Prediction of SH3 Domain Binding Motifs

Presented by: Siba IsmaelSupervised by: Mazen AhmadUniversity of Saarland

Saarbrücken, 17.10.08

Outline 2

Outline

SH3 motif and proline-rich domains- Motivation to find SH3 domains binding sites-Why proline-rich domains?

Binding Free Energy Method: What flanking sequences govern binding specificity

Materials and Methods; Bioinformatics

Results of Prediction

Conclusions and Outlook

Introduction- SH3 Motif and Proline-Rich Domains

3

SH3 DomainsMotivation- Assembly Comprise 60 residues

Play assembly and regulatory roles.

Assembly role: example; Grb2

Cascade: Growth factor receptor tyrosine kinase Grb2 SOS Ras MAPK

- Play roles in cell growth and differentiation

Introduction- SH3 Motif and Proline-Rich Domains

4

SH3 DomainsMotivation- Regulation Regulation: example; Src

Built-in SH2+SH3: inactivation (autoinhibition)

Disruption: External SH2 and SH3 domains interaction-result in kinase activation

SH3 interactions: week- typical dissociation constant- essential for reversible switching mechanism.

Introduction- SH3 Motif and Proline-Rich Domains

5

Repetitive Proline-Rich Sequences

in many cases, thought to function as docking sites for signaling modules

found in the context of larger multidomain signaling proteins.

Binding: assembly and targeting of protein complexes involved in:- cell growth

- cytoskeletal rearrangements- transcription - postsynaptic signaling processes

play a regulatory role and autoinhibitory interactions

Introduction- SH3 Motif and Proline-Rich Domains

6

Repetitive Proline-Rich Sequences

Why proline in interaction modules?

Proline: unique amino acid in: - constraints on dihedral angles imposed by cyclic side chain- its resulting secondary structural preferences

Introduction- SH3 Motif and Proline-Rich Domains

7

Repetitive Proline-Rich Sequences Why proline in interaction modules?

propensity to form a polyproline type II (PPII) helix.- extended left-handed helical structure with three residues per turn.

- useful recognition motif: - carbonyls point out from the helical axis into solution - restricted backbone: entropy cost of binding reduced

- twofold rotational pseudosymmetry:

- two binding possibilities

- orientational switching differing domain function

Introduction- SH3 Motif and Proline-Rich Domains

8

Repetitive Proline-Rich Sequences Why proline in interaction modules?

The only naturally occuring N-substituted amino acid:

- sequence-specific recognition without high-affinity interaction.

- specific and low affinity interactions: - reversibility - intracellular signalling

Stable cis conformation- high kinetic barrier- rate limiting step

Introduction- SH3 Motif and Proline-Rich Domains

9

Proline-Rich Sequences vs. SH3 Interaction

PxxP motif: flanked by different specificity elements:- K/RxxPxxP and PxxPxK/R classes of ligand motif- single recognition surface: two N- to C-terminal orientations ligand binding

SH3 fold: two antiparallel β sheets at right angles.- in fold RT and n-Src loops: flanking specificity pockets

Aromatic SH3 groove PPII helix ridges (a pair of residues)

10

So how to detect the binding affinity to SH3 domains?

Computational Analysis!!!Solvation Energy!!

„BIOPHYSICS“

Binding Free Energy Components 11

Binding Free Energy mechanical energy to disassemble a whole into

separate parts scalar

Binding free energy cycle:- in terms of transfer free energiesWhy? - from a homogeneous dielectric environment (interactions: Coulomb's law)- to an inhomogeneous dielectric environment:

differing internal and external dielectric constants.

GGGGGbind 1243 )( GGG coulsolvbind

Binding Free Energy Components 12

Binding Free EnergySolvation Energy Contribution Solvation energy for the complex and each of its parts

But how to calculate solvation energy?

GGGsolv 24

proteinsolv

ligandsolvcomplexsolvsolv

G

GGG

Remember!! This stands for Coulombic

Binding Free Energy Components 13

Binding Free EnergySolvation Energy Contribution

Full solvation energy cycle

- Step 1: Total Solvation

- Step 2: charging of the solute in solution inhomogeneous presence of mobile ions. -Step 3: attractive solute-solvent dispersive interaction - Step 4: repulsive solute-solvent interaction

- Steps 5 and 6: null steps. - but used to offset unwanted energies

charging of the solute in vacuum homogeneous absence of mobile ions.

Binding Free Energy Components 14

Binding Free EnergySolvation Energy Contribution

APBS??

GGG npsolv

GGGp 62

)11

(8 0

2

inoutBornp a

qG

AVpG 4

dyyyuGG att )()()(53

)( 534 GGGGn

ACC??

Binding Free Energy Components 15

Binding Free EnergyIncluding Coulombic Contribution

the sum of pairwise Coulombic interactions:- for all atoms in the molecule - for a particular uniform dielectric

Coulomb‘s Law:

Potential Dielectric Energy:

ligandcoulproteincoul

complexcoulcoul

GG

GGG

1

221

4

1

r

qqF

r

qqU 21

12 4

1

Coulomb??

