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Stability of the core domain of p53 Insights from MD simulations

Stability of the core domain of p53 Insights from MD simulations. A Madhumalar, Derek Smith, Chandra Verma Bioinformatics Institute (A-STAR) Singapore. P53 : the guardian angel. Vogelstein, Lane, and Levine Nature 408: 307-310 (2000). core domain of p53. Background.

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Stability of the core domain of p53 Insights from MD simulations

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

  2. P53 : the guardian angel Vogelstein, Lane, and Levine Nature 408: 307-310 (2000)

  3. core domain of p53 Background • major tumor suppressor in mammals which protects cells against stress. • Majority of tumor derived p53 mutations map to the core, DNA binding domain • P53 is unstable compared to its homologues p63, p73 transcription factors T Y • 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

  4. L3 H1 S6 L2 S7 S4 S9 S8 S10 S3 S1 S2’ S2 L1 H2 Crystal structure of core domain of p53 dimer interacting with DNA (2AHI) • residues from helix H2 , loops L1, L3 interacts with DNA

  5. R175 G245 R249 R248 R273 R282 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

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

  7. Sequence alignment of core domain of P53, P63 & P73 P63 P73 P53 P63 P73 P53 hot spot sites of P53 (residues making contacts with DNA) Residues when mutated, increases the stability of P53

  8. So how do we study this Exptlly: Urea indd unfolding Temp studies Computnlly MD simulations Reaction paths 12.3/12.8 11.3/11.5

  9. Superposition of core domains of p53,p63,p73 • Core domains of p63, p73 were modeled based on homology • 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)

  10. p53 p63 p73 RMSD (Å) Root Mean Square Deviations (RMSD) • Wild type • double mutant

  11. p53 • Wild type • double mutant p63 p73 • Radius of gyration (Å) Radius of gyration

  12. Simulated fluctuations.. relative to Xtal

  13. L1 S6&s7 S7&s8 L2 L3 S7&s8 S6&s7 S7&s8 p53 L2 L1 L3 S6&s7 L1 L2 S7&s8 S7&s8 L2 S6&s7 p63 L1 L3 S6&s7 L1 L2 L1 L2 p73 S6&s7 S7&s8 L3 S7&s8 S6&s7 L1 L2 S1 L1 S2S2’S3S4L2H1S5S6S7S8L3 S9S10H2 Root Mean Square Fluctuations (RMSF)

  14. coverage of phase space pc1(x-axis) and pc2(y-axis) dp53 p53 dp63 p63 dp73 p73

  15. 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

  16. 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

  17. Sequence alignment showing the distribution of Tyrosine (coloured in blue) p63_human AIPSNTDYPGPHSFDVSFQQSSTAKSATWTYSTELKKLYCQIAKTCPIQIKVMTPPPQGA 223 p73_human VIPSNTDYPGPHHFEVTFQQSSTAKSATWTYSPLLKKLYCQIAKTCPIQIKVSTPPPPGT 173 p53_human SVPSQKTYQGSYGFRLGFLHSGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGT 155 :**:. * *.: * : * :*.****.* ***. *:*::**:*****:*: * :.** *: p63_human VIRAMPVYKKAEHVTEVVKRCPNHELSREFNEGQIAPPSHLIRVEGNSHAQYVEDPITGR 283 p73_human AIRAMPVYKKAEHVTDVVKRCPNHELGRDFNEGQSAPASHLIRVEGNNLSQYVDDPVTGR 233 p53_human RVRAMAIYKQSQHMTEVVRRCPHHERCSDSDG--LAPPQHLIRVEGNLRVEYLDDRNTFR 213 :***.:**:::*:*:**:***:** : **..******** :*::* * * p63_human QSVLVPYEPPQVGTEFTTVLYNFMCNSSCVGGMNRRPILIIVTLETRDGQVLGRRCFEAR 343 p73_human QSVVVPYEPPQVGTEFTTILYNFMCNSSCVGGMNRRPILIIITLEMRDGQVLGRRSFEGR 293 p53_human HSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVR 273 :**:******:**:: **: **:******:********* *:*** .*::***..** * p63_human ICACPGRDRKADEDSI 359 p73_human ICACPGRDRKADEDHY 309 p53_human VCACPGRDRRTEEENL 289 :********:::*:

  18. distribution of Tyrosine in the p53 wild type core domain Tyr126 Tyr220 Tyr236 Tyr163 Tyr205

  19. 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, 6.8 14.5, 20.6, 8.6 F238 P63(F,I) 4-5 dp63(Y,T) 7.5 F238 p73(F,I) 4-5 dp73(Y,T) 7.5

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

  21. Thanks!

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