Crystallization and preliminary X-ray diffraction analysis of the complex of Kunitz-type tamarind trypsin inhibitor and porcine pancreatic trypsin.
ABSTRACT The complex of Tamarindus indica Kunitz-type trypsin inhibitor and porcine trypsin has been crystallized by the sitting-drop vapour-diffusion method using ammonium acetate as precipitant and sodium acetate as buffer. The homogeneity of complex formation was checked by size-exclusion chromatography and further confirmed by reducing SDS-PAGE. The crystals diffracted to 2.0 angstrom resolution and belonged to the tetragonal space group P4(1), with unit-cell parameters a = b = 57.1, c = 120.1 angstrom. Preliminary X-ray diffraction analysis indicated the presence of one unit of inhibitor-trypsin complex per asymmetric unit, with a solvent content of 45%.
Acta Cryst. (2009). F65, 1179–1181doi:10.1107/S1744309109041694
Acta Crystallographica Section F
Crystallization and preliminary X-ray diffraction
analysis of the complex of Kunitz-type tamarind
trypsin inhibitor and porcine pancreatic trypsin
Sakshi Tomar, Dipak N. Patil,
Manali Datta, Satya Tapas,
Preeti, Anshul Chaudhary,
Ashwani K. Sharma, Shailly
Tomar* and Pravindra Kumar*
Department of Biotechnology, Indian Institute of
Technology, Roorkee, Roorkee 247667, India
Correspondence e-mail: firstname.lastname@example.org,
Received 12 September 2009
Accepted 12 October 2009
The complex of Tamarindus indica Kunitz-type trypsin inhibitor and porcine
trypsin has been crystallized by the sitting-drop vapour-diffusion method using
ammonium acetate as precipitant and sodium acetate as buffer. The homo-
geneity of complex formation was checked by size-exclusion chromatography
and further confirmed by reducing SDS–PAGE. The crystals diffracted to 2.0 A˚
resolution and belonged to the tetragonal space group P41, with unit-cell
parameters a = b = 57.1, c = 120.1 A˚. Preliminary X-ray diffraction analysis
indicated the presence of one unit of inhibitor–trypsin complex per asymmetric
unit, with a solvent content of 45%.
Proteases are proteinaceous enzymes that catalyze the hydrolytic
cleavage of the peptide bond. These enzymes play a vital role in
nonspecific digestion of intracellular and extracellular proteins and
are specifically required for the proteolytic cleavage of inactive pre-
cursors, the activation of zymogens, the processing of hormones and
neuropeptides, the activation of receptors, protein translocation
through membranes etc. (Neurath, 1984). One class of proteases are
the serine proteases (EC 3.4.21), which are thus classified owing to
the presence of a serine residue in their active site. These proteolytic
enzymes are actively involved in various physiological processes such
as the coagulation of blood, complement system activation, fertili-
zation, the hypersensitive response, microsporogenesis, cell prolif-
eration and differentiation and signal transduction via protein
degradation/processing (Neurath, 1986; Anta ˜o & Malcata, 2005).
With proteases being involved in so many important functions, the
significance of the antagonist becomes equally crucial. Moderation of
activity is performed by a class of ubiquitous moieties called protease
inhibitors (PIs). Various classes of inhibitors have been studied
extensively as they have the potential to act as therapeutic agents.
Among these, serine protease inhibitors have been the most studied
and have been isolated from various sources including Leguminosae
seeds (Macedo et al., 2000; Macedo & Xavier-Filho, 1992; Mello et al.,
2003; Souza et al., 1995). Serine proteinase inhibitors are particularly
effective against insects and foraging herbivores and have therefore
become a prominent target for use in pest control (Reckel et al.,
1997). Legume seeds contain various PIs that have been classified
into several families, one of which, the Kunitz-type inhibitors, are one
of the best characterized families of plant serine protease inhibitors.
In general, Kunitz-type inhibitors are low-molecular-weight proteins
of approximately 20 kDa with low cysteine content, resulting in 1–2
intrachain disulfide bonds, and a single reactive site.
