Modulation of inhibitory activity of xylanase - α-amylase inhibitor protein (XAIP): binding studies and crystal structure determination of XAIP- II from Scadoxus multiflorus at 1.2 Å resolution
© Kumar et al; licensee BioMed Central Ltd. 2010
Received: 28 August 2010
Accepted: 20 November 2010
Published: 20 November 2010
Plants produce a wide range of proteinaceous inhibitors to protect themselves against hydrolytic enzymes. Recently a novel protein XAIP belonging to a new sub-family (GH18C) was reported to inhibit two structurally unrelated enzymes xylanase GH11 and α-amylase GH13. It was shown to inhibit xylanase GH11 with greater potency than that of α-amylase GH13. A new form of XAIP (XAIP-II) that inhibits α-amylase GH13 with a greater potency than that of XAIP and xylanase GH11 with a lower potency than that of XAIP, has been identified in the extracts of underground bulbs of Scadoxus multiflorus. This kind of occurrence of isoforms of inhibitor proteins is a rare observation and offers new opportunities for understanding the principles of protein engineering by nature.
In order to determine the structural basis of the enhanced potency of XAIP-II against α-amylase GH13 and its reduced potency against xylanase GH11 as compared to that of XAIP, we have purified XAIP-II to homogeneity and obtained its complete amino acid sequence using cloning procedure. It has been crystallized with 0.1 M ammonium sulphate as the precipitating agent and the three-dimensional structure has been determined at 1.2 Å resolution. The binding studies of XAIP-II with xylanase GH11 and α-amylase GH13 have been carried out with surface plasmon resonance (SPR).
The structure determination revealed that XAIP-II adopts the well known TIM barrel fold. The xylanase GH11 binding site in XAIP-II is formed mainly with loop α3-β3 (residues, 102 - 118) which has acquired a stereochemically less favorable conformation for binding to xylanase GH11 because of the addition of an extra residue, Ala105 and due to replacements of two important residues, His106 and Asn109 by Thr107 and Ser110. On the other hand, the α-amylase binding site, which consists of α-helices α6 (residues, 193 - 206), α7 (residues, 230 - 243) and loop β6-α6 (residues, 180 - 192) adopts a stereochemically more favorable conformation due to replacements of residues, Ser190, Gly191 and Glu194 by Ala191, Ser192 and Ser195 respectively in α-helix α6, Glu231 and His236 by Thr232 and Ser237 respectively in α-helix α7. As a result, XAIP-II binds to xylanase GH11 less favorably while it interacts more strongly with α-amylase GH13 as compared to XAIP. These observations correlate well with the values of 4.2 × 10-6 M and 3.4 × 10-8 M for the dissociation constants of XAIP-II with xylanase GH11 and α-amylase GH13 respectively and those of 4.5 × 10-7 M and 3.6 × 10-6 M of XAIP with xylanase GH11 and α-amylase GH13 respectively.
Plants produce a wide range of proteinaceous inhibitors that protect them from the unwanted hydrolytic effects of endogenous enzymes as well as from those of infecting micro-organisms. Recently, a new inhibitor protein with two independent binding sites designated as XAIP (Xylanase and α-amylase inhibitor protein) was isolated from Scadoxus multiflorus . This protein showed sequence homologies of 48% with heavamine, another plant protein with chitinase activity , 39% with concanavalin (con-B)  and 11% with narbonin . The latter two did not act as chitinases while their precise functions are still unkonown. XAIP also showed a 36% sequence homology with XIP-I (xylanase inhibiting protein) that inhibits xylanases GH10 and GH11. It also lacks chitinase-like activity [5, 6]. Structurally, they all adopt (β/α)8 barrel fold. Because of an extra α-helix α8' in the structures of these proteins, they all are classified into a sub-family of glycosyl hydrolyses 18C (GH18C) as a part of the larger family of GH18 proteins that consists of mainly chitinases  and various other proteins of unknown functions [3, 4, 8]. The proteins of sub-family GH18C show significant sequence variations while they adopt an overall similar scafolding. These proteins differ greatly in their functional specificities [9, 10]. We report here a new form of XAIP (XAIP-II) which inhibits xylanase GH11 with a reduced potency whereas it binds to α-amylase with a considerably enhanced binding affinity as compared to XAIP . The two forms, XAIP-II and XAIP show a sequence homology of 87% while 13% sequence variations occur mostly in the regions of ligand binding sites. The detailed structure determination of XAIP-II has allowed us to examine the reasons for the lack of chitinase activity, loss of carbohydrate binding capability, reduction in xylanase specific activity and significant increase in the potency of α-amylase inhibition.
