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
Trang 1strength and specificity of wheat xylanase inhibitors
TAXI-IA and TAXI-IIA
Annick Pollet1, Stefaan Sansen2, Gert Raedschelders3, Kurt Gebruers1, Anja Rabijns2,
Jan A Delcour1and Christophe M Courtin1
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.
Trang 2Endo-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 molemole-cular 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 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-TAXI-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 His374TAXI-IA imidazole ring is located directly between the two catalytic glutamate residues of ANX and makes additional interactions with Asp37ANX, Arg115ANX and Tyr81ANX In the )1 glycon subsite, contacts are made between Phe375TAXI-IA and Thr376TAXI-IA and the xylanase, while, in subsite )2, Leu292TAXI-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
Trang 3power 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 (LNiNj, LHdCk, LHfCs, LHhCy and LCzCterm)
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 LCzCterm
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˚ (Pro116BSX
Ccto Trp9BSXNe1) 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 Thr110BSX side-chain, rotated 102 around
the v1-torsion angle, results in the loss of a hydrogen
bond with the side-chain of Gln127BSX, which
subsequently is involved in a close interaction with the
main-chain carbonyl oxygen of Phe375TAXI-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 Tyr174BSX(subsites
+1 and +2) and Tyr88BSX(subsite +3) [23] Driven by
the presence of TAXI-IA, the aromatic side-chain of
Tyr174BSX is pushed back to be re-oriented parallel to
the xylanase surface, stabilized in its newly acquired
posi-tion by the Asn63BSXside-chain that underwent a similar conformational change Asn63BSX in turn pushes Tyr88BSX outwards, from pointing into the substrate-binding cleft towards the solvent As a result of the new orientation of Tyr174BSX, the side-chain of Gln175BSXis
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 LNiNj, LHdCk, LHfCs,
LHhCy and LCzCterm 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.
Inhibition by
References TAXI-I TAXI-II
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.
Trang 4BSX hand, with a net lengthening of 3.1 A˚ of the distance between Pro116BSX Cc at the tip of the thumb and Trp9BSX Ne1 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 Phe377rTAXI-IIA oxygen and Gln127BSX Ne1 as the main driving force The posi-tion of Tyr174BSX in the aglycon subsites (subsites +1 and +2), however, is different from that in the BSXÆTAXI-IA complex In the BSXÆrTAXI-IIA com-plex Tyr174BSX is highly stabilized through a hydro-phobic stacking interaction with Pro375rTAXI-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 Tyr174BSX is most aberrant Whereas
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 LCzCtermis 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-IBSXÆTAXI-IA 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.
Trang 5plexation with TAXI-IA results in the burial of
65 A˚2 of the Tyr174BSX solvent-accessible surface,
upon rTAXI-IIA binding Tyr174BSX is much better
stabilized by interaction with Pro375rTAXI-IIA, burying
100 A˚2 For Tyr88BSX, the re-orientation and
stabiliza-tion in its new posistabiliza-tion 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 Pro294rTAXI-IIA,
compared with that of Leu292TAXI-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 His374TAXI-IA is located directly between the two
catalytic glutamate residues of BSX (Fig 3A) In this
position, the Ne2 atom of the imidazole side-chain is
highly stabilized through hydrogen-bonded contacts
with Glu172BSX Oe2 (2.9 A˚), Glu172BSX Oe1 (3.0 A˚)
and Tyr80BSX Of(2.8 A˚), while the more positive Nd1
atom is involved in a weak electrostatic interaction with
the negatively charged Glu78BSX Oe2 over a distance
of 3.7 A˚ and in a water-bridged contact with the Pro116BSX main-chain O Moreover, the main-chain His374TAXI-IA N is tightly bonded to Asn35BSX Nd2 (2.6 A˚) and the main-chain Phe375TAXI-IA O is hydro-gen-bonded to Gln127BSXNe2(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 His374TAXI-IA Ne2 atom nearly coincides with the xylose C1 atom in subsite)1, and, in subsite )2, five Leu292TAXI-IA atoms (N, Ca,
Cb, Cc and Cd1) get close to the atomic positions of C5, O5, C1, C2 and O2 of the xylose in subsite )2 In this way, Leu292TAXI-IA accomplishes an efficient bur-ial of the hydrophobic surface of Trp9BSX, constituting subsite )2, resulting in a tight binding through a sig-nificant hydrophobic effect Furthermore, as a conse-quence of the conformational changes of Tyr174BSX and Tyr88BSX in the aglycon subsites of BSX, induced upon binding of TAXI-IA, additional interactions can
be observed Contacts between Gln187TAXI-IA main-chain O and Tyr174BSX Of (2.9 A˚), Gln187TAXI-IA main-chain O and Asn63BSX Nd2 (3.2 A˚), and
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.
