1. P 53 M ISSENSE M UTATIONS : S TRUCTURAL AND F UNCTIONAL P ERTURBATIONS TO DNA B INDING Bich-Chau Van Department of Biochemistry and Molecular Biology Professor Ray Luo

2. H UDSON AND A BELLA http://www.hudsonandabella.org

3. P RESENTATION O UTLINE 1. Why are we interested in p53? 2. What is p53? Functions Structure Regulation Wild type and mutants 3. Project Goal Methods Results and discussion

4. P 53 IS I MPORTANT B ECAUSE … Acted upon by viral proteins (such as Adenovirus E1B, SV40 large T antigen, HPV E6) Germline mutation leads to Li-Fraumeni Syndrome which predisposes the individual to many kinds of tumors (Vousden, KH, Balint, E. (2001). British Joul. Of Cancer, 85 (12): 1813- 1823) 50% of human cancers contain mutations in p53

5. P 53 IS … A transcription factor with tumor suppressor function Encoded by the tumor suppressor gene TP53 Expressed at very low levels in normal cells Short half-life of 6-30 minutes depending upon cell or tissue type=> unstable Optimal DNA binding sequence: RRRCWWGYYY where R is a purine, W is A or T and Y is a pyrimidine Olivier, M., et al. (2002). Human Mutation, 19 : 607-614

6. O THER F UNCTIONS Activate cellular differentiation, senescence (Vousden, K. H. (2000). Cell, 103 : 691-694.) Inhibit angiogenesis, maintain genetic stability through DNA repair gene induction (Vogelstein, B., Lane, D., Levine, A.J. (2000). Nature, 408 : 307-310.) New functions in stem cells: suppression of pluripotency and inhibition of stem cell self- renewal (Hede, S. M., et al. (2010). Journal of Oncology, 2011.)

7. S TRUCTURE OF P 53 Have 393 amino acids Active as a homotetramer

8. S TRUCTURE OF P 53 Have 393 amino acids Active as a homotetramer 5 major, interdependent domains within each subunit: 1. Transcriptional activation domain (residues 1- 63) 2. Proline- rich domain (residues 64- 92) 3. Specific DNA- binding (core) domain (residues 94- 292) 4. Tetramerization domain (residues 326- 355) 5. C- terminal domain (residues 363- 393) Prives, C., Hall, P. (1999). J. Pathol, 187 : 112-126.

9. S TRUCTURE OF P 53 http://www-p53.iarc.fr

10. P 53 C ORE D OMAIN AND DNA B INDING I NTERFACE

12. V IRAL P ROTEIN B INDING S ITES http://www.ncbi.nlm.nih.gov/books/NBK21551/figure/A7159

13. W HICH L EADS TO THE A CTIVATION OF P 53 P ROTEIN ?

14. P OSITIVE R EGULATION Regulate p53 stability by inhibiting or degrading Mdm2 protein Mdm2 : an E3 ubiquitin ligase

15. M DM 2 AND P 53 I NTERACTION

16. P OSITIVE R EGULATION

17. N EGATIVE R EGULATION

18. P 53 N ETWORK Vogelstein, B., Lane, D., Levine, A.J. (2000). Nature, 408 : 307-310. G1 arrest G2 arrest Mitochondria- independent Mitochondria- dependent DNA repair

19. DNA B INDING D OMAIN M UTATIONS 50% of human cancers contain mutations in p53, 95% of which is in the DNA- binding domain (DBD) 75% of DBD mutations are single missense mutations. Hot spot mutations: at residues 175, 245, 248, 249, 273, 282 Olivier, M., et al. (2000). Human Mutation, 19 : 607-614

20. T YPE OF C ANCERS DUE TO M ISSENSE M UTATIONS Vogelstein, B., Lane, D., Levine, A.J. (2000). Nature, 408 : 307-310.

21. P 53 M ISSENSE M UTATIONS Dominant negative Mutants reside in cytoplasm Stable MutationsUnstable Mutations ΔΔG≤ 1 kT, at least 98% folded at 37 ̊C> 3 kT, at most 85% folded at 37 ̊C Most common amino acids before mutation Q, N, K, D, S, T, AY, I, C, V, F, W Most common amino acids after mutation I, T, L, VW, R, F Side chain exposure to solvent Most are exposed 82% exposed by more than 20% Most are buried in the interior of protein 93% buried by more than 80% 86% buried by more than 90% Location of mutationsMany far from DNA binding region Most DNA contact mutations Some DNA contact mutations Mutations that cause structural clash Mutations that disrupt Zn-binding sites Tan, Y., Luo, R. (2009). PMC Biophysics, 2 :5.

22. S IDE C HAIN E XPOSURE Unstable sitesStable sites Tan, Y., Luo, R. (2009). PMC Biophysics, 2 :5.

23. M ECHANISMS FOR P 53 I NACTIVATION BY M ISSENSE M UTATIONS Loss of stability and/ or Loss of DNA contact, protein- protein contact with other domains, tetramer subunits or binding partners, or Zn contact Loss of stability and DNA contact are main mechanisms. Tan, Y., Luo, R. (2009). PMC Biophysics, 2 :5.

24. DNA B INDING S URFACE M UTANTS R248 R273 Directly affect DNA binding Recognized by PAb 1620 R175 G245 R249 R282 Indirectly affect DNA binding Recognized by PAb240 DNA contact Structural

25. L OCATION OF M UTATIONS

26. G OALS OF THE P ROJECT Identify the structural effects of three common missense mutations (G245S, R249S, R273H) on the p53 DNA binding interface Effects of these mutations on DNA binding affinity of p53 Long-term goal: to develop better pharmaceutical interventions to restore wild-type p53 functions in many mutant proteins

27. M ETHODS Conduct Molecular Dynamics simulations of wild-type and mutant p53 proteins in both the presence and absence of DNA Collect trajectories to determine the structural effects of the mutations Analyze DNA binding affinities of wild-type and mutant p53 with Molecular Mechanics Poisson- Boltzmann Surface Area (MM-PBSA) method