Results and Discussion
Determination of KD by surface plasmon resonance
Quality of the model
At the end of the refinement, the values of Rcryst and Rfree factors were 15.8% and 18.6% respectively. The overall geometry of the XAIP-II structure refined to 1.2Å resolution was good with a molprobity  score of 85 percentile. The average thermal B-factor was 15.8Å2 while the 92.3% residues were found in the most favored regions of the Ramachandran plot  as indicated by PROCHECK .
Overall structure of XAIP-II
Comparisons of the structures of XAIP-II and XAIP
Both molecules lack chitinolytic activity. Both structures differ significantly in the regions of two interaction sites corresponding to xylanase GH11 binding site and α-amylase GH13 binding site. The structures show that the carbohydrate binding channel is relatively less obstructed in XAIP-II than that in XAIP. The binding constant of XAIP-II with xylanase GH11 is less than that of XAIP while the binding affinity with α-amylase GH13 is considerably enhanced.
Carbohydrate binding channel
Binding with xylanase GH11
Binding with α-amylase GH13
The XAIP-II and XAIP are two forms of a protein that possess two independent interaction sites and inhibits two structurally unrelated enzymes xylanase GH11 and α- amylase GH13. Both xylanase GH11 and α-amylase GH13 hydrolyze plant related polymers xylan and starch respectively. Similarly, both XAIP-II and XAIP adopt stable TIM barrel folds. However, interestingly with only a few differences in their amino acid sequences on the common TIM barrel framework, the binding affinities of XAIP-II and XAIP varied significantly. XAIP-II binds to xylanase GH11 with an affinity lower than that of XAIP. The most important interactions of XAIP with xylanase GH11 are provided by His106 whereas the corresponding Thr107 in XAIP-II does not provide comparable interactions. The positioning of His106 in XAIP is a result of a unique conformation of the tripeptide Gly105-His106-Ser107 with two important intra tripeptide hydrogen bonds, Ser107 N --- O Gly105 = 3.0Ǻ and Ser107 Oγ --- O His106 = 2.7Ǻ. In contrast, the corresponding residue in XAIP-II is Thr107 which is positioned unfavorably due to an entirely different conformation of the corresponding tripeptide. The observed interactions clearly show that XAIP binds to xylanase GH11 more favourably than that of XAIP-II. On the other hand, XAIP-II inhibits the function of α-amylase GH13 with a considerable higher potency as shown by the dissociation constant of 3.4 × 10-8 M whereas XAIP inhibits it with a lower potency as the dissociation constant in this case is 3.6 × 10-6 M. In this regard, it is pertinent to note here that the amino acid changes between XAIP-II and XAIP have been observed primarily in the binding segments including loops α3-β4 and α4-β5 for the interactions with xylanase GH11 and in loops β6-α6 and β7-α7 and α-helices α6 and α7 for the interactions with α-amylase GH13. The insertion of an extra residue alanine at position 105 in the α3-β4 loop and replacements of His106 by Thr107 and Asn98 by Lys98 reduced both structural and chemical complementarities in XAIP-II with respect to the binding site in xylanase GH11 resulting in the formation of a less number of interactions with xylanase GH11. On the other hand the replacements of Gly191 by Ser192, Glu194 by Ser195, Glu231 by Ser232 and His236 by Glu237 made XAIP-II more compatible for the binding with α-amylase GH13 as compared to XAIP.
The existence of isoforms of enzymes is well known  and very often various organisms alter amino acid sequences in enzymes for altering their stereochemical arrangements for preventing the unwanted inhibitions of their functions. In this regard, the present example is one of the rare cases where the inhibitor proteins are found in more than one forms for protecting the host from the undesirable effects of hydrolytic enzymes presumably by alternating potencies so as to address the variations in the concentrations of enzymes Xylanase GH11 and α-amylase GH13.
We have determined the structure of a new form of xylanase GH11 and α-amylase GH13 inhibitor protein (XAIP-II) at 1.2Ǻ resolution. The XAIP-II structure with slightly altered stereochemistry of its two independent binding sites shows that it binds to xylanase GH11 less favorably as compared to XAIP . Whereas its α-amylase binding site shows stronger interactions as compared to those of XAIP. This specific interchange of inhibitory potencies demonstrates the potential of protein engineering by nature and allows us to have a deeper insight into the principle of selective replacements and insertions of amino acid residues in the proteins.
