Retinoblastoma Binding
Protein
Geoff Carney-Knisely '18 and Liz Eder '17
Contents:
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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