Identifying specificity-altering

allosteric mutations in the PDZ domain

using novel computational tools

Pia Wahi-Singh
Dr. Banu Ozkan Lab, Department of Biophysics
Arizona State University
&
Student of BASIS Ahwatukee High School – 11th grade
 Background: PDZ domain
• The PDZ protein domain is a ubiquitous interaction domain
important in
– Regulatory signal transduction pathways
– Anchoring receptor proteins
– Transport
• It is a universal outlet of many protein residue networks that wire
diverse functions in our cells.

A schematic
representation of the
postsynaptic terminal
illustrating PDZ
domains (red) in
synaptic proteins and
their interaction
partners. (Noury et al,
Science 2003)

1
 Diseases related to PDZ domain
• Alteration of PDZ domain’s expression due to
harmful mutations is associated with several
diseases:
– Alzheimer’s, Parkinson’s, depression, and a
number of cancers
• Mutations affect the binding affinity of PDZ
domain. Disease causing mutation

Strong binding Decrease in binding

Disease related mutant

Wild Type
2
 Classic Allostery
Enzyme
Effector
Allosteric Active
Site Site Substrate

Inhibition Activity

Enzyme reaction Substrate Enzyme reaction

obstructed cannot bind proceeds

3
 Allostery within proteins: Between amino acid residues

Residue location that

is allosterically linked
Enzyme
to the active site

Active
Site Substrate

Mutation at Inhibition Activity

allosteric site

Enzyme reaction Substrate

obstructed Enzyme reaction
cannot bind proceeds

4
 Allosteric connection between binding pocket and a
distal site can alter binding
• What is hindering the outbreak of the PDZ domain, a novel drug target, is
the missing technology that can incorporate the role of conformational
dynamics in binding affinity.
– Incorporating the effect of allosteric mutations on conformational
dynamics is especially difficult
• Computational methods developed in the Ozkan lab are able to correctly
simulate PDZ conformational dynamics and predict the affect of allosteric
mutations.

Allosteric mutation

Strong binding
Change in binding

Wild Type mutant 5

 Goal of the project
• Given the difficulty of modeling PDZ conformational
dynamics and the difficulty of experimentally finding
appropriate allosteric residues, I want to explore the
conformational space in depth.
• The comprehensive set of tools developed in the Ozkan
lab allows me explore this.

• Current goal: To determine allosteric mutations in the

PDZ domain that will alter its binding affinity and
specificity.

6
 Methods
1. Will determine residues that are allosterically linked to binding
site
Using: Dynamic Coupling Index

2.Will identify what the likely mutation is for each linked residue
Using: Coevolutionary Analysis

3.Will introduce this mutation in the PDZ domain protein

Using: PyMOL’s mutagenesis function

4. Will model the binding with docking simulation

Using: ABP-Dock

5.Will analyze change in binding affinity and repeat process

for each identified residue

Will identify a specificity-changing allosteric mutation

7
 1. Dynamic Coupling Index (DCI)
• Quantifies the resilience of each residue Distant location (red) allosterically
upon perturbation of the binding site coupled to binding site (blue)
residues
• Sites distant from the binding site that have
high DCI values are called allosterically
coupled sites
• Mutations at allosteric spots often influence
the conformational dynamics and allosteric
regulation of a protein, and are thus often
linked to diseases

High allosteric coupling to binding site

> 0.8%

8
 2. Co-evolution
• Co-evolution of amino acids can be utilized to further determine if two residues
are linked
• After choosing residue sites, evolutionary data tells what mutations would likely
occur • I will use MISTIC (Mutual
Information Server to Infer
Coevolution) for the co-
evolutionary analysis

Image adapted from: Marks et al., 2011

9
 Compare allosteric coupling with co-evolutionary
coupling to identify residues linked to binding spot

Example of a chosen residue: shows both high %DCI and high coevolutionary
linkage, meaning that residue 46 is allosterically and coevolutionarily coupled to
binding site residues.
10
 3. Mutagenesis
• I will introduce the mutation using pyMOL’s
mutagenesis function
Example:

H372 H372A 11
 The method Adaptive BP-Dock can capture the
allosteric effect of a mutation on binding
• Adaptive BP-Dock is a tool to model the
interactions between a protein and a
peptide/ligand.
• I will employ this docking technique to
predict binding on PDZ domain.