Purification of XAIP-II from Scadoxus multiflorus
The purification of XAIP-II was carried out using the modified procedure of Kumar et al. . The samples of underground bulbs of Scadoxus multiflorus were collected from the nurseries located in the podhills of Himalayas. The bulbs were cut into small pieces and pulverized with the help of liquid nitrogen in a ventilated hood. The suspended material was dissolved in the extraction solution containing 0.2 M NaCl and 50 mM phosphate buffer, pH 7.2. An amount of 2.5 g of polyvinylpyrolidine (PVPP)/100 ml was added to the sample. The homogenate was centrifuged at 8000 g for 45 min at 4°C and the supernatant was collected. The ammonium sulphate was gradually added to the supernatant to make it to 80% saturation with constant stirring. This was incubated overnight on ice and centrifuged at 7000 g for 30 min. The pellet was removed and resuspended in the amount of 50 mM phosphate buffer, pH 7.2. It was dialyzed repeatedly against the same buffer with frequent changes for removing the salt. The dialyzed suspension was centrifuged at 8000 g for 30 min and the supernatant was loaded on DEAE-Sepharose A-50 column (50 × 2 cm) which was equilibrated with 50 mM phosphate buffer, pH 7.2. The protein was eluted using a linear gradient of 0.0 - 0.5 M NaCl in 50 mM phosphate buffer, pH 7.2. The second and third peaks in the elution profile were collected and pooled. These two peaks were identified with high and low inhibitions of xylanase (Penicillium furniculosum). The fraction that showed low inhibition of xylanase GH11 was collected and freeze-dried. This fraction was further gel filtered using Sephadex G-50 column (150 × 1 cm) with 50 mM phosphate buffer, pH 7.2 at a flow rate of 6 ml/hour. The first peak was collected, pooled and lyophilized. The sequence of the first 20 amino acid residues from N-terminus 1Gly-Ser-Leu-Asp-Ile-Ala-Val-Tyr-Trp-Gly-Gln-Ser-Phe-Asp-Glu-Arg-Ser-Asn-Glu-Ala20 was determined using automatic protein sequencer PPSQ21A (Shimadzu, Kyoto, Japan).
Complete nucleotide sequence determination
In order to obtain the complete amino acid sequence of XAIP-II, the bulbs were sliced into small pieces and crushed into powder with Liquid Nitrogen. The total RNA was extracted using TRIZOL Reagent (Invitrogen, Carlsbad, USA). The cDNA synthesis was carried out from 10 ng of RNA with reverse transcription kit (Fermentas, Burlington, Canada) according to the manufacturer's instructions. The gene was amplified from the cDNA using a pair of primers. The forward primer 5'-GGCAGTCTGGACATCGCCGTC-3' derived from the N-terminal amino acid sequence of Gly-Ser-Leu-Asp-Ile-Ala-Val and the reverse primer 5'-CACGCCTTTGCCGAGGATCTT-3' obtained from the C-terminal amino acid sequence, Lys-Ile-Leu-Gly-Lys-Gly-Val were prepared. The PCR product was cloned in pGEMT-easy vector (Promega, Madison, USA) and the nucleotide sequence was obtained using ABI Prism 7000 (Applied Biosystem, Foster City, USA). The nucleotide sequence has been submitted in Genbank with an ID code of HM474410.
