI-I and rTAXI-IIA in complex with BSX Prior to crystallization trials, the protein solutions were concentrated to approximately 10 mgÆml )1 . Crystals of the BSXÆTAXI-I complex were grown using the hanging-drop vapor-diffusion method at 277 K, with a reservoir solution containing 0.22 m ammonium sulfate and 25% (w ⁄ v) poly- ethylene glycol 4000 in a sodium acetate buffer (0.1 m,pH 4.6). For the BSXÆrTAXI-IIA complex, a fine-tuned condi- Inhibition specificity of TAXI-type inhibitors A. Pollet et al. 3924 FEBS Journal 276 (2009) 3916–3927 ª 2009 The Authors Journal compilation ª 2009 FEBS tion of 18% (w ⁄ v) polyethylene glycol 4000, 0.18 m ammo- nium sulfate, in 0.1 m sodium acetate buffer pH 4.6, pro- moted the growth of cube-shaped crystals, suitable for X-ray diffraction data collection. Crystals of complexes were cryoprotected by soaking for 30 s in a drop containing the crystallization condition to which 20% glycerol was added. Data collection, structure solution and refinement of the BSXÆTAXI-IA complex A high-quality diffraction data set was collected at 100 K using an ADSC Q4R charge-coupled device (CCD) detec- tor at the European Synchrotron Radiation Facility (ESRF, Grenoble, France) on beam line ID14-EH1. Inten- sity data were indexed and integrated using mosflm [29] and scaled using scala [30]. The packing density for one inhibitor–enzyme complex molecule in the asymmetric unit of these crystals was 2.6 A ˚ 3 ÆDa )1 , corresponding to an approximate solvent content of 51.7% [31]. The TAXI-I model (PDB code 1T6E) [21], together with the Bacil- lus circulans xylanase structure (PDB code 1C5H) [32], were used in molecular replacement searches in order to obtain a first model of this protein–protein complex. In two consecutive molecular replacement protocols, the posi- tions of TAXI-I (first) and the xylanase were determined using CNS [33]. Initial rigid-body least-square minimiza- tion was followed with cycles of maximum-likelihood refinement, as implemented in REFMAC [34], refining individual percentage factors after applying a translation, libration and screwrotation (TLS) correction (two TLS groups, i.e. one for each molecule in the asymmetric unit, 20 parameters each), with intermittent manual re-adjust- ments. Ramachandran statistics indicated that 87.0% of the residues are in the most favored regions and the remaining residues are in the additionally allowed regions. Table 3 lists further data-collection and refinement statis- tics. Based on well-defined electron density for residues Gly380 and Leu381 it could be concluded that TAXI-IA was present in the complex structure, while TAXI-IB was not. Therefore, the naming ‘TAXI-IA’ was used through- out the manuscript. Data collection, structure solution and refinement of the BSXÆrTAXI-IIA complex Diffraction data were collected at 100K on a MAR Research CCD area detector (165 mm) using synchrotron radiation at the BW7A beamline (DESY, Hamburg, Germany). Data were processed using mosflm [29] and scala [30]. According to Matthews [31] coefficient calcula- tions, the unique and repeating environment in the crystals consisted of two inhibitor–enzyme complex molecules. A packing density of 2.6 A ˚ 3 ÆDa )1 and an approximate solvent content of 51.5% were calculated for these crystals. Table 3 lists further data-collection and refinement statistics. Because of the very high degree of sequence homology between TAXI-IA and rTAXI-IIA (86.4%), on the one hand, and complete sequence identity for BSX, on the other, molecular replacement was the method of choice to obtain preliminary phases for calculating the first BSXÆrTAXI-IIA electron density maps. The complete BSXÆTAXI-IA model was used as a template for rotation and translation searches in the auto-MR mode of the program molrep [35]. Refine- ment of the model thus obtained was initiated by rigid-body fitting followed by cycles of maximum-likelihood refinement using REFMAC [34], with intermittent minor manual re-adjustments. In silico mutations using the molecular visualization program O [36] of the template molecule TAXI-IA, in order to match the rTAXI-IIA sequence, was performed only when the electron density maps unambigu- ously indicated to do so. To this end, electron density maps were calculated after the amino acid of interest was mutated to an alanine. Five short rTAXI-IIA portions, invariably turn-regions located at the surface, could not be unequivo- cally retrieved in the electron density (i.e. residues 43–48, 70–80, 225–228, 264–268 and 336–342). As none of these residues is involved in the interaction with the xylanase, the lack of coordinates for these rTAXI-IIA amino acids did not hamper the protein–protein interface analysis. The same holds true for the residues Arg387 rTAXI-IIA –Ser388 rTAXI-IIA – Thr389 rTAXI-IIA at the C-terminus. Acknowledgements We acknowledge the European Synchrotron Radiation Facility and the EMBL Grenoble Outstation for pro- viding support for measurements at the ESRF under the European Union ‘Improving Human Potential Pro- gramme’. Furthermore, we gratefully acknowledge the beam line scientists at EMBL ⁄ DESY for assistance and the European Union for support of the work at EMBL Hamburg. The ‘Instituut voor de aanmoediging van Innovatie door Wetenschap en Technologie in Vlaanderen’ (IWT Vlaanderen) (Brussels, Belgium) is thanked for project funding. This study is also part of the Methusalem programme ‘Food for the Future’ at the Katholieke Universiteit Leuven. References 1 Fierens K, Gils A, Sansen S, Brijs K, Courtin CM, Declerck PJ, De Ranter CJ, Gebruers K, Rabijns A, Robben J et al. (2005) His374 of wheat endoxylanase inhibitor TAXI-I stabilizes complex formation with gly- coside hydrolase family 11 endoxylanases. FEBS J 272, 5872–5882. A. Pollet et al. Inhibition specificity of TAXI-type inhibitors FEBS Journal 276 (2009) 3916–3927 ª 2009 The Authors Journal compilation ª 2009 FEBS 3925 [...]