The complex of porcine pancreatic trypsin with Kunitz-type
soybean trypsin inhibitor (SKTI) has been characterized by X-ray
crystallography. Superposition of the complexed (PDB code 1avu)
and uncomplexed (PDB code 1avw) forms of SKTI showed obvious
changes in the reactive loop and the N-terminal region of the inhi-
bitor, with reduction ofthe B factor upon complex formation (Song &
Suh, 1998). In the case of another important protease inhibitor,
# 2009 International Union of Crystallography
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bovine pancreatic trypsin inhibitor (BPTI), the reactive-loop region
and C-terminal region were found to coordinate to trypsin (Capasso
et al., 1998). This highlights the involvement of different regions of
protease inhibitors in interactions with the counterpart proteases,
enabling different inhibition mechanisms. The ability of a protease
inhibitor to interact with a protease depends on the structural
framework of the surface residues of the inhibitor. Some Kunitz-type
inhibitors can inhibit a range of proteases, whereas others are
protease-specific (He et al., 2008; Milstone et al., 2000). Modelling of
the structure of Erythrina caffra trypsin inhibitor (ETI) based on
SKTI confirmed the role of the surface residues as the main factor in
selection of the interacting partner (Song & Suh, 1998). Although a
large number of sequences of trypsin inhibitors are available in
sequence databases, basic knowledge of the mechanism of their
inhibitory action remains elusive. Therefore, determination of three-
dimensional structures of Kunitz inhibitor–protease complexes
should elucidate the molecular mechanism of Kunitz-type protease
inhibitors. The structural and molecular-interaction information
provided by these structures could subsequently be employed in the
design of potential novel protease inhibitors.
In an attempt to study protease–inhibitor interaction, a Kunitz-
type proteinase inhibitor from the seeds of Tamarindus indica was
cocrystallized with porcine trypsin. Here, we report the crystallization
and preliminary X-ray diffraction analysis of this complex of a
Kunitz-type proteinase inhibitor with trypsin.
2. Experimental procedure
2.1. Complex formation and purification
Tamarind trypsin inhibitor (TTI) from the seed kernel of T. indica
was purified according to a previously published protocol (Patil et al.,
2009). Porcine pancreatic trypsin was obtained from Sigma–Aldrich.
The complex was prepared by mixing 50 ml purified Kunitz-type
protease inhibitor at a concentration of 10 mg ml?1in a buffer
solution containing 100 mM Tris pH 7.4 with 50 ml porcine trypsin at a
concentration of 10 mg ml?1in a buffer solution containing 100 mM
Tris pH 7.4 and 40 mM CaCl2. The protease–inhibitor mixture was
then incubated at 277 K for 2 h. For purification of the protease–
inhibitor complex , 100 ml of the mixture was loaded onto a Superdex-
200 GL 10/300 (GE Healthcare) size-exclusion column using a 100 ml
sample loop at 0.5 ml min?1on an A¨KTA Purifier FPLC system (GE
Healthcare). Elution of the protein was detected by UVabsorbance
at 280 nm. The size-exclusion column was calibrated with Gel
Filtration Calibration LMW standards (GE Healthcare; blue dextran,
thyroglobulin, ferritin, aldolase, conalbumin and ovalbumin) for
determination of the void volume, construction of the standard curve
and estimation of the molecular weight of the complex. The eluted
fractions corresponding to the molecular weight of the protease–
inhibitor complex were analyzed on 15% reducing SDS–PAGE. The
fractions containing the trypsin–TTI complex were pooled and
concentrated to 5 mg ml?1at 277 K using an Amicon Ultra-4 10 kDa
cutoff concentrator (Millipore). The protein concentration was
determined with the Bio-Rad protein-assay kit using bovine serum
albumin (BSA) as a standard.
Crystallization was performed by the sitting-drop method at 293 K
using Crystal Screens I and II and Salt Screen (Hampton Research,
USA) for initial screening in 96-well crystallization plates (Hampton
Research, USA). Drops were prepared by mixing 2 ml protein solu-
tion with 2 ml precipitant solution and were equilibrated against
50 ml reservoir solution. Tamarind inhibitor–porcine trypsin complex
crystals grew under various conditions, but the best quality crystals
grew in 4 M ammonium acetate and 0.1 M sodium acetate trihydrate
2.3. Data collection and analysis
Crystals were mounted in cryoloops (Hampton Research, USA)
and flash-cooled by direct immersion in liquid nitrogen prior to X-ray
diffraction analysis. Data were collected on a MAR 345dtb image-
plate system using Cu K? radiation generated by a Bruker Microstar-
H rotating-anode generator operated at 45 kV and 60 mA and
equipped with Helios optics. Data were collected as 90 images with a
crystal-to-detector distance of 170 mm and 1?oscillation per image.
The time of exposure was 10 min. The crystal diffracted to 2.0 A˚
resolution. The diffraction data were processed and scaled with the
AutoMAR program (Bartels & Klein, 2003).
3. Results and discussion
was found to possess trypsin-inhibitory activity (Patil et al., 2009).
Tomar et al.
? Trypsin–tamarind trypsin inhibitor complex
Acta Cryst. (2009). F65, 1179–1181
15% SDS–PAGE. Lane 1, porcine trypsin; lane 2, tamarind trypsin inhibitor TTI;
lane 3, the major gel-filtration peak fraction containing the complex between TTI
and trypsin; lane 4, protein molecular-weight markers (kDa).
A crystal of the complex between tamarind trypsin inhibitor (TTI) and trypsin. The
longest dimension of a typical crystal was between 80 and 100 mm.
