Transcript 1 Crystallographic structure analysis of Chitinase enzyme from

Crystallographic structure analysis of Chitinase enzyme
from Corms of Crocus vernus
Dr. Ahmed Akrem
Bahauddin Zakariya University, Multan
29.04.14
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Importance of Chitinase
 Chitinases catalyze the hydrolysis of chitin1
 Chitinases occur in a wide range of organisms, including plants, animals, viruses,
bacteria, fungi and insects, and play a variety of roles in these organisms.2
 Plant chitinases are a structurally diverse group with respect to their physical
properties, enzymatic activities and localization.3
 Chitin is an unbranched homopolymer of 1,4-linked N-acetyl-d-glucosamine.4
 Chitin is not a component of mammalian cells; it occurs widely elsewhere in nature
and is abundant in human pathogens.
 More than 75% of the industrial enzymes are hydrolases.5
1Bishop
2
et al., 2000; 2Brunner et al., 1998; 2Hoell et al., 2005; 3Collinge et al., 1993; 4Butt & Sultan, 2010;
5Leishola
et al., 2005
Aims and Objectives
1.
Isolation and purification of the chitinase protein from C. vernus
2.
Crystallization of the purified protein
3.
X-ray diffraction data collection

3D molecular structure determination
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Protein Crystallization
Non-recombinant
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Nextal.com
Crocus vernus
 Genus Crocus belongs to family Iridaceae.
 A perennial flowering plant found in Central and Southern Europe,
North Africa, Middle East, Central Asia to China.
 Most expansive spice “Saffron” is from Crocus sativus L.
 Chitinases & Lectins are ´´ Defense-related plant proteins``.
 Plant chitinases are a structurally diverse group.3
 So far no crystal structure of this plant is deposited in the Protein Data
Bank.
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3Collinge
et al., 1993,
Crude protein porfile
Crocus vernus
118kDa
66.2kDa
45.0kDa
35.0kDa
25.0kDa
18.4kDa
Crude Protein profile from corm on
SDS-PAGE
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14.4kDa
Purified Chitinase
 Final optimized Mono S chromatogram on 12% SDS-PAGE produces lectin
contaminations.
 Size exclusion chromatography produces 30 kDa protein bands with identical pattern
under reduced and non-reduced conditions.
 Partial N-terminal sequence blast showed 50% identity with the already reported
chitinases.11
 TLFVEYIGYPLFSGVKFSDVPINPE ITKFQ
Mono S peak
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11Akrem
et al., 2011
Size exclusion
chromatogram
L1: Reduced,
L2: Non-reduced
PVDF blot
Purification techniques/Instruments
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 Protein characterization
• N-terminal amino acid sequencing
• MALDI/TOF Mass spectrometry
• SDS-PAGE
 Protein Purification
• Ammonium sulfate precipitation
• Dialysis
• Column Chromatography
• Gel filtration
• Ion exchanger columns (Cation/Anion: Isoelectric pH
or pI)
Crystallization of Chitinase
 Dynamic Light Scattering (DLS) measurement of the 30 kDa purified protein
monodispersive and monomeric protein solution.
 PCT™ was performed to optimize the protein concentration.
 Protein crystallized at concentration of 16 mg ml -1.
 Vapor diffusion method
 Crystal with dimensions of 0.625 × 0.370 × 0.1 mm: Scale bar, 0.5 mm.
 0.1M CHES, pH 9.0 and 20% (w/v) PEG 800011
Monodisperse
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11Akrem
et al., 2011
RH = 2.6nm
Thin sheet
showing
Vapor Diffusion Methods
Hanging Drop
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Sitting Drop
Protein Crystallization
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Metastable
Soluble aggregate formation but no nucleation
Nucleation
Critical nuclei formation and crystal growth
Precipitation
No nucleation. Growth of amorphous precipitate
Phase Diagram
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Crystallization Machinery
Nanodrop
(Protein quantification)
Zinsser Pipetting Robot (Digilab
Genomic Solution, Germany)
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Dynamic Light Scattering
(Monodispersity)
UV-microscope
Protein/Salt Crystals
VIS-microscope
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UV-microscope
Best is to go for diffraction image
1mm size approx.
Diffraction to 3D
Single Crystal
X-ray Bombarment
3D Structure
Crystallographic Softwares
Diffraction Image
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X-ray Diffraction Data
 Diffractometer
 Rotating anode
 Synchrotron: the best ultimate choice
 A synchrotron is a particular type of cyclic particle accelerator in which the magnetic
field (to turn the particles so they circulate) and the electric field (to accelerate the
particles) are carefully synchronized with the travelling particle beam
 Deutsches Elecktronen Synchrotron (DESY), Hamburg, Germany
 Approx. 1000 scientists from more than 30 countries around the world are working (2008)
 Few countries in the world are enjoying this facility
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Diamond, UK
ESRF, France
DESY, Germany
Crystal Mounting
 Nylon loops to fish out crystals
 Goniohead
 X-ray gun
 Cryonozzle
 Microscope
 Beamstop
 Detectors
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Bioinformatics
 Imosflm; Scala
 Denzo; Scalepack
 CCP4i Suite
 Molecular Replacment; Molrep, Phaser, Mrbump
 Homer
 COOT
 Refmac5
 Protein Data Bank
 Pdbsum
 Pdb goodies
 Chimera
 Pymol
 Auto-Rickshaw
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X-ray diffraction Data
Table 6: Statistics for the native crystal11
Space group
C2
Unit-cell parameters (Å,°)
a = 172.3, b = 37.1, c = 126.4 Å, β = 127o
VM (Å3/ Da)
2.7
Solvent content (%)
54.2
Resolution range (Å)
25.0 - 2.1 ( 2.2 - 2.1 )
Total Reflections
14,0335 (20369)
Unique reflections
36468 (5230)
Redundancy
3.8 (3.9)
Average I/σ (I)
17.2 (6.8)
R merge* (%)
6.2 (19.4)
Data Completeness (%)
96.6 (96.0)
Values in parentheses are for the highest resolution shell.
*R merge =∑hkl ∑i | Ii (hkl) – <I (hkl)> | ⁄ ∑hkl∑i Ii (hkl), where <I (hkl)> is the mean intensity of the
observations Ii (hkl) of reflection hkl.
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11Akrem
et al., 2011
mtz and pdb files
 Out put of first processing is a single mtz file of few MB
 Electron density map
 Second important file is pdb file based on sequence homology from Protein Data
Bank (PDB) like INAR for Narbonin vicia
 MR strategy to solve the phase information
 Sequence identity of atleast 40%
 Clustalw 2, www.pdb.org
 Pdbgoodies input page
 Phase information and Coordinate information
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GH18 Type Chitinase
 Phase problem was solved by Molecular Replacement (MR) using the narbonin
structure (PDB code: 1NAR) as search model and the program Molrep.
 Chitinases catalyze the hydrolysis of chitin.
 Chitinases occur in a wide range of organisms including plants, animals, viruses,
bacteria, fungi and insects.
 In glycosyl hydrolases, they are classified into family 18 and family 19 chitinases.13
 Family 18, in their catalytic domain, possesses a common α, β-TIM barrel fold.
 Matthews’s coefficient calculations indicated two molecules per asymmetric unit.
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13Henrissat
& Bairoch, 1993
TIM Barrel
 Triose Phosphate Isomerase (TIM)
 Main feature of TIM barrel is an eight stranded parallel β-barrel making a core surrounded
by α- helices.
 The cavity of the TIM barrel in CVC is filled with aromatic and polar residues.
 The catalytic motif of CVC is directed into the cavity of TIM barrel.
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Sequence Alignment
 16% sequence identity between CVC
and Hevamine (2HVM).14
 33% sequence identity between CVC
and Narbonin.
 Hevamine: a plant endochitinase
isolated from rubber tree (Hevea
brasiliensis).
 All three proteins are sharing a TIM
barrel structure.
 Catalytic motif is DXDXE
 Two consensus sequences have been
highlighted through blue squares.
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14Terwisscha
van Scheltinga et al., 1996
Sequence alignment between
CVC, Heavime & Narbonin
Structure Alignment
 The superposition of the Cα atoms of CVC with that of other members of the family
gives a rmsd of 3.5 and 3.6 Å for models 2HVM, 1NAR respectively.
 The catalytic motifs for all structures are on similar position in the TIM barrel.
 Narbonin, due to lack of an Aspartate in the catalytic motif, cannot show chitinase activity.
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Summary
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A 30 kDa chitinase protein was purified from Crocus vernus corm.
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Single suitable size crystals were developed from pure enzyme
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Already the native Chitinase structures have been deposited at the Protein Data Bank
with ID code 3SIM.
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Thanks for kind attention
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