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.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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