Binding Free Energy Components 16

Binding Free Energy Entropy Entropy: a measure of the

unavailability of a system’s energy to do work

- measure of the randomness of molecules in a system - central to the second law of thermodynamicsSpontaneous changes Entropy (isolated systems)

Binding Free Energy Components 17

Binding Free Energy van der Waals van der Waals force: attractive or

repulsive forces between molecules and per molecule:

not covalent bonds or electrostatic interaction of ions, but:

- permanent dipole–permanent dipole forces

- permanent dipole–induced dipole forces

- instantaneous induced dipole-induced dipole

Binding Free Energy Components 18

Poisson-Boltzmann Equation Differential equation – describes electrostatic interactions between

molecules in ionic solutions

models implicit solvation (continuum solvation )

solution.in ions theto

r position ofity accessibildependent -position for thefactor a :r)(

re temperatuthe:T constant,Boltzmann :k

proton, a of charge the:q ion, theof charge the:z

solute thefrominfinity of distance aat iion theofion concentrat :c

solute theofdensity charge :r)(

potential ticelectrosta the:r)( ,dielectricdependent -position the:r)(

B

i

i

f

Methods 19

Methods APBS Package: Adaptive Poisson–Boltzmann Solver:

- numerical solution for the Poisson-Boltzmann equation - modeling biomolecular solvation In my work:* apbs: electrostatic potential and polar solvation* acc: SASA calculation: „solvent accessible surface area“ nonpolar solvation* coulomb: coulombic interactions in vacuum

Pdb2pqr Package: platform-independent utility - converts protein files in PDB format to PQR format

Methods 20

Methods PQR file: PDB file temperature and occupancy columns;

replaced by the per-atom charge (Q) and radius (R)

Jackal: package for protein structure modeling scap: protein side-chain program: predicts side-chain conformations and side chains of a whole

protein and in mutates specified residues in a protein

R language Package: Statistical Language environment

Methods 21

Methods To predict a binding motif of length 10:

- chose the crystal structure of the peptide APSYSPPPPP complexed with the Abl SH3 domain - mutate it to other sequences

Try: predicton of 10 very good out of the 600 candidates, and 15 of the nonbinders almost all have a PxxP domain!

late with tempcompare

motifsdifferent 1-20 toReduce - 8

!late! with tempcompare

motifsdifferent 1-20 :Total - 10

motif?! PxxP thehave all

Fix P at P0 and P3

Methods 22

Methods From

literature: Binding Free Energy Difference to the base sequence with the following mutations:

23

Results

Results 24

Correlation?!

Correlation :

0.4530898

Correlation:

0.9534504 Correlation:

0.722554

Results 25

Reproducibility?!

Without vdW or entropy

correlation:0.5435262

For both

Correlation: 0.3357690Second compared to base

sequence Why not much good?

Results 26

Peptide Binding-Solvation Polar

Easier barrier to break for binders

Results 27

Coulombic Interactions

Mean Coulombic Energy is less for binders!

Results 28

Nonpolar Solvation Contribution

Neglicted effect!

Results 29

van der Waals Contribution

Major contribution to binding specificity

Results 30

Entropy Contribution

Most non-Binders Lost more Entropy upon Binding than did Binders!

Results 31

Binding Free Energy

Less Binding Free Energy for Binders!Easier barrier to break

Results 32

Separation of Binders from non-bindersPrediction

Linear Discriminant Analysis!

Results 33

From LiteratureBinders

NonbindersSequence Gpred

SKKEMQPTHP 19.6

ASQKMEPRAP 43.3

WELSSQPTIP 26.3

LAPASTPTSP 13.6

ASTPTSPSSP 11.4

SSPGLSPVPP 13.8

RGVLIEPVYP 38.9

DEPNLEPSWP 26.4

RLVGARPLLP 24.6

RTESEVPPRP 26.6

LASRPLPLLP 20.1

ISQRALPPLP 30.8

ITMRPLPALP 17.3

RSGRPLPPIP 32.7

KWDSLLPALP 17.4

YWDMPLPRLP 4.2

YYQRPLPPLP 9.1

YFSRALPGLP 8.8

SLWDPLPPIP 15.2

DPYDALPETP 28.6

Results 34

Results concerning Prediction!

Proline preferece in the binding motif- Available experimental measurements at positions P3, P0, P−3, and P−5: - Particularly important for the peptide binding: - conserved Pro residues at P3 and P0: strong binding affinity (PxxP- work here) - residues at P−3, and P−5: the binding specificity (the other work)

Other residues, especially hydrophobic (Phe, Leu, Met, Val, and Trp), also favored

Conclusions and Outlook 35

Conclusion and Outlook!

Binding free energy: - nice method predictiong binding preferences- easy to deal with data

Can be used in prediction of different sets of protein-ligand interaction prediction

High throughput results in the fields of medicine, pharmacy, and biology

36

References Tingjun Hou, Ken Chen, William A McLaughlin, Benzhuo Lu, and Wei Wang.

Computational Analysis and Prediction of the Binding Motif and Protein Interacting Partners of the Abl SH3 Domain

Wikipedia T.Geyer, Dynamic Cell Simulation Jackal: supported by National Science Foundation and National Institute of Health;

developed in Honig Lab Baker NA, Sept D, Joseph S, Holst MJ, McCammon JA. APBS: Electrostatics of

nanosystems: application to microtubules and the ribosome. Proc. Natl. Acad. Sci. USA 98, 10037-10041 2001.

Dolinsky TJ, Nielsen JE, McCammon JA, Baker NA. PDB2PQR: an automated pipeline for the setup, execution, and analysis of Poisson-Boltzmann electrostatics calculations. Nucleic Acids Research, 32, W665-W667 (2004).

R: Regulatory Compliance and Validation Issues A Guidance Document for the Use of R in Regulated Clinical Trial Environments

Google Machine Search

37