The Retinoblastoma Protein

1. The Retinoblastoma Protein Its Structure and Its Role in Cancer By Ariel Lefkovith

2. Retinoblatoma Breast Cancer Cervical Cancer Colo-rectal cancer Endometrial cancer Head & neck cancer Liver cancer Lung cancer Malignant carcinoid Malignant glioma Urothelial cancer Malignant lymphoma Malignant melanoma Ovarian cancer Pancreatic cancer Prostate cancer Renal cancer Skin cancer Stomach cancer Testis cancer Thyroid cancer Cancers

3. Background • It is a protein product of the RB1 gene. • A nuclear phosphoprotein. • Gene cloned in 1986. • Small cell lung carcinoma and Retinoblastoma are cancers in which the RB1 gene is mutated and RB1 loss of heterozygosity, forming Rb-/Rb-, is found in other cancers. • A tumor suppressor because of its control over the cell cycle. • Some viral oncoproteins can bind to pRb to deactivate it.

4. pRB Role in the Cell Cycle • One of the factors needed to pass the restriction point from G1 phase to S phase. • pRB is bound to the E2F transcription factor. • pRB is phosphorylated by different cyclin-Cdk complexes at different stages of the cell cycle. • In G0 quiescent cells pRB is unphosphorylated and unbound. • In early G1 pRB is bound to E2F and becomes hypophosporylated by cyclin D-Cdk 4/6. • In late G1 pRB is bound to E2F and becomes hyperphosphorylated by cyclin E-Cdk 2. • The hyperphosphorylation of the pRB inactivates it, releasing E2F.

5. pRB In the Cell cycle Key G0= G0 quiescent stage G1= Gap 1 Phase S= Synthesis Phase Red circle= phosphates pRB= retinoblastoma protein

6. Structure • Three main Domains: N-terminal Domain, C-terminal Domain, and Pocket Domain. • The N-terminal domain has 379 residues. • The Pocket is made of 406 residues. • The C-terminal domain is a 143 residue domain. • Has 16 phosphorylation sites.

7. The Pocket • Made up by A and B domains. • The two main structures are the A and B cyclin-box folds of five α helices each. • There are also 8 other α helices, a beta hairpin, and an extended tail. • The A-B Interface is held together by a hydrophobic core, created by 20 side chains and hydrogen bonds between the back bone and side chains. • E2F binds in the pocket. • Many of the mutations are found in the pocket. • The LxCxE site is on the B box.

8. N-Terminal Domain • Structure is hard to crystallize. • Has a globular structure formed by an A lobe and a B lobe. • Similar structure to the Pocket Domain. • The two lobes have separate hydrophobic cores. • It is hypothesized to be associated with familial retinoblastoma and could be involved with protein-protein interactions. α2 Lobe A α3 α1 α6 α5 α4 α13 α7 η1 α8 α12 α11 α10 Lobe B α9 PDB ID # 2qdj, x-ray diffraction

9. The C-terminal Domain • Structure hard to crystalize. • This domain is necessary if pRB is stop cell progression by binding to E2F. • This domain binds to the E2F-DP heterodimer. • Phosphorylation also deactivates this domain from binding to the E2F-DP complex. • Part of this domain can bind to the LxCxE site on the pocket of the protein.

10. C-terminal Domain Fragment bound to E2F-DP • The E2F-DP complex forms an intermolecular coiled coil, an intermolecular β sandwich, and 5 α helices. • The β sandwich has a hydrophobic core. • The fragment of RbC forms a strand-loop-helix structure and a tail. • The strand-loop of RbC binds to the β sandwich, and the tail loops around one end. • RbC binds to the E2F-DP complex through hydrogen bonds and van der Waals interactions.

11. C-Terminal Domain Bound to E2F-DP Red-RbC fragment Blue-DP Green-E2F Coiled Coil β sandwich PDB ID # 2aze, x-ray diffraction

12. Binds in the pRB pocket domain. E2F binds residues on α4, α5, α6, α8, and α9 from A and α11 from B. The ends of E2F make the most contact with pRB. E2F binds to pRB by its main chain, which hydrogen bonds to pRB’s side chain. Tyr(411), Leu(424), Phe(425), Glu(419), and Asp(423) are the five most important E2F residues. pRB bound to E2F

13. E2F Bound to pRB Pocket E2F Key Red is Domain A of the pocket. Green is Domain B of the pocket. PDB ID #1o9k, x-ray diffraction

14. pRB • Domain A is red. • Domain B is neon green. • E2F Residues • Tyr(411) is orange. • Asp(423) is blue. • Glu(419) is light blue. • Leu(424) is magenta. • Phe(425) is green • Tyr(411)-E2F has a phenolic ring in a hydrophobic pocket of pRB, and its hydroxyl group makes a hydrogen bond to Glu(554)-pRB. • Leu(424)-E2F and Phe(425)-E2F interact hydrophobicly with pRB at Lys(530)-pRB and Phe(482)-pRB • Glu(419)-E2F, which forms a hydrogen bond with a water molecule and Thr(645)-pRB. • Asp(423)-E2F, which forms a salt bridge with Arg(467)-pRB. • Asp(423)-E2F and Glu(419)-E2F point outward, unlike the other three residues that point inward.

15. LxCxE site • Is a shallow groove found on the B box of the pocket domain. • The groove is formed by α17, α14, α15 on the sides, the hydrophobic core of the B box, and α16 and α18 on the ends. • HPV E7, T antigen, and E1A oncoproteins all have the LxCxE motif and bind to the site to deactivate pRB.

16. pRB Bound to HPV E7 Pocket Domain A E7 Pocket Domain B PDB ID # 1gux, x-ray diffraction

17. HPV E7 bound at LxCxE • Alternating Leu 22, Cys 24, Glu 26 (Green), and Leu 28 side chains point into the groove. • The sidechains that point in the box interact by van der Waals forces and hydrogen bonds. • Asp 21, Tyr 23, Tyr 25, Gln 27, and Asn 29 don’t bind because they point out of the box (Magenta). • Leu 22, Leu 28, and Cys 24 bind in hydrophobic pockets of the groove (Orange).

18. E7 Bound to the LxCxE site Glu 26 Leu 22 Asn 29 Cys 24 Leu 28 Asp 21 Tyr 23 Tyr 25 Gln 27 Key Orange residues point inward and bind in the hydrophobic pockets. Magenta residues point outward. Green residue points inward. PDB ID # 1gux, x-ray diffraction

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