P53 MISSENSE MUTATIONS: STRUCTURAL AND FUNCTIONAL

PERTURBATIONS TO DNA BINDING

Bich-Chau Van

Department of Biochemistry and Molecular Biology

Professor Ray Luo

HUDSON AND ABELLA

http://www.hudsonandabella.org

PRESENTATION OUTLINE

1. Why are we interested in p53?2. What is p53? Functions Structure Regulation Wild type and mutants3. Project Goal Methods Results and discussion

P53 IS IMPORTANT BECAUSE…

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

P53 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

OTHER FUNCTIONS 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.)

STRUCTURE OF P53 Have 393 amino acids Active as a homotetramer

STRUCTURE OF P53 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.

STRUCTURE OF P53

http://www-p53.iarc.fr

P53 CORE DOMAIN AND DNA BINDING INTERFACE

VIRAL PROTEIN BINDING SITES

http://www.ncbi.nlm.nih.gov/books/NBK21551/figure/A7159

WHICH LEADS TO THE ACTIVATION OF P53

PROTEIN?

POSITIVE REGULATION Regulate p53 stability by inhibiting or

degrading Mdm2 proteinMdm2: an E3 ubiquitin ligase

MDM2 AND P53 INTERACTION

POSITIVE REGULATION

NEGATIVE REGULATION

P53 NETWORK

Vogelstein, B., Lane, D., Levine, A.J. (2000). Nature, 408: 307-310.

G1 arrest

G2 arrest

Mitochondria- independent

Mitochondria- dependent

DNA repair

DNA BINDING DOMAIN MUTATIONS

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

TYPE OF CANCERS DUE TO MISSENSE MUTATIONS

Vogelstein, B., Lane, D., Levine, A.J. (2000). Nature, 408: 307-310.

P53 MISSENSE MUTATIONS Dominant negative Mutants reside in cytoplasm

Stable Mutations Unstable Mutations

ΔΔG ≤ 1 kT, at least 98% folded at 37 C F > 3 kT, at most 85% folded at 37 C F

Most common amino acids before mutation

Q, N, K, D, S, T, A Y, I, C, V, F, W

Most common amino acids after mutation

I, T, L, V W, R, F

Side chain exposure to solvent

Most are exposed82% 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 mutations Many far from DNA binding regionMost 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.

SIDE CHAIN EXPOSURE

Unstable sites Stable sites Tan, Y., Luo, R. (2009). PMC Biophysics, 2:5.

MECHANISMS FOR P53 INACTIVATION BY MISSENSE

MUTATIONS 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.

DNA BINDING SURFACE MUTANTS

R248 R273 Directly affect DNA

binding Recognized by PAb

1620

R175 G245 R249 R282 Indirectly affect DNA

binding Recognized by

PAb240

DNA contact Structural

LOCATION OF MUTATIONS

GOALS OF THE PROJECT 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