Retinoblastoma Binding Protein

Geoff Carney-Knisely '18 and Liz Eder '17


I. Introduction

In order for a cell to divide and proliferate, it first must progress through growth phases G1 and G2 and replicate its entire genome in S phase.  Retinoblastoma Protein (Rb) is a cell-cycle regulatory protein. Rb is a G1 checkpoint protein that can prevent entry into S phase and induce cell-cycle arrest. Click here to see Rb's role in the cell-cycle. To advance to S phase, the cell needs stimulation from CDKs during only the first two-thirds of G1. This point is termed the Restriction (R) point. Rb is the R point switch that signals progression into S phase (Giacinti and Giordano, 2006).

Rb interacts with E2F transcription factors to regulate cell-cycle progression by controlling the transcription of E2F-dependent genes. Specifically, Rb binds to E2F to inhibit cell division.

In eukaryotes, E2F transcribes cell cycle activators and suppressors. Cancerous cells, which proliferate without cellular control, often contain mutations in Rb that render it unfunctional and unbound to E2F. Thus, Rb is known as a tumor suppressor protein. When E2F is unbound, the E2F genes that trigger S-phase are transcribed and cell growth continues. Binding occurs between the E2F transactivation site and a cleft in the Rb pocket domain.

Rb remodels chromatin by recruiting histone deacetylases like HDAC1, among other proteins. The recruitment of HDACs works to repress E2F genes, as hypoacetylated histones are generally at promoters of inactive genes.

Our tutorial displays a tetramer of the Rb small pocket, with each monomer containing an A and B domain but not the flexible linker between the two domains.

II. General Structure

Retinoblastoma protein contains a large and small pocket. The large pocket is comprised of both the small pocket and the C-terminal domain. Rb domains A and B, connected by a spacer, make up the small pocket. Domain A is composed of 11 helices and domain B is composed of 8 helices and one beta sheet.

The domains are connected by a flexible linker, which allows for alterations of the pocket domain conformation. (Burke et al.) The phosphorylation of the sites in the C terminus by cyclin-CDK complexes alters the conformation of Rb so that E2F binding is inhibited and Rb itself is inactivated. (Burke et al.). In Rb's inactive, phosphorylated form, E2F is unbound and free to transcribe genes activating DNA synthesis. Click here. for a schematic of Rb structure.

The small pocket is followed by the C terminus domain and preceded by two structural domains. Rb domains A and B form the binding site for E2F and other proteins, including oncoproteins, cyclin, and HDAC.

III. E2F-1 Binding

Our tutorial focuses on the interactions of Rb domains A and B with E2F-1 residues 409-426 located within the E2F-1 pocket-binding E2F-1 domain.  contains four domains: a cyclin-CDK-binding domain, a DNA-binding domain, a dimerization partner domain, and a transcriptional activation domain. Within the transcriptional activation domain is the pocket-binding domain that binds directly with the cleft formed by Rb domains A and B.

The E2F peptide makes contact with five alpha-helices of domain A and one alpha-helix of domain B.  

Of nine highly-conserved E2F-1 residues, five have been shown to be critical for Rb binding and result in weaker binding when mutated. A hydrophobic pocket is created by the phenol ring of Tyr411 interacting with Ile536, Ile532, Ile547, and Phe413 , while the hydroxyl group of Tyr411 forms hydrogen bonds with Glu554 . Additional hydrophobic interactions occur between Leu424 and Phe425 of E2F , and between Leu424, Leu415, Phe425 of E2F and Lys530 of RbA .

Within the Rb B domain, Lys652 and Lys653 hydrogen bond with the peptide backbone of the E2F C-terminus . Despite multiple contact points between E2F and Rb A and B domains, the binding of two proteins does not results in significant structural change of the small pocket.

Click here for a summary of interactions between RbA, RbB, and E2F

IV. C-terminal Domain

V. Biomedical Significance

Retinoblastoma is a type of cancer that originates in the retina of the eye and is common in children. Rb can be considered an oncogene, but both copies of the Rb gene must be damaged for tumor growth to begin. Rb is often inactivated in human cancers, such that its checkpoint abilities cannot suppresses tumors and cells proliferate. CDK phosphorylation destabilizes the Rb-E2F complex, and with unbound E2F cells can enter S phase.

While phosphorylation by CDKs is one contributor to Rb inactivation, mutations in the protein can also be detrimental to its function and cancerous. In cancers like retinoblastoma, osteosarcoma, and small-cell lung carcinoma, Rb inactivation is a result of mutation or deletion. Another hypothesis for modified checkpoint control in carcinogenesis involves the regulation of Rb by ICBP90, a transcription factor. Cancer cells overexpress ICBP90, which down regulate Rb by binding to the Rb promoter when methylated. Lower levels of Rb as a result of transcriptional regulation by ICBP90 lead to the cell favoring S phase.

Rb has also been shown to have a role in differentiation of the eye, brain, muscle, and liver, among others. Lee et al., 1992, showed that Rb-deficient mice die early on in gestation and suffer defects to the central nervous system and hematopoietic system.

VI. References

Burke JR, Hura GL, Rubin SM. 2012. Structure of inactive retinoblastoma protein reveal multiple mechanisms for cell cycle control. Genes Dev 26:1156-1166.

Dick FA. 2007. Structure-function analysis of the retinoblastoma tumor suppressor protein- is the whole a sum of its parts? Cell Division 2:26.

Giacinti C and Giordano C. 2006. RB and cell cycle progression. Oncogene 25:5220-5227.

Lee C, Chang JH, Lee HS, Cho Y. 2002. Structural basis for the recognition of the E2F transactivation domain by the retinoblastoma tumor suppressor. Genes Dev 16:3199-3212.

Munger K. 2003. Clefts, grooves, and (small) pockets: the structure of the retinoblastoma tumor suppressor in complex with its cellular target E2F unveiled. PNAS 100(5):2165-2167.

Munro S, Carr SM, La Thangue NB. 2012. Diversity within the pRb pathway: is there a code of conduct? Oncogene 31:4343-4352.

Weinberg RA. 1995. The retinoblastoma protein and cell cycle control. Cell 81:323-330.

Xiao B, Spencer J, Clements A, Ali-Khan N, Mittnacht S, Broceno C, Burghammer M, Perrakis A, Marmorstein R, Gamblin SJ. 2003. Crystal structure of the retinoblastoma tumor suppressor protein bound to E2F and the molecular basis of its regulation. PNAS 100(5): 2363-2368.

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