Crystal Structure of the Retinoblastoma Protein

Molecular Cell
Article
Crystal Structure of the Retinoblastoma Protein
N Domain Provides Insight into Tumor Suppression,
Ligand Interaction, and Holoprotein Architecture
Markus Hassler,1,2 Shradha Singh,1,6 Wyatt W. Yue,2 Maciej Luczynski,1 Rachid Lakbir,1
Francisco Sanchez-Sanchez,4 Thomas Bader,3,5 Laurence H. Pearl,2,* and Sibylle Mittnacht1,*
1Cancer Research UK Centre for Cell and Molecular Biology
2Section of Structural Biology
Chester Beatty Laboratories, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK
3Inserm, U567, Paris 75014, France
4A´ rea de Gene´ tica, Facultad de Medicina/Centro Regional de Investigaciones Biome´ dicas, Universidad de Castilla-La Mancha,
02006 Albacete, Spain
5Institut Cochin, Universite´ Paris Descartes, CNRS (UMR 8104), Paris 75019, France
6Present address: Protein Science, Syngenta, Jealott’s Hill International Research Station, Bracknell, Berkshire RG42 6EY, UK.
*Correspondence: laurence.pearl@icr.ac.uk (L.H.P.), sibylle.mittnacht@icr.ac.uk (S.M.)
DOI 10.1016/j.molcel.2007.08.023
SUMMARY
The retinoblastoma susceptibility protein, Rb,
has a key role in regulating cell-cycle progres-
sion via interactions involving the central
‘‘pocket’’ and C-terminal regions. While the
N-terminal domain of Rb is dispensable for
this function, it is nonetheless strongly con-
served and harbors missense mutations found
in hereditary retinoblastoma, indicating that dis-
ruption of its function is oncogenic. The crystal
structure of the Rb N-terminal domain (RbN),
reveals a globular entity formed by two rigidly
connected cyclin-like folds. The similarity of
RbN to the A and B boxes of the Rb pocket do-
main suggests that Rb evolved through domain
duplication. Structural and functional analysis
provides insight into oncogenicity of mutations
in RbN and identifies a unique phosphorylation-
regulated site of protein interaction. Addition-
ally, this analysis suggests a coherent confor-
mation for the Rb holoprotein in which RbN
and pocket domains directly interact, and
which can be modulated through ligand binding
and possibly Rb phosphorylation.
INTRODUCTION
Loss of signaling involving the retinoblastoma tumor sup-
pressor protein (Rb) is common and important in cancer
development. Some tumor viruses inhibit Rb, and muta-
tions in the Rb gene are associated with oncogenic trans-
formation (Classon and Harlow, 2002). Rb is a member of
the ‘‘pocket’’ protein family implicated in the regulation of
cell proliferation. In its hypophosphorylated form, Rb inter-
acts with and represses E2F/DRTF transcription factors,
impeding the G1/S transition. During late G1, Rb is
phosphorylated at multiple sites by cyclin D/cdk4 and
cyclin E/cdk2 kinases, which abrogates Rb’s repressive
interaction with E2Fs and allows cell-cycle progression
(Mittnacht, 2005).
Besides its G1/S inhibitory function, Rb is involved in
differentiation, prevention of cell death, and control of tis-
sue fate, via its ability to activate transcription factors such
as ATF-2, MyoD, Runx2, C/EBP, and glucocorticoid (GR)
and androgen (AR) receptors and to recruit SWI/SNF
chromatin-remodeling activity (Mittnacht, 2005). Rb’s
ability to activate gene transcription is regulated indepen-
dently of its ability to inhibit E2F, by a mechanism involving
the tripartite motif protein 27/ret finger protein (TRIM27/
RFP), which stabilizes the E1A-like inhibitor of differentia-
tion (EID-1) (Krutzfeldt et al., 2005). EID-1, itself an Rb-
binding protein degraded in an Rb-dependent manner,
inhibits p300/CBP in vitro and may interfere with activating
chromatin modifications in vivo (Miyake et al., 2000).
Regulation of G1/S progression is primarily a function of
the conserved central pocket (aa 379–792) and C-terminal
region (aa 792–928) of Rb. The N-terminal region (aa 1–
378), although well conserved among Rb orthologs and
paralogs, has been studied far less. However, a significant
number of mutations in this region occur in retinoblas-
tomas, strongly implicating it in tumor suppression. Other
work suggests that its integrity is critical for rescue of both
the developmental defects and increased tumor suscepti-
bility in Rb-deficient mice, but this finding is controversial
(Goodrich, 2003).