Therefore, an attempt was made to prepare a protein–protein
complex between porcine trypsin and TTI. For complex formation,
porcine trypsin and TTI were mixed and the mixture was incubated at
277 K for 2 h. As described above, size-exclusion chromatography
was employed to purify the protease–inhibitor complex from the
incubated mixture of TTI and trypsin. The molecular weight of the
major elution peak from a Superdex-200 GL 10/300 (GE Healthcare)
size-exclusion column was calculated using a standard curve and was
estimated to be ?44 kDa, which corresponded to the expected
molecular weight of the TTI–trypsin protein complex. Additionally,
to confirm the presence of the complex in the major elution peak,
peak fractions were run on reducing 15% SDS–PAGE and protein
bands corresponding to trypsin (?23 kDa) and TTI (?21 kDa) were
observed (Fig. 1). The fractions containing the complex were subse-
quently pooled andconcentrated to15 mg ml?1. The complex crystals
of TTI and trypsin were obtained in 20 d at 293 K by vapour diffusion
of a 4 ml sitting drop against 50 ml reservoir solution containing 4 M
ammonium acetate and 0.1 M sodium acetate trihydrate pH 4.6
(Fig. 2). Cryoprotection was achieved by the high concentration of
ammonium acetate. Diamond-shaped crystals were obtained that
belonged to the tetragonal space group P41and diffracted to 2.0 A˚
The unit-cell parameters were found to be a = b = 57.1, c= 120.1 A˚,
? = ? = ? = 90?, with one binary complex of porcine trypsin and
tamarind inhibitor per asymmetric unit. This corresponds to a crystal
volume per unit molecular weight (VM) of 2.26 A˚3Da?1, given the
molecular weight of 44 kDa for the protein complex, with a solvent
content of 45% (Matthews, 1968). The data-collection statistics are
summarized in Table 1.
The authors are grateful to and thank the Macromolecular Crys-
tallographic Facility (MCU) at IIC, IIT Roorkee for data collection.
DNP thanks MHRD, Government of India, MD and ST thank
AICTE and Preeti thanks CSIR for financial support.
Anta ˜o, C. M. & Malcata, F. X. (2005). Plant Physiol. Biochem. 43, 637–650.
Bartels, K. S. & Klein, C. (2003). The AUTOMAR Manual, v.1.4. Norderstedt,
Germany: MAR Research GmbH.
Capasso, C., Rizzi, M., Menegatti, E., Ascenzi, P. & Bolognesi, M. (1998). J.
Mol. Recognit. 10, 26–35.
He, Y. Y., Liu, S. B., Lee, W. H., Qian, J. Q. & Zhang, Y. (2008). Peptides, 29,
Macedo, M. L. R., Matos, D. G. G., Machado, O. L. T., Marangoni, S. &
Novello, J. C. (2000). Phytochemistry, 54, 553–558.
Macedo, M. L. R. & Xavier-Filho, J. (1992). J. Sci. Food Agric. 58, 55–58.
Matthews, B. W. (1968). J. Mol. Biol. 33, 491–497.
Mello, M. O., Tanaka, A. S. & Silva-Filho, M. C. (2003). Mol. Phylogenet. Evol.
Milstone, A., Harrison, L., Bungiro, R., Kuzmic, P. & Cappello, M. (2000). J.
Biol. Chem. 275, 29391–29399.
Neurath, H. (1984). Science, 224, 350–357.
Neurath, H. (1986). J. Cell. Biochem. 32, 35–49.
Patil, D. N., Preeti, Chaudhry, A., Sharma, A. K., Tomar, S. & Kumar, P. (2009).
Acta Cryst. F65, 736–738.
Reckel, R. K., Kramer, K. J., Baker, J. E., Kanost, M. R., Fabrick, J. A. &
Behnke, G. A. (1997). Advances in Insect Control. The Role of Transgenic
Plants, edited by N. Carozzi & M. Koziel, pp. 157–183. London: Taylor &
Song, H. K. & Suh, S. W. (1998). J. Mol. Biol. 275, 347–363.
Souza, E. M. T., Mizuta, K., Sampaio, M. U. & Sampaio, C. A. M. (1995).
Phytochemistry, 39, 521–525.
Acta Cryst. (2009). F65, 1179–1181 Tomar et al.
? Trypsin–tamarind trypsin inhibitor complex
Data-collection and processing statistics.
Values in parentheses are for the highest resolution shell.
Unit-cell parameters (A˚,?)
a = b = 57.1, c = 120.1,
? = ? = ? = 90
No. of observed reflections
No. of unique reflections
of reflection hkl.
ijIiðhklÞ ? hIðhklÞij=P
iIiðhklÞ, where Ii(hkl) is the ith observa-
tion of reflection hkl and hI(hkl)i is the weighted average intensity for all observations i