Trang 6Gln190TAXI-IA Ne2 and Tyr88BSX Of (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
Asp320TAXI-IA Od2 and Asp121BSX Od2 (3.4 A˚), and
Glu354TAXI-IA Oe1 and Arg122BSX Nf2 (2.8 A˚)
Asp11BSXOd2, located in the outer finger region,
inter-acts with Arg371TAXI-IANe(3.7 A˚)
BSXÆrTAXI-IIA
Although the interactions between the inhibitor key
residues His376rTAXI-IIA and Phe377rTAXI-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
His376rTAXI-IIA imidazole side-chain is
hydrogen-bonded with its Ne2 atom to the acid⁄ base catalyst
Glu172BSX Oe2 (2.9 A˚) and Glu172BSX Oe1 (2.9 A˚),
while the positive Nd1 atom points towards the
nega-tively charged nucleophile Glu78BSX Oe2 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 His376rTAXI-IIANd and
Pro116BSX main-chain O, a hydrogen bond between
main-chain His376rTAXI-IIA N and Asn35BSX Od2
(2.9 A˚), and an interaction between main-chain
Phe377rTAXI-IIAO hydrogen-bonded to Gln127BSX Ne2
(3.1 A˚)
In the BSXÆrTAXI-IIA complex, however, Tyr80BSXis
no longer involved in a contact with His376rTAXI-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 Leu292TAXI-IAcoincides with
the xylose moiety bound in the)2 subsite, in the case of
rTAXI-IIA, Pro294rTAXI-IIA is responsible for the
substrate mimicry in this subsite The envelope
confor-mation of Pro294rTAXI-IIA(with N, Ca, Ccand Cd
copla-nar, and Cb 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
Pro294rTAXI-IIA conformation maximizes the burial of
the Trp9BSXside-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 Gln189rTAXI-IIA main-chain O
and Asn63BSXNd2(3.1 A˚), and between Gln192rTAXI-IIA
Ne2 and Tyr88BSX Of (2.3 A˚) (Fig 4B) Also, several
interactions are made between Arg122BSX in the thumb region and rTAXI-IIA, in particular with Asp322rTAXI-IIAOd2(4.1 A˚), Glu356rTAXI-IIAOe1(2.8 A˚) and Lys317rTAXI-IIAmain-chain O (2.8 A˚) Finally, inter-action is observed between Arg373rTAXI-IIA Ne and Asp11BSX Od2 (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.
Trang 7active 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 Glu127BSX in the)1
glycon subsite, involved in a hydrogen-bonding
inter-action with Phe375TAXI-IA and Phe377rTAXI-IIA, and of
Asp11BSX, which interacts with Arg371TAXI-IA and
Arg373rTAXI-IIA, resulted in TAXI insensitivity [3,4]
Xylanase mutants, where thumb-region residues
Arg122BSX and Asp121BSX, 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 acsensi-tivities [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 ANXÆTAXI-IA and
BSXÆTAXI-IA complexes In contrast to the
conformational change observed in TAXI-IA for this
His374TAXI-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 Asp37ANX has the lowest
pKa 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 His374TAXI-IA upon complexation with the inhibitor Re-orientation
of the histidine allows charge complementarity between the positively charged Nd1 atom of the imid-azole side-chain and the negatively charged Asp37ANX [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 Nd atom of the imidazole side-chain of His374TAXI-IA and the negatively charged Asp37ANX
Od2 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
D11Y ⁄ F ⁄ K >> 100
B
Xylanase
Inhibition (IC50) a
a The IC50 value is defined as the half-maximal inhibitory concen-tration under the conditions of the assay [3] [4].
Trang 8that the lower the pH optimum of the xylanase (i.e.
the lower the pKa 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 Asn35BSX with the corresponding
Asp37ANX 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
LCzCtermin 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 Pro294rTAXI-IIA– as
a result of the ring structure – shares more equivalent
positions with the xylose)2 sugar ring atoms compared
with the Leu292TAXI-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 Trp9BSX by Pro294rTAXI-IIA
than by the more voluminous Leu292TAXI-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 Pro294rTAXI-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 Pro294rTAXI-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 Trp9BSX was
exchanged for Tyr10ANX, was no longer inhibited by rTAXI-IIA and displayed a lower TAXI-I sensitivity (Table 3), illustrating the incompatibility between Pro294rTAXI-IIAand Tyr10ANXand the tighter binding between Pro294rTAXI-IIA and Trp9BSX than between Leu294TAXI-IA and Trp9BSX This confirms the crucial role of Leu294TAXI-IAand Pro294rTAXI-IIAfor 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
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
Unique reflections a 20136 (2965) 44570 (955) Completeness of all data (%)a 98.0 (98.0) 97.5 (97.5)
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 freec 0.181 ⁄ 0.239 0.211 ⁄ 0.266 Number of atoms
Root mean square deviations d
a Values in parentheses are for the highest resolution shell.
b R sym ¼ R h R j <IðhÞ> IðhÞj
=R h R j <IðhÞ>, where <I(h)> is the mean intensity of symmetry-equivalent reflections.cR cryst =R free ¼ R F j j o j
F c
j jj=R F j o j, where F o and Fcare the observed and calculated struc-ture factors, respectively d Root mean square deviations relate to the Engh and Huber parameters.
Trang 9lyst 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
Trang 10condi-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˚3ÆDa)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˚3ÆDa)1and 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 Arg387rTAXI-IIA–Ser388rTAXI-IIA– Thr389rTAXI-IIAat 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
...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... 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. .. funiculosum xylanase XynC, for which a pH optimum of results from a combination of an aspartate and a tryptophan residue Both our criteria on the strength and specific-ity of the inhibition, however,