• PDZ domain (PSD95) shown in green

• Bound peptide shown in magenta

PDB ID: 1BE9

12
4. Adaptive BP-Dock algorithm
• Induced fit docking
approach
• Integrates perturbation
response scanning (based
on elastic network
model) with the docking
protocol of RosettaLigand
1000
40

Depiction of an elastic network model

Image adapted from: Ozkan et. al., 2016
13
 PDZ domain specificity
• PDZ domain proteins have two categories based on their
binding specificity to the peptide/ligand

Protein:
Peptide:
N
Class I Class II
Consensus of Peptide:
P-3
X X
P-2 T or S Φ
P-1 X
X
P0 Φ Φ
C X : any amino acid
Φ : hydrophobic amino acid (V, Y, F, L, I)

14
 Adaptive BP-Dock correctly captures the
effect of mutation on PDZ domain
• In a 2016 study by Ranganathan et. al., class switching and class bridging mutations to
an originally class I PDZ domain in synaptic protein PSD-95 were identified:
H372A – switches specificity from class I to class II
G330T – bridges specificity (protein binds to both class I and II peptides)
H372A and G330T – switches specificity from class I to class II

Results of Preliminary Tests:

Predicted binding energy (kcal/mol) Experimental data for mutations
-7.6 -7.4 -7.2 -7 -6.8 -6.6 -6.4
-9
R=0.84
-10

-11 ln(Kd) (kcal/mol)

-12

-13

-14

Image adapted from: Ranganathan et. al., 2016

-15

We can capture the change in binding affinity and change in

15
specificity due to a mutation!
 Significance of Identifying Allosteric,
Specificity-Altering Mutations
• Clinical implications
– Identifying specificity-altering allosteric mutations elucidates
another pathway for disease-causing malfunction
– Utilization as a screening tool to guide disease diagnosis.
– Synthetic drugs with high binding affinity to the diseased
PDZs can be created, which will bind and thus cause the
same effect as if the proper ligand/peptide had binded to a
wild type PDZ (thereby renewing the function)
• Tool for drug design/discovery to wire new protein
networks and to modulate disease-related proteins.

16
 Significance: Drug design
Wild Type
Mutation: Inhibition

Substrate
cannot
bind

✕
Understanding of
Desired the allosteric
Effect mutation’s effect
on PDZ specificity
Mutation: Inhibition guides creation of
Normal Cellular Function! synthetic
ligands/peptides
Drug Binds (drugs) which will
exhibit high binding
Desired affinity to the
Effect mutated PDZ

17
 Acknowledgments
• Dr. S.Banu Ozkan, Arizona State University
• I. Can (John) Kazan, Arizona State University

18
 References
• Bolia, Ashini, and S. Banu Ozkan. “Adaptive BP-Dock: An Induced Fit Docking Approach for Full
Receptor Flexibility.” Journal of Chemical Information and Modeling 56, no. 4 (April 25, 2016): 734–
46. https://doi.org/10.1021/acs.jcim.5b00587.
• Butler, Brandon Mac, S. Banu Ozkan, Sara Vaiana, Giovanna Ghirlanda, Robert Ros, and Arizona
State University. “Protein Conformational Dynamics In Genomic Analysis.” In ASU Electronic Theses
and Dissertations. Arizona State University, 2016. http://hdl.handle.net/2286/R.I.41277.
• Facciuto Florencia, Cavatorta Ana L., Valdano Marina Bugnon, Marziali Federico, and Gardiol
Daniela. “Differential Expression of PDZ Domain‐containing Proteins in Human Diseases –
Challenging Topics and Novel Issues.” The FEBS Journal 279, no. 19 (September 17, 2012): 3538–48.
https://doi.org/10.1111/j.1742-4658.2012.08699.x.
• Larrimore, Katherine E., I. Can Kazan, Latha Kannan, R. Player Kendle, Tameem Jamal, Matthew
Barcus, Ashini Bolia, et al. “Plant-Expressed Cocaine Hydrolase Variants of Butyrylcholinesterase
Exhibit Altered Allosteric Effects of Cholinesterase Activity and Increased Inhibitor Sensitivity.”
Scientific Reports 7, no. 1 (September 5, 2017): 10419. https://doi.org/10.1038/s41598-017-10571-
z.
• Marks, Debora S., Lucy J. Colwell, Robert Sheridan, Thomas A. Hopf, Andrea Pagnani, Riccardo
Zecchina, and Chris Sander. “Protein 3D Structure Computed from Evolutionary Sequence
Variation.” PLOS ONE 6, no. 12 (December 7, 2011): e28766.
https://doi.org/10.1371/journal.pone.0028766.
• Raman, Arjun S., K. Ian White, and Rama Ranganathan. “Origins of Allostery and Evolvability in
Proteins: A Case Study.” Cell 166, no. 2 (July 14, 2016): 468–80.
https://doi.org/10.1016/j.cell.2016.05.047.

19