Several cellular proteins have been reported to interact
with the Rb N-terminal region. Yeast two-hybrid studies
identified MCM7 (Sterner et al., 1998), a component of
the replication origin recognition complex (ORC), and
p84N5/pThoc1 (Durfee et al., 1994), a death domain
protein involved in mRNA splicing and transport (Li et al.,
2005). Other identified partners include Sp1 (Udvadia
et al., 1995), TFIID/TAF1 (Shao et al., 1997), and the
Molecular Cell 28, 371–385, November 9, 2007 ª2007 Elsevier Inc. 371

interferon-responsive protein p202 (Choubey and Lengyel,
1995). Most recently, transcriptional coactivators ASC-2
(Goo et al., 2004) and GRIP-1/SRC-2 (Batsche et al.,
2005) were found to associate with the N-terminal region
of Rb, consistent with its recognized role in gene activation.
Toward a functional understanding of the Rb N-terminal
region, we determined the crystal structure of its prote-
ase-resistant core, encompassing residues 40–355 of
human Rb. The structure and associated analysis reveal
unexpected insight into the evolution of the pocket protein
family and suggest mechanisms by which RbN contrib-
utes to tumor suppression. Importantly, we identify (1)
a key site of protein interaction in RbN, regulated by phos-
phorylation, and provide evidence for (2) a closed Rb
holoprotein conformation in which the RbN and pocket
domains interact directly and (3) the modulation of this
interaction through ligand binding.
RESULTS
Structure Determination
As attempts to crystallize the entire N-terminal region (aa
1–370) of human Rb were unsuccessful, we identified
a subconstruct amenable to crystallization by partial prote-
olysis (Figure 1A and Figure S1, in the Supplemental Data
available with this article online). In line with previous
observations (Hensey et al., 1994), limited tryptic digestion
of Rb residues 1–370 generated two fragments with
approximate sizes of 24.7 and 10.9 kDa (Figure S1A).
Mass spectrometry and N-terminal sequencing identified
three polypeptides encompassing residues 46–251, 263–
355, and 266–355 (data not shown), indicating trimming
from both termini and excision of an internal arginine
(Arg)-rich linker (aa 251–266) connecting the 24.7 and
10.9 kDa fragments. The two fragments copurified,
indicating that they remained associated (Figure S1B). A
refined RbN construct (aa 40–355) combined with limited
tryptic digestion reproducibly yielded diffracting crystals
containing both fragments (Figure S1C). The structure
was determined by single-wavelength anomalous diffrac-
tion (SAD) and was refined to 2.0 A˚ resolution (Table 1).
Overall Structure
RbN has a globular structure consisting of tandem cyclin-
like folds: lobe A formed by helices a1, a2, a3, a4, and a5
and lobe B formed by the C-terminal 3.5 turns of a6, and
helices a7, a8, a10, and a11 (Figure 1B). A C-terminal seg-
ment (aa 313–355) containing helices a12, h1, and a13
packs tightly onto helices a5, a6, and a7 and occupies
the space between the two lobes, positioning the visible
C terminus (Arg355) between lobes A and B.
Both cyclin folds superimpose well (<2 A˚ rmsd between
Ca atoms) with the canonical folds present in cyclins (An-
dersen et al., 1996; Brown et al., 1995) (Figure S2C) and
Figure 1. The Rb N-Terminal Region
Consists of Two Cyclin Folds
(A) Schematic of Rb domains (top) and the
trypsin-resistant RbN core (bottom). Arrow-
heads indicate sites of tryptic cleavage; pro-
tease-resistant fragments are in cyan and
orange. The positioning of cyclin fold helices
for fold A (dark blue) and fold B (red) is
indicated. Putative phosphorylation sites are
marked.
(B) Ribbon representation of the RbN structure
colored as in (A). Disordered or absent resi-
dues are indicated by dashed lines.
372 Molecular Cell 28, 371–385, November 9, 2007 ª2007 Elsevier Inc.
Molecular Cell
Crystal Structure of the RbN Domain

TFIIB (Nikolov et al., 1995). The architecture of RbN is
reminiscent of the Rb pocket, which also consists of
tandem cyclin folds (Lee et al., 1998). Structure-guided
sequence alignment reveals an above-average identity
(17.75%) between equivalent residues of the B folds of
RbN and the Rb pocket (Figures S2B and S2C), indicating
that these folds are remote homologs and suggesting that
Rb probably arose through duplication of an ancestral
cyclin fold pair.
