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.1 Rb is a G1 checkpoint protein that can prevent entry into S phase and induce cell-cycle arrest.1, 2 Click here to see Rb's role in the cell-cycle. To advance to S phase, the cell needs stimulation from CDKs only during the first two-thirds of G1.3 This point is termed the Restriction (R) point.3, 4 Rb is the R point switch that signals progression into S phase.3

Rb interacts with E2F transcription factors to regulate cell-cycle progression by controlling the transcription of E2F-dependent genes.3 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 nonfunctional and unbound to E2F.3 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.5 Phosphorylation of the N-terminus of Rb can destabilize E2F-Rb interactions and cause E2F release.

In addition to its role in E2F sequestration, Rb also remodels chromatin by recruiting histone deacetylases such as HDAC1. The recruitment of HDACs works to repress E2F genes, as hypoacetylated histones are generally at promoters of inactive genes.6

Our tutorial displays two crystal structures of Rb. The first is 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.1 Each monomer is highlighted a different color. Within a single monomer, we will focus on the small pocket domain, and then the N-terminus. In the small pocket domain, RbA is green, RbB is blue, and E2F is yellow. The second crystal structure is of the N-terminus of Rb. The entire N-terminus is highlighted in cyan, separately from small pocket. Lobe A of RbN is blue and lobe B is orange.

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 .1

The domains are connected by a flexible linker, which allows for alterations of the pocket domain conformation.2 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.2 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-terminal domain and preceded by two structural domains.1 Rb domains A and B form the binding site for E2F and other proteins, including oncoproteins, cyclin, and HDAC.2, 6

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 domain. E2F-1 contains four domains: a cyclin-CDK-binding domain, a DNA-binding domain, a dimerization partner domain, and a transcriptional activation domain.1, 5 Within the transcriptional activation domain is the pocket-binding domain that binds directly with the cleft formed by Rb domains A and B.5

The E2F peptide makes contact with five alpha-helices of domain A and one alpha-helix of domain B .1

Of nine highly-conserved E2F-1 residues, five have been shown to be critical for Rb binding and result in weaker binding when mutated.1 These five are Tyr411, Phe413, Leu415, Leu424, and Phe425. A hydrophobic pocket is created by the phenol ring of Tyr411 and Phe413 of E2F interacting with Ile536, Ile532, Ile547 of RbA , while the hydroxyl group of Tyr411 of E2F forms hydrogen bonds with Glu554 in RbA . 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 in RbB 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.1, 7

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

IV. N-terminal Domain

RbN is a globular domain that contains two cyclin folds, lobes A and B, connected by a single helix that is shared between lobes . Lobes A and B are each composed of five helices; A is helices 1-5 and B is helices 6,7,8,10, and 11. Helix 6 extends from lobe A yet its C-terminus is considered the first helix of lobe B . Beyond these two lobes, the RbN C-terminal domain has helices 12 and 13 that help with packing in between the two lobes.8

RbN is hypothesized to be a distant homolog of the Rb pocket, and could have been a result of duplication event. However there is no similar site for E2F binding in RbN like in Rb pocket, as the lobe A and B interface is different due to the cyclin-like folds. Our tutorial includes residues 52-244 and 270-355 of RbN. Crystallization resulted in two fragments, which are cleaved within lobe B at its arginine-rich linker (residues 251-266).8

Cyclin-CDK phosphorylation sites include Ser230, Ser249,Thr252, and Thr356/Thr373.8, 9 The crystallization of RbN only includes the residue Ser230 . Only the phosphorous atom in the phosphate group is shown. Mutations in the Rb N-terminus are carcinogenic, and there are two notably well-conserved residue patches of RbN: Lys122, Asp332, Arg334, Asp340 , and Met208, Leu212, Val213, Ile214 . The former conserved patch (CP1) is predominantly polar, and the latter conserved patch (CP2) is predominantly hydrophobic.8 Met208 is not highlighted in this tutorial as it was not included in the crystal structure.

Specific mutations that have been identified in retinoblastoma patients include several missense mutations in exons 4, 5, 7, and 9: Glu72Gln, Glu137Asp, Ile185Thr, Leu220Val, Thr307Ile, and Gly310Glu . Many of these mutations lead to destabilization of the Rb holoprotein. Exons 4, 7, and 9 are also critical for the core structure, and deletions of any of these exons leads to major misfolding and oncogenicity. Mutations in Thr307 and Gly310 were not highlighted as these amino acids were not present in the crystal structure.8

V. Biomedical Significance

In 2003, Rb had been proven to be the only known gene whose mutation was both "necessary and sufficient" for oncogenesis in humans.9 Its namesake 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.3 Rb is often inactivated in human cancers, such that its checkpoint abilities cannot suppresses tumors and cells proliferate.3, 10 CDK phosphorylation destabilizes the Rb-E2F complex, allowing cells with unbound E2F to enter S phase.2

While phosphorylation is one contributor to Rb inactivation, mutations in the protein can also be detrimental to its function and cancerous.3, 7 In cancers like retinoblastoma, osteosarcoma, and small-cell lung carcinoma, Rb inactivation is a result of mutation or deletion.3 Another hypothesis for modified checkpoint control in carcinogenesis involves the regulation of Rb by ICBP90, a transcription factor. Cancer cells overexpress ICBP90, which down-regulates Rb by binding to the Rb promoter. Lower levels of Rb as a result of transcriptional regulation by ICBP90 lead to the cell favoring S phase.3

Rb also plays a role in differentiation of the eye, brain, muscle, and liver, among others.3, 10 Rb-deficient mice die early on in gestation and suffer defects to the central nervous system and hematopoietic system.11

VI. References

(1) 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.

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

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

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

(5) 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.

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

(7) 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.

(8) Hassler M, Singh S, Yue WW, Luczynski M, Lakbir R, Sanchez-Sanchez F, Bader T, Pearl LH, Mittnacht S. 2007. Crystal structure of the retinoblastoma

(9) Goodrich DW. 2003. How the other half lives, the amino-terminal domain of the retinoblastoma tumor suppressor protein. J Cell Physiol 197:169-180.

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

(11) Lee EY, Chang CY, Hu N, Wang YC, Lai CC, Herrup K, Lee WH, Bradley A. 1992. Mice deficient for Rb are nonviable and show defects in neurogenesis and haematopoiesis. Nature 359:28-294.

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