Surface plasmon resonance studies of XAIP-II with xylanase GH11 and α-amylase GH13 enzymes
The method of surface plasmon resonance (SPR) was used for studying the binding properties of XAIP-II with xylanase GH11 [, PDB ID: 1TE1] and α-amylase GH13 [19, PDB ID: 1BLI]. All the SPR measurements were performed at 25°C using the BIAcore-2000 apparatus (Pharmacia Biosensor AB, Uppsala, Sweden) in which a biosensor-based system has been used for the real time specific interaction analysis. The sensor chip CM5, the amine coupling kit containing N-hydroxysuccinimide (NHS), N-ethyl-N'-3 (diethylaminopropyl) carbodiimide (EDC) and ethanolamine hydrochloride (Pharmacia Biosensor AB, Uppsala, Sweden) were used in the experiment. The running buffer used was 10 mM HBS-EP (pH 7.4) containing 0.005% surfactant P20. The sensor chip CM5 (disposable sensor chip, the surface of which was covered with a thin gold layer coated with carboxy-methyl dextran residue for covalent protein immobilization) was purchased from Pharmacia Biosensor AB (Uppsala, Sweden). The immobilization of XAIP-II was carried out at a flow rate of 10 μl/min at 25°C using amine coupling kit. The dextran on the chip was equilibrated with running buffer and carboxymethylated matrix was activated with an EDC/NHS mixture containing 210 μl of XAIP-II (80 μg/ml) in 10 mM sodium acetate (pH 4.6) was injected and unreacted groups were blocked by injecting ethanolamine. The SPR signal for immobilized XAIP-II was found to be 1254 RUs. Three different concentrations of the ligands, α-amylase and xylanase, 1.8 μM, 3.6 μM and 5.4 μM were prepared in 10 mM HBS-EP buffer (pH 7.4). These samples were then injected separately in two different flow cells, one with immobilized XAP-II and the other without XAIP-II as a reference to remove nonspecific binding with the surface of the chip in different cycles at a flow rate of 10 μl/min at 25°C. The dissociations of these ligands were induced by 10 mM HBS-EP buffer (pH 7.4). The rate constants KA and KD were obtained by fitting the primary sensorgram data using the BIA evaluation 3.0 software. The regeneration of the ligand bound to the surface of the protein was carried out using 0.1 mM NaOH. The kinetic parameters were obtained using the BIA evaluation software package. The association (kon) and dissociation (koff) rate constants for the ligand binding to XAIP-II were calculated and the value of the dissociation constant (KD) value was determined by the mass action relation KD = koff /kon.
Crystallization of XAIP-II
The freshly purified samples of XAIP-II were dissolved in 20 mM phosphate buffer pH 7.2 to a final protein concentration of 20 mg/ml. The protein was crystallized with hanging drop vapour diffusion method at 293K using 24 well Limbro crystallization plates. The protein drops of 10 μl were equilibrated against the reservoir solution containing 0.1 M ammonium sulphate, 15 mM phosphate buffer, pH 7.2, 0.1 M sodium acetate and 10% (w/v) PEG 6000. The crystals grew to the maximum dimensions of 0.3 × 0.15 × 0.10 mm3 within a period of three weeks.
X-ray intensity data collection
Data collection and refinement statistics
Unit cell dimensions
Number of molecules in the unit cell
Resolution range (Å)
36.0 - 1.2
The range of the highest shell
1.24 - 1.20
Total number of measured reflections
Number of unique reflections
Completeness of data (%)
Rfree (%) 5% of reflections
Water oxygen atoms
Phosphate ion atoms (1)
Atoms from PEG
R.m.s.d in bond lengths (Å)
R.m.s.d in bond angles (°)
R.m.s.d in torsion angles (°)
B-factors (Å 2 )
B-factor from Wilson plot (Å2)
Mean B-factor for main chain atoms (Å2)
Mean B-factor for side chain and water atoms (Å2)
Mean B-factor for all atoms(Å2)
Ramachandran's ϕ, ψ map
Residues in the most favoured regions (%)
Residues in the additionally allowed regions (%)
Residues in the generously allowed regions (%)
Structure determination and refinement
The structure of XAIP-II was determined with molecular replacement method using coordinate of XAIP (PDB: 3D5H) as the search model. It was refined using the options of rigid body refinement, simulated annealing and energy minimization with program CNS  using data in the resolution range of 36.0 to 1.2Å. The conformations of loops were particularly examined by inspecting the composite OMIT maps using the programs REFMAC5 from CCP4 program suite  and COOT . The refinement steps were repeated with intermitant manual building of the model. The positions of water oxygen atoms were determined manually using difference Fourier (|Fo - Fc|) maps on the basis of peak height and distance criteria. The water molecules whose thermal factors were 50Å2 or above after refinement were removed from the list. Further model building and refinement cycles resulted in an Rcryst of 0.158 and Rfree of 0.186 for 62,459 reflections from 36.0 to 1.2Å resolution. The average value of thermal B factor for all the atoms was 15.8Å2. The refinement statistics is given in Table 1.
xylanase - α-amylase inhibitor protein
The authors acknowledge the grant from the Department of Science and Technology (DST), New Delhi. TPS thanks the Department of Biotechnology, Ministry of Science and Technology New Delhi for the award of Distinguished Biotechnology Research Professorship to him. NS and MS thank Council of Scientific and Industrial Research, New Delhi for the award of Senior Research Associateships.
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