... Tyr10ANX and the tighter binding between Pro294rTAXI-IIA and Trp9BSX than between Leu29 4TAXI-IA and Trp9BSX This confirms the crucial role of Leu29 4TAXI-IA and Pro294rTAXI-IIA for inhibition specificity In summary, the first interaction of the inhibitors with the xylanase active site can be identified as the interaction between the residue on position 374 or 376 of TAXI-IA or TAXI-IIA, respectively, and the xylanase. .. funiculosum xylanase XynC, for which a pH optimum of 5 results from a combination of an aspartate and a tryptophan residue Both our criteria on the strength and specificity of the inhibition, however, indicate an increased susceptibility of this xylanase for inhibition by TAXI, which is in line with the determined inhibition specificity [26] (Table 1) The elucidation of the molecular architecture of complexes of. .. complexes of TAXI-IA and TAXI-IIA with xylanases considerably contributes to the understanding of TAXI-type xylanase inhibition The structures hold key information on the features of TAXI that are indispensable for the inhibitory action Combined with mutational and biochemical data from previous studies, structural analysis of the xylanase TAXI complexes provides an integrated view on the inhibition of xylanases... inhibition of xylanases by TAXI-type inhibitors Materials and methods Production and purification of xylanases and xylanase inhibitors TAXI-I (i.e a mixture of TAXI-IA and TAXI-IB) was purified from wheat whole meal (cv Soissons) by cationexchange chromatography and affinity chromatography [27] The production (in P pastoris), and purification, of recombinant TAXI-IIA (rTAXI-IIA) were carried out as described... Delcour JA, Van Campenhou S et al (2005) Molecular identification of wheat endoxylanase inhibitor TAXI-II and the determinants of its inhibition specificity Biochem Biophys Res Commun 335, 512–522 3 Sørensen JF & Sibbesen O (2006) Mapping of residues involved in the interaction between the Bacillus subtilis xylanase A and proteinaceous wheat xylanase inhibitors Protein Eng Des Sel 19, 205–210 4 Bourgois... extremely acidophilic and acid-stable xylanase: biased distribution of acidic residues and importance of Asp37 for catalysis at low pH Protein Eng 11, 1121– 1128 12 Debyser W, Derdelinckx G & Delcour JA (1997) Arabinoxylan solubilization and inhibition of the barley malt xylanolytic system by wheat during mashing with wheat wholemeal adjunct: Evidence for a new class of enzyme inhibitors in wheat J Am Soc... refinement statistics Because of the very high degree of sequence homology between TAXI-IA and rTAXI-IIA (86.4%), on the one hand, and complete sequence identity for BSX, on the other, molecular replacement was the method of choice to obtain preliminary phases for calculating the first BSXÆrTAXI-IIA electron density maps The complete BSX TAXI-IA model was used as a template for rotation and translation searches... respectively, and the xylanase amino acid constituting the )2 subsite Except for TAXI-IIA (Pro294), the TAXI residue on position 292 ⁄ 294 is a leucine The nature of the amino acid constituting subsite )2 has been shown to be important for the pH optimum of the xylanase For ‘acidic’ xylanases, glycon subsite )2 corresponds to a tyrosine, while a tryptophan is found for ‘alkaline’ xylanases [11] Hence, for the... subsite is formed by a tyrosine Therefore, the presently defined criteria for the TAXI -inhibition specificity indicate a weak or absent inhibition by TAXIIIA When probing these xylanases for their sensitivity against TAXI-type inhibitors, they were indeed less sensitive towards TAXI -inhibition, because they are not, or are only weakly, affected by TAXI-II type inhibitors [18,19,24] (Table 1) For xylanase. .. using mosflm [29] and scala [30] According to Matthews [31] coefficient calculations, the unique and repeating environment in the crystals consisted of two inhibitor–enzyme complex molecules A ˚ packing density of 2.6 A3ÆDa)1 and an approximate solvent Inhibition specificity of TAXI-type inhibitors content of 51.5% were calculated for these crystals Table 3 lists further data-collection and refinement statistics . Identification of structural determinants for inhibition strength and specificity of wheat xylanase inhibitors TAXI-IA and TAXI-IIA Annick. residues Leu292 of TAXI-IA and Pro294 of TAXI-IIA with the )2 glycon subsite of the xylanase is shown to be critical for both inhibition strength and specificity.