Stability of the core domain of p53 Insights from MD simulations A Madhumalar, Derek Smith, Chandra Verma Bioinformatics Institute (A- STAR) Singapore
Feb 11, 2016
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!