In-Silico Approach to Investigate

Death Domains Associated with

Nanoparticle-Mediated Cellular
Responses

PROJECT DONE BY: SARAH YOUSUF & NIDA MOKASHI

MENTORED BY: MS. HUSSAINA BANU
 INTRODUCTION
Nanotech - promising approach towards targeted drug therapy
Nanoparticles, through the NALP3 inflammasome, mediate
apoptotic cell death
NALP3 inflammasome formed as a result of association of
ASC PYD and NALP3 PYD and the CARDs between ASC and
Caspase-1

D.D superfamily - comprising of a large no:of protein

interaction molecules - play a major role in apoptosis,
inflammation, necrosis & immune cell signalling pathway
Any aberration in the superfamily’s functioning could result in
human diseases like cancers, neurodegenerative diseases &
immunological disorders
 SIGNIFICANCE
Out of several protein complexes,
PYD domains are exceptional in that
they aggregate under in-vivo
conditions
This has made structural elucidation
and study of their interactions
challenging
Hence, we have used computational
methods to study interactions between
NALP3 & ASC and NALP3 & ASC2
The comparative study helps
understand the effectiveness of the
activated inflammasome
Further, structural similarities will
help understand inflammasome
assembly
High resolution crystal
structures of the proteins were
obtained from the Protein
Database (PDB) website
Proteins were simulated using
VMD and NAMD
Simulated proteins were
submitted to the online docking
software HADDOCK to study
the protein-protein interactions
Molecular visualization
software, CHIMERA, was used
to study and compare the
interaction by NALP3 PYD &
ASC PYD and NALP3 PYD &
ASC2 PYD proteins
RESULTS & DISCUSSION
Some of the characteristic features that were analysed include :
1. The binding site on PYD of ASC2 and binding site on PYD of
NALP3
2. Binding site’s amino acids of NALP3-ASC and NALP3-ASC2
complexes
3. Hydrogen bond distances between the key amino acids
4. Role of hydrophilic residues in protein-protein interaction
5. Involvement of the H1 & H6 helices of NALP3 protein in
binding
 1 BINDING ENERGY & Z- SCORE
In order to study the interactions, the simulated proteins were
submitted to HADDOCK
The binding energies and Z-scores were then analysed

GREATER
High binding energy INTERACTIO
NALP3 – ASC N
Protein complex
Low Z - score HIGHER
AFFINITY
 PROTEIN COMPLEXES

NALP3-PYD/ASC-PYD NALP3-PYD/ASC2-PYD
protein complex protein complex
 BINDING SITES ON THE PYRIN
2 DOMAIN(PYD) OF ASC PROTEIN

Active amino acids from previous

studies has shown that Glu13, Lys21,
Arg41, Asp48 & Asp51are involved in
the interaction between NALP3 and Any mutation in any
one of the 5 key
ASC amino acids affects
Our study revealed that although the the intensity of the
above mentioned amino acids are interaction,
indeed involved in the NALP3-ASC especially Glu13
interaction, the same can’t be said for
the case of the NALP3-ASC2 interaction
Only Lys21, Asp48 & Asp51 were
involved in the NALP3-ASC2
interaction
List of key ASC amino acids interacting with the NALP3 protein in
NALP3-ASC protein complex and NALP3-ASC2 protein complex.
ASC-PYD interacting amino acids
ASC2-PYD interacting amino acids
(purple chain) Glu 13, Lys 21, Arg 41,
(green chain) Glu 13 and Arg 41 away
Asp 48 and Asp 51 close to the NALP3
from the NALP3 protein (orange chain)
protein (orange chain)
 BINDING SITES ON THE PYD
3
OF NALP3 PROTEIN

On comparison, NALP3-ASC is well-bound as

almost 10 amino acids of NALP3 are associated with
binding to the ASC protein

However, only 6 amino acids of NALP3 protein are

involved in the interaction with ASC2
List of NALP3 amino acids interacting with the key amino acids of
ASC & ASC2 proteins
 BINDING SITES AMINO ACIDS OF PYD OF
4 NALP3-ASC & NALP3-ASC2 PROTEIN
COMPLEXES

Ten key amino

acids of NALP3-
PYD protein
mainly interact
with five key
amino acid of ASC-
PYD protein
 Six key amino
acids of NALP3-
PYD protein
mainly interact
with three key
amino acid of
ASC2-PYD protein
 H-BOND DISTANCE BETWEEN KEY AMINO ACIDS
5 OF PYD OF NALP3 & ASC/ASC2 PROTEIN
COMPLEXES

Supports the fact that NALP3-ASC is more closely

bound that NALP3-ASC2 protein complex
 ROLES OF HYDROPHILIC RESIDUES IN
6 PROTEIN-PROTEIN INTERACTION

Previous studies have shown that there are 2

hydrophobic clusters involved in the interaction

However, our study has revealed that in addition to

the hydrophobic clusters, there are several hydrophilic
residues that are involved in the protein-protein
interaction
 H1 & H6 HELICES’ ROLE OF NALP3
7 PROTEIN IN PROTEIN BINDING

One of the 2 previously mentioned hydrophobic clusters

is the core comprising of 6 helices & fine loops

Our study has shown that the amino acids forming the
H1 and H6 helices of NALP3 are extensively involved in
the interaction between the complexes
 FUTURE PROSPECTS

 Computational studies provide in-depth investigation &

analysis of the proteins & their interactions at a Nano –
scale.

 The information thus obtained could prove to be beneficial

in the effective diagnosis and treatment of diseases.

 Several diseases such as cancer, Alzheimer's, Cystic

fibrosis etc. can be studied at their Nano – level.
 CONCLUSION

Understand how PYD domains interact

Insight into molecular mechanisms in forming the

NALP3 inflammasome complex

New information uncovered will help understand the

role of hydrophilic residues in interaction
ACKNOWLEDGEMENT

We would like to acknowledge

Dr. FIRDOS ALAM KHAN,
chairperson of School of Life
Sciences, Manipal University, Dubai

Dr. GEETHA VASANTHAKUMAR,

HITS labs, Dubai

Ms. RENUKA SEENIVASAN, HCT,

Abu Dhabi
for their encouragement!