Báo cáo khoa học: Identification of structural determinants for inhibition strength and specificity of wheat xylanase inhibitors TAXI-IA and TAXI-IIA doc

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:
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 ()
(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.
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