While RbN and the Rb pocket regions are both formed
by a pair of cyclin folds, the connection between these
pairs and their juxtaposition differ significantly (Figure 1B
and Figure S2A). The RbN lobes are rigidly connected via
a single long helix a6, which projects from the end of
lobe A, with its C-terminal half providing the first helix of
lobe B. The hydrophobic cores of lobes A and B are entirely
separate, suggesting that they may fold independently of
each other. This is in line with observations that Rb variants
lacking all or part of lobe A stably accumulate in cells and
can provide tumor suppressor activity (Sanchez-Sanchez
et al., 2007; Xu et al., 1994). The cyclin fold arrangement
also means that the interface that provides the E2F dock-
ing site in the Rb pocket (Lee et al., 2002; Xiao et al.,
2003) is not recapitulated in RbN.
In contrast to lobe A, lobe B contains substantial exten-
sions connecting helices a6 and a7, and helices a8 and
a10 (Figure 1B). The connection between a6 and a7 forms
a well-ordered hairpin loop (aa 173–188) projecting from
Table 1. Data Collection and Processing Statistics
Crystal 1 Crystal 2
Data Collection ID29, ESRF ID14-2, ESRF
Wavelength 0.97940 0.93300
Space group C222 C222
Unit cell a = 72.34, b = 107.09, c = 98.61 a = 72.30, b = 106.65, c = 98.19
Resolution (A˚ ) 60–2.55 (2.69–2.55) 51.1–2.0 (2.11–2.00)
Observations (N) 180,430 (26,716) 104,577 (15,127)
Unique reflections (N) 12,855 (1,864) 25,840 (3,714)
Redundancy 14.0 (14.3) 4.0 (4.1)
Completeness (%) 100 (100) 99.5 (99.4)
Rmerge 0.124 (0.565) 0.059 (0.304)
I/sigmaI 23.5 (5.9) 14.7 (3.9)
Anomalous completeness 100 (100)
Anomalous redundancy 7.4 (7.4)
SAD Analysis
Number of heavy atom sites 5
Resolution 15.0–2.8
FOM (after DM) 0.67
Refinement Statistics
Resolution range (A˚ ) 51.1–2.0
Reflections 24,550
Total atoms 2,431
Water molecules 266
R factor (last shell: 2.00–2.08) 0.214 (0.24)
Rfree (last shell: 2.00–2.08) 0.267 (0.29)
Rmsd
Bonds (A˚ ) 0.019
Angles (
) 1.657
B factor main-chain bond (A˚ 2
) 1.392
B factor side-chain bond (A˚ 2
) 3.645
Highest shell values are in parentheses.
Molecular Cell

Figure 2. RbN Domain Functional Surfaces
(A) Surface of RbN colored by sequence conservation in metazoa (human, mouse, chicken, frog, newt, trout, and rainbow ﬁsh). Prominent conserved
residues are indicated.
(B and C) Lattice contacts indicate sites for protein interaction. Residues involved in lattice interactions are labeled. (B) Helix a13 of a symmetry-
related molecule packs through hydrophobic interactions onto the conserved patch CP2 (see [A]). (C) The crystal contact involving the projection
features a ‘‘handshake’’ interaction motif involving exposed hydrophobic residues.
(D and D0
) Superposition of cyclin folds from RbN (N-A and N-B) with those of the Rb pocket (P-A and P-B) (1GUX). Structural alignments were
produced by superposition of 40–57 residues within the cyclin fold-forming helices (rmsd of Ca atoms 2.00 A˚ for all four folds). For clarity, pair-
wise superpositions between N-B and P-B (rmsd of Ca atoms = 1.51 A˚ [D]) and between N-A and P-A (rmsd of Ca atoms = 2.00 A˚ [D0
]) are shown.
Cyclin fold helices are colored. Interaction partners involving the canonical protein-binding ‘‘cyclin wedge’’ in the four cyclin folds, including E7 pep-
tide for P-B (D) and intramolecular helices a6 and a9 for N-A and P-A, respectively (D0
), are in yellow.
(E) Conservation between the LxCxE-binding surface and equivalent in N-B. Conserved residues in N-B isostructural to those involved in LxCxE bind-
ing by P-B are in green. Remaining residues are colored as in (A). Dotted red lines depict disordered areas near the cyclin wedge. Putative cdk phos-
phorylation sites S249 and T252 are indicated.
