Stability of the core domain of p53Insights from MD simulations

A Madhumalar, Derek Smith, Chandra Verma

Bioinformatics Institute (A-STAR) Singapore

Vogelstein, Lane, and Levine Nature 408: 307-310 (2000)

P53 : the guardian angel

Background

YT

P53 is unstable compared to its homologues p63, p73 transcription factors

The double mutations in the p53 core domain (Y236, T253), as observed in its homologous p63 (F,I) and p73(F,I) increases the stability (Canadillas et al, PNAS, 2006, 103:2109) To understand the stability of the core domains of p53,p63 and p73 and its double mutants using Molecular Dynamics

major tumor suppressor in mammals which protects cells against stress.

core domain of p53

Majority of tumor derived p53 mutations map to the core, DNA binding domain

residues from helix H2 , loops L1, L3 interacts with DNA

S2

S2’S3

S10

S9S4

S1

S8

S7S6

H2

L1

L3H1

L2

Crystal structure of core domain of p53 dimer interacting with DNA (2AHI)

Six hot spot mutations found in ~40% of human cancer R175, G245, R248, R249, R273, R282

R282

R273

R248

R249

R175G245

3 classes of mutations:

dna-contact mutants, minor affects on stability/folding

vicinity of dna binding surface local changes destab < 2kcal/mol

global unfolding of beta sandwich > 3 kcal/mol

Six hot spot mutations found in ~40% of human cancer R175, G245, R248, R249, R273, R282

R282

R273

R248

R249

R175G245

P53 is very unstable > 25*

Last class of mutants highly populated

Can one rescue them and if so why?

need to activate stable p53 to cause apoptosis….

Tyler Jacks and Scott Lowe restored p53 in mouse tumours and killed them

Sequence alignment of core domain of P53, P63 & P73

hot spot sites of P53 (residues making contacts with DNA) Residues when mutated, increases the stability of P53

P63P73P53

P63P73P53

So how do we study this

Exptlly: Urea indd unfoldingTemp studies

Computnlly MD simulations

Reaction paths

12.3/12.8

11.3/11.5

Superposition of core domains of p53,p63,p73

To understand the dynamics of core domains, MD simulations were carried out on p53, p63, p73 core domains and its double mutants of p53(F236, I253), p63(Y238,T255) p73(Y238,T255)

Core domains of p63, p73 were modeled based on homology

RMSD (Å)

p53

p63

p73

Root Mean Square Deviations (RMSD)

- Wild type- double mutant

Radius of gyration (Å)

p53

p63

p73

- Wild type- double mutant

Radius of gyration

Simulated fluctuations..relative to Xtal

L1

S7&s8

S6&s7

L2

L1

L1 L2S7&s8

S1 L1 S2 S2’ S3 S4 L2 H1 S5 S6 S7 S8 L3 S9 S10 H2

S6&s7

S6&s7

S6&s7

S7&s8

S7&s8

L3

L3

L3L1

L2

L2

L2 S7&s8S6&s7L3

L1

S7&s8

S6&s7

L2

L1

S7&s8

S6&s7L2

p73

p53

p63

L1

Root Mean Square Fluctuations (RMSF)

coverage of phase space pc1(x-axis) and pc2(y-axis)

p53

p63

p73

dp53

dp63

dp73

Summary so far..

Double mutant doesn’t affect the overall structure of the core domain in all three cases as seen from the RMSD and Radius of gyration

Consistent difference in the fluctuations of Loop L1(observed in NMR),L2, and loops connecting S5 & S6, S7 &S8 is observed

Sampling of phase space seems higher for p63,p73 compared to p53

Sampling of phase space also indicates that the overall mobility has been increased in the case of double mutant of p53 and decreased in the double mutant of p63,p73

Structural plasticity and stability

What is stability?

Urea induced unfolding as a fn of temp…

So how does urea get in?

Can we look at certain types of motions that may give us a clue

Sequence alignment showing the distribution of Tyrosine (coloured in blue)

p63_human AIPSNTDYPGPHSFDVSFQQSSTAKSATWTYSTELKKLYCQIAKTCPIQIKVMTPPPQGA 223p73_human VIPSNTDYPGPHHFEVTFQQSSTAKSATWTYSPLLKKLYCQIAKTCPIQIKVSTPPPPGT 173p53_human SVPSQKTYQGSYGFRLGFLHSGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGT 155 :**:. * *.: * : * :*.****.* ***. *:*::**:*****:*: * :.** *:p63_human VIRAMPVYKKAEHVTEVVKRCPNHELSREFNEGQIAPPSHLIRVEGNSHAQYVEDPITGR 283p73_human AIRAMPVYKKAEHVTDVVKRCPNHELGRDFNEGQSAPASHLIRVEGNNLSQYVDDPVTGR 233p53_human RVRAMAIYKQSQHMTEVVRRCPHHERCSDSDG--LAPPQHLIRVEGNLRVEYLDDRNTFR 213 :***.:**:::*:*:**:***:** : **..******** :*::* * *p63_human QSVLVPYEPPQVGTEFTTVLYNFMCNSSCVGGMNRRPILIIVTLETRDGQVLGRRCFEAR 343p73_human QSVVVPYEPPQVGTEFTTILYNFMCNSSCVGGMNRRPILIIITLEMRDGQVLGRRSFEGR 293p53_human HSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVR 273 :**:******:**:: **: **:******:********* *:*** .*::***..** *p63_human ICACPGRDRKADEDSI 359p73_human ICACPGRDRKADEDHY 309p53_human VCACPGRDRRTEEENL 289 :********:::*:

Tyr163

Tyr126

Tyr205

Tyr220

distribution of Tyrosine in the p53 wild type core domain

Tyr236

Barriers of rotation (kcal/mol) for core Tyr/Phe in wild type and double mutants

Y236

P53 (Y, T) 11.1 F,T 11.0 Y,I 4.0 F,I 3.9

p53 (dp53)12.4 (15.5), 20.4 (21.2), 17.6 (18.8), 6.5 (6.9)

6.8, 16.4, 6.814.5, 20.6, 8.6

F238P63(F,I) 4-5dp63(Y,T) 7.5

F238p73(F,I) 4-5dp73(Y,T) 7.5

Summary

The decrease in the rate of flipping of aromatic ring in the double mutant suggest that there is increase in the local packing

Sampling of phase space seems higher for p63,p73 compared to p53

Sampling of phase space also indicates that the overall mobility has been increased in the case of double mutant of p53 and decreased in the double mutant of p63,p73

Thanks!Thanks!