Transcript The Retinoblastoma Protein

The Retinoblastoma Protein
Its Structure and Its Role in Cancer
By Ariel Lefkovith
Cancers
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Retinoblatoma
Breast Cancer
Cervical Cancer
Colo-rectal cancer
Endometrial cancer
Head & neck cancer
Liver cancer
Lung cancer
Malignant carcinoid
Malignant glioma
Urothelial cancer
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Malignant lymphoma
Malignant melanoma
Ovarian cancer
Pancreatic cancer
Prostate cancer
Renal cancer
Skin cancer
Stomach cancer
Testis cancer
Thyroid cancer
Background
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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.
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.
The Cycle
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.
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.
5
10
2
3
7
13
6
4
17
11
18
12
8
9
14
16
1
15
The helices in Domain A are in red.
The helices in Domain B are in green.
The 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.
Lobe A
Lobe B
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.
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.
Red-RbC fragment
Blue-DP
Green-E2F
pRB bound to E2F
• 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
•Domain A is red.
•Domain B is neon green.
E2F Residues
•Tyr(411) is orange.
•Asp(423) is blue.
•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.
•Glu(419) is light blue.
•Leu(424) is magenta.
•Phe(425) is green
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.
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).