Molecular Cell

the body of the protein. The conformation of this projection
is stabilized by its symmetry-related counterpart in the
crystal lattice (Figure 2C). The region connecting a8 to
a10 (aa 230–271) forms an elaborate coil and helix a9,
before running into the proteolytically labile Arg-rich linker
(aa 251–266) whose excision is required for crystallization.
The structure of RbN contradicts previous predictions
from yeast two-hybrid and bioinformatics studies (Bork
et al., 1997; Hensey et al., 1994; Yamane et al., 2000,
2001), which identified two independently folded subdo-
mains, the more amino-terminal of which was deemed
to contain a BRCT-like fold. Our structure shows that the
two segments generated by limited proteolysis are in
fact intimately intertwined parts, forming a single struc-
tural entity, so that interaction studies based on one or
other of these in isolation must be viewed with consider-
able caution.
RbN Domain Functional Surfaces
While the precise biochemical function of the Rb N-ter-
minal region is not known, it displays distinctive patterns
of sequence conservation, and mutation of this region is
associated with familial retinoblastoma (see below), indi-
cating important functional roles. Several lines of structural
evidence identify potential functional surfaces on RbN.
Two clusters of conserved and surface-exposed resi-
dues are evident (Figure 2A). One comprises an extensive
patch of predominantly polar residues including Lys122,
Asp332, Arg334, and Asp340 (conserved patch [CP] 1)
(Figure 2A, left). A second (CP2), on the opposite side,
consists of a cluster of prominent hydrophobic residues,
including Met208, Leu212, Val213, and Ile214 (Figure 2A,
right), which forms lattice contact with a hydrophobic he-
lical segment in a13 from a symmetry-related molecule
(Figure 2B).
A third site involves the projection (aa 173–188), which
contains a number of moderately conserved surface-ex-
posed hydrophobic residues, including Leu174, Pro177,
Ile181, and Ile185, that make a ‘‘handshake’’ interaction
with their symmetry equivalents in the crystal (Figure 2C).
Both lattice contact sites (CP2 and the projection) score
significantly as interaction interfaces with the MSDPisa
tool (p values = 0.055 and 0.084, respectively) (Krissinel
and Henrick, 2005), supporting their potential involvement
in protein-protein binding.
Finally, superimposition of RbN lobe B (N-B) with pocket
B (P-B) (see Figure S2) identifies an unoccupied ‘‘cyclin
wedge’’ in N-B (Figure 2D). In other cyclin structures this
area, formed by the third, fourth, and fifth cyclin helices,
mediates high-affinity ligand interactions (Jeffrey et al.,
1995; Nikolov et al., 1995). Most notably, in the context
of P-B, it facilitates binding of the LxCxE motif common
to many Rb ligands (Lee et al., 1998). Comparison of the
LxCxE-binding site in the Rb pocket with the correspond-
ing surface in N-B reveals considerable similarity. Several
residues in the N-B cyclin wedge structurally equivalent to
those that coordinate the LxCxE peptide in P-B show
strong conservation (e.g., Lys228, Lys289, and Tyr292)
(Figure 2E and Figure S2B). However, the N-B wedge is
shorter and too small to accommodate a residue isostruc-
tural to the glutamic acid in the LxCxE motif. The hydro-
phobic recess, which accommodates the leucine in P-B,
is deeper in the RbN cyclin wedge, potentially providing
space for a larger hydrophobic residue. An unstructured
loop (aa 301–311) connecting helices a11 and a12 lies
adjacent to the this recess, most likely providing one of
its edges. The Arg-rich linker, removed by proteolysis and
not present in the structure (see above; Figure 1), lies at
the opposite side and probably provides the other edge.
This linker contains two known cyclin-dependent kinase
phosphorylation sites, indicating a possible mechanism
for regulating this putative binding site (see Figure 2E).
The area analogous to the cyclin wedge in the A lobe is
occupied by the N-terminal half of helix a6, and thus is un-
available for interactions. This mirrors the situation in the
Rb pocket A, where the cyclin wedge likewise is engaged
by an intramolecular helix (Figure 2D0
).
In summary, the cumulative evidence suggests a mini-
mum of four candidate functional surfaces within RbN,
any or all of which could be involved in protein-protein
interactions (Figure 2F).
