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BIOINFORMATICSPROJECT

BY: MIT KOTECHA

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CONTENTS…. 

Aim Introduction

Softwares used for modeling

Sequence alignment & homologymodeling

Validation of model

Docking Docking Results

Conclusions

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Structure prediction of chitinase(enzymethat degrades chitin layers) isolated fromthe pathogen Aphanocladium Album ,using

web based bioinformatics tools ,molecularmodeling softwares and servers.

To find effective inhibitor for this enzyme by

docking.

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INTRODUCTION Degrades chitin layer by breaking down the glycosidic

bonds.

Mostly used for digestion purposes, this enzyme isfound in a wide category of organisms includingorganisms as primitive as bacteria & fungi and asadvanced as human beings.

Most often the chitinase in pathogens falls underfamily 18.

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Clinical references associate chitinase with variousallergies (both in animals and plants )including

asthma for which exposure to high levels of chitinase isobserved to be a cause (though the reason for infectionis not clear yet.

In microorganisms, chitinase is found as to be a

primary metabolite , hence inhibiting chitinase willinhibit the growth of the microorganisms. Therefore

 we can form antibiotic drugs via inhibiting chitinase.

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Aphanocladium Album ,though a weak pathogen, is

associated with the brown spots growing on the caps

of mushrooms.And so this particular fungus is found to be

pathogen of another fungus.

Pathogen Activity of the

Given Fungus

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SOFTWARES

Modeling involves sequence alignment (single or multiple

depending on the requirements) followed by homology modelling orthreading(2’ structure prediction). 

From sequence alignment (BLAST) we can find out whether or

not the target sequence has a crystal structure assigned in PDB.

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Homology Modeling (automated)can be done by SWISS MODEL,

PHYRE, GENO3D .

 The acquired models are to be verified for validation.

 This can be done either by VERIFY-3D or by Ramachandran plot

(wincoot).

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Sequence of the query protein obtained from protein

database of NCBI( www.ncbi.nlm.nih.gov) .

This sequence is put into BLAST programme ofNCBI and the closest homologue is determined to be“crystal structure of a chitinase CrChi1 from thenematophagous fungus Clonostachys rosea”( pdb

code 3G6L).

Sequence alignment and

homology modelling

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BLAST indicates 2 things.

1.The target sequence doesn’t have a 

known structure.2.For the homology modelling ,if to be

done manually , the protein with max

sequence identity(70%). After BLAST we need to do the homology

modelling.

As mentioned earlier the automatedhomology modeling was performed by usingthree programs: SWISS MODEL, PHYRE,and GENO3D.

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The validation for the structure models obtained fromvarious software tools (SWISS MODEL, PHYRE,GENO3D), was done by analyzing the psi-phiRamachandran plot generated by wincoot.

The compatibility of the predicted structures with thegiven sequence was also verified using VERIFY-3D.

The entire process of verification pointed towards thestructure predicted by swiss-model.

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Molecular docking is a widely-used

computational tool for the study of molecular

recognition, which aims to predict the bindingmode and binding affinity of a complex formedby two or more constituent molecules withknown structures.

•An important type of molecular docking isprotein-ligand docking because of its therapeuticapplications in modern structure-based drugdesign.

DOCKING

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Inhibitor is a substance that inhibits the action of anenzyme.

By finding inhibitors to the given protein and making aprotein- inhibitor complex helps a lot in antibiotic drugpreparation.

To calculate the exact position where the inhibitorshould bind ,having minimum energy ineractions , we

use computational docking.

For the protein of interest , the suitable ligands arefound to be methyl xanthine derivatives (Caffeine,Threophylline) & allosamidin.

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We docked three different ligands with theobtained 3D structure of the given proteinusing Hex.

This was done to find the energy interactionsbetween the ligands and the given protein.

The functions performed by Hex in order tofind the best possible protein-ligandinteraction are: SPF Transform, FFT streric scan,FFT Final Search , MM Refinement, TotalDockings.

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DOCKING RESULTS… 

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DOCKING RESULTS (contd…) 

Energy Interactions of Each Complex

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DOCKING CONCLUSIONS The comparision of the total energies of interaction shows

that the best inhibitor(ligand) for the given proteinmolecule is Allosamidin.

 Allosamidin has the minimum amount of interactionenergy ,i.e. the interaction between the given protien andallosamidin is most feasible as compared to other ligandsdocked.

Hence ,out of the given 3 molecules, Allosamidin turns out

to be the most potent inhibitor.  APPLICATION :This process is used to make Anti-fungal

drugs these days by inhibiting the chitinase function infungi.