RbN Domain Alterations in Cancer
A significant number of mutations that map to RbN have
been detected in retinoblastoma patients. These include
in-frame deletion of amino acids encoded by exons 4, 5,
7, and 9 (Dryja et al., 1993; Jakubowska et al., 2001; Lefe-
vre et al., 2002; Sanchez et al., 2000) as well as missense
mutations Glu72Gln, Glu137Asp, Ile185Thr, Leu220Val,
Thr307Ile,andGly310Glu(Blanquetetal.,1993,1995;Bric-
hard et al., 2006; Liu et al., 1995; Lohmann et al., 1997).
The structure of RbN shows that protein segments en-
coded by exons 4, 7, and 9 form integral parts of the pro-
tein core (Figure 3A). Loss of these would cause gross
misfolding, which could trigger triage of the mutant protein
into misfolding-associated degradation. In line with this,
we found that full-length Rb proteins with deletion of these
exons display increased turnover in cultured cells, and
some are only detected following proteasome inhibition
(Figure S3). Destabilization of the Rb holoprotein by exon
4, 7, or 9 deletion provides a satisfactory explanation for
the oncogenicity of these mutations. Significantly, we also
observed some increased turnover of an Rb construct
lacking the N-terminal region (RbDN, aa 379–928), indicat-
ing a general role of RbN in holoprotein stabilization.
In contrast, loss of exon 5 could be accommodated
without gross effects on the overall fold. The residues en-
coded by this exon form two turns of the long, central helix
(F) Putative functional surfaces in RbN. Ribbon representation for the RbN structure in a color gradient from blue (aa 51) to red (aa 355). The relative
position of individual functional surfaces is indicated.
Molecular Cell

Figure 3. Tumorigenic Mutations in RbN
(A) Exon deletions and point mutations associated with cancer are shown. Deletions of exon 4 (purple), exon 7 (orange), exon 9 (blue), and exon 5
(green) are indicated. Residues associated with point mutations (red) are shown as stick models.
(B) Conservation and accessibility of RbN residues. Gray bars indicate conservation among Rb homologs as in Figure 2A. Circles show accessibility
(red, highly accessible; black, buried). Dashed lines in the secondary structure assignments indicate residues disordered or absent in the crystals.
a helices corresponding to the cyclin fold are labeled. Sites of cancer-associated mutations are colored as in (A).
Molecular Cell

6 and part of the projection (Figure 3). Deletion of exon 5
removes the projection but otherwise is unlikely to disturb
the continuity of the RbN fold. Consistent with the limited
structural impact predicted, an exon 5-deleted Rb protein
accumulates in cells at similar levels as, and clears with a
half-life indistinguishable from, wild-type Rb (Figure S3A).
Furthermore, like wild-type RbN, but unlike the other exon
deletion mutants, exon 5-deleted RbN can be expressed
in E. coli and purified as a stable soluble protein whose cir-
cular dichroism (CD) spectrum is indistinguishable from
wild-type (Figure S8). In consequence, the link between
loss of exon 5 and cancer argues for a specific role of
the projection in tumor suppression. Of note, the retino-
blastoma-associated missense mutation Ile185Thr maps
within the projection. Ile185 forms hydrophobic contacts
with several residues, including its counterpart in a sym-
metry-related molecule (Figure 2C). Mutation to threonine
introduces a polar side chain disrupting these hydropho-
bic interactions, and may affect function in a similar way
as the loss of exon 5.
The structure provides no explanation why missense
mutations Glu72Gln and Glu137Asp might be oncogenic.
Both are surface exposed, and the mutations would have
no structural effects (Figure 3A) but might conceivably ab-
rogate a protein contact; however, this is not evident from
the structure.
Thr307Ile and Gly310Glu mutations map in the disor-
dered loop (aa 301–311) between a11 and a12 (Figure 3)
adjacent to the N-B cyclin wedge (refer to Figure 2E) and
might affect the interaction of ligands. Leu220Val lies in
the hydrophobic core of lobe B, at the interface of helices
a8, a10, and a11 (Figure 3A), and the mutation generates
a gap in the closely packed interface, which would perturb
the orientation of these helices, with effects on the binding
of potential cyclin wedge ligands.
Thus, while a number of cancer-associated RbN muta-
tions clearly act through global destabilization of the Rb
holoprotein, others generate subtle surface changes,
most notably affecting the hydrophobic projection and
the B cyclin wedge. Both features are likely sites of pro-
tein-protein interaction (see above), which implicates
these sites and their interactions in the tumor suppressor
activity of Rb.
Protein Interactions Involving RbN
We assessed the in vitro interaction with RbN of a number
of previously reported proteins, using purified compo-
nents and GST affinity precipitation (Figure 4A). While we
were unable to recapitulate binding of MCM7 (Sterner
et al., 1998) (data not shown), binding was seen for
p84N5 (aa 379–675) (Durfee et al., 1994) and GRIP-1 (aa
1236–1392) (Batsche et al., 2005). We also confirmed an
interaction between RbN and the rest of the Rb protein
(RbDN, aa 379–928), previously suggested by yeast two-
hybrid screen (Hensey et al., 1994). In addition, we identi-
fied an interaction with the E1A-like inhibitor of differenti-
ation, EID-1 (MacLellan et al., 2000; Miyake et al., 2000).
This interaction was seen with either EID-1 or RbN fused
to GST, and EID-1 bound RbN to the same degree as
p/CAF (Figure S4A). KD for the RbN-EID-1 interaction
was determined as 8 mM by isothermal titration calorim-
etry (ITC) (Figure S4B). Significantly, an Rb deletion con-
struct lacking RbN showed reduced sensitivity to the
inhibitory effects of EID-1 as determined by its ability to
coactivate GR driven transcription (Figure S5), implying
that the RbN-EID-1 interaction contributes to EID-1’s abil-
ity to modulate Rb activity in cells. Both RbDN and EID-1
precipitated RbN more readily than GRIP-1 or p84N5,
while GST or an irrelevant GST protein did not bind RbN,
confirming that the interactions are selective (Figure 4A).
Taken together, these data show that RbN facilitates inter-
actions with an array of heterologous proteins and can
engage in homotypic interactions with a region C-terminal
to RbN.
The Interaction of EID-1 with RbN
While to the best of our knowledge the RbN-EID-1 interac-
tion has not been reported, an EID-1 segment containing
an LxCxE motif has previously been shown to bind to an
Rb pocket construct (379–885) (MacLellan et al., 2000;
Miyake et al., 2000). We observed a similar interaction
in vitro between EID-1 and the central Rb pocket (aa 379–
792) (Figure 4B). Both independent interactions of EID-1
with RbN and the Rb pocket were confirmed using di-
methyl-superimidate (DMS) crosslinking and antibody-
facilitated Foerster resonance excitation transmission
(FRET) (Figures S6 and S7). Binding to both the Rb pocket
and RbN has been noted for other ligands, including GRIP-
1, ASC-2, TAF1, and p202, and appears to be a common
theme among RbN-binding proteins (Batsche et al.,
2005; Choubey and Lengyel, 1995; Goo et al., 2004; Shao
et al., 1997).
To analyze the interaction between EID-1 and RbN, we
performed targeted mutagenesis of putative functional
surfaces of RbN. Specifically, we (1) mutated the con-
served residues Leu212, Val213, and Ile214 to affect
CP2, (2) generated a construct corresponding to the onco-
genic exon 5 deletion (aa 168–181) which compromises
the projection, and (3) replaced the Arg-rich linker (aa
250–263) with a 6 residue glycine-rich linker (polyG). CD
spectroscopy documented appropriate overall folding of
all mutants (Figure S8). Mutations in CP1 significantly im-
paired solubility (data not shown), suggesting that the
mutant proteins did not fold correctly, and precluded their
inclusion in the analysis.
Using GST affinity precipitation, we found that EID-1 re-
tained binding to RbN CP2 and exon 5 mutants, suggest-
ing that these features are not required (Figure 4C). In con-
trast, replacement of the Arg-rich linker led to complete
loss of binding, strongly implicating this feature in the in-
teraction with EID-1. Involvement of the adjacent cyclin
wedge was not confirmed, as targeted or tumor-derived
point mutations in this feature did not abrogate EID-1
binding (Figure S9A). However, like attempts to mutate
the LxCxE-binding cleft in the Rb pocket (Chan et al.,
2001; Dahiya et al., 2000; Dick et al., 2000), it is possible
Molecular Cell

Molecular Cell

that single-residue changes in the RbN cyclin wedge are
insufficient to abrogate ligand binding. Our results do,
however, show that the various cancer-associated mu-
tants tested do not target EID-1 binding.
As the Arg-rich linker encompasses Thr252 and Ser249,
two cdk phosphorylation sites, we asked whether phos-
phorylation might affect EID-1 binding to RbN. Using anti-
body-FRET, we found that phosphorylation by either
cyclin A/cdk2 or cyclin B/cdk1, which both modified the
respective sites, abrogated RbN interaction with EID-1,
revealing a mechanism of regulation by cdk phosphoryla-
tion and corroborating the involvement of the linker in EID-
1 binding (Figures 4D and 4D0
). The Arg-rich linker and cdk
phosphorylation sites in RbN are not conserved in the Rb
homologs p107 and p130, and a p130 N domain construct
did not bind EID-1 (Figure 4E). In contrast to RbN, phos-
phorylation of the Rb pocket did not affect its interaction
with EID-1 (Figure S10).
EID-1 Regions Interacting with Rb
To delineate the region(s) of EID-1 that interacts with Rb,
we probed an array of serially overlapping 25-mer pep-
tides of the entire EID-1 sequence (Figure 5A), generated
with SPOT technology (Frank and Overwin, 1996). EID-1
is a natively unfolded protein as verified by 1D NMR (Fig-
ure S11A) and therefore ideally suited for array analysis.
Probing with RbN revealed two discontinuous footprints
covering residues 142–169 and residues 152–180 of
EID-1, respectively (Figure 5A and Figure S11B). When
probed with the Arg-rich linker mutant (polyG), the second
footprint was absent, suggesting that residues within this
sequence facilitate interaction with the linker. The Rb
pocket displayed a different footprint, involving peptides
spanning the central and acidic regions of EID-1, and
a C-terminal cluster encompassing the EID-1 LxCxE motif,
consistent with previous suggestions (MacLellan et al.,
2000; Miyake et al., 2000) (Figure 5A). Notably, the RbN
footprints were not bound by the Rb pocket, showing
that the two regions engage nonoverlapping segments
of EID-1. Using a second peptide array, covering the two
RbN interacting footprints on EID-1, but with serial Ala
substitutions in single and multiple consecutive residues
(Figure 5A0
and Figure S11C), we defined a minimal motif
of Phe166 and Val170 (FxxxV), plus a triplet of residues
N-terminal to these (FIE), as essential for RbN interaction
(Figures S11B and S11C).
Competition experiments verified this site, with a pep-
tide spanning the FxxxV motif, but not an Ala-substituted
mutant (see Figure 5B), out-competing the interaction of
EID-1 with RbN (Figure 5C and Figure S12A). Consistent
with the selectivity of this motif, the FxxxV peptide did
not affect binding of EID-1 to the Rb pocket. Conversely,
a peptide spanning the LxCxE motif competed binding
of EID-1 to the Rb pocket, but did not affect its interaction
with RbN (Figure 5C0
and Figure S12B). Together these
results show that EID-1 utilizes nonoverlapping peptide
motifs to interact simultaneously with RbN, via the Arg-
rich linker, and with the Rb pocket, most probably via
the LxCxE-binding site.
The Rb Homotypic Interaction and Its Interplay
with EID-1
Data presented above indicate a homotypic interaction
between RbN and the remainder of the Rb protein,
RbDN. Using different Rb subconstructs, we identified
the central Rb pocket region (aa 379–792), excluding the
spacer located between the A and B subdomains, as
sufficient for this interaction (Figures 6A–6B0
and
Figure S13).
To determine if any of the potential interaction surfaces
of RbN are essential for binding to the Rb pocket, we
tested the available RbN mutants using GST affinity pre-
cipitations. All mutants bound the Rb pocket equally well
(Figure 6C and Figure S9B), including the Arg-rich linker
mutant, which is unable to bind EID-1 (Figure S9A). This
linker mutant was also defective in binding p84N5 and
GRIP-1 (Figure S14), indicating that these heterologous
ligands interact with RbN in a similar way, but dissimilar
to the Rb pocket. Furthermore, cdk phosphorylation of
RbN, which affected its ability to bind EID-1 (see
Figure 4D), did not affect its interaction with the Rb pocket,
corroborating the distinct nature of these interactions
and indicating that the RbN-Rb pocket interaction is not
regulated by phosphorylation of RbN (Figure 6D). Similar
Figure 4. RbN Interacts with EID-1
In vitro protein-binding assays with purified proteins. Immunoblots were probed with antibodies as indicated.
(A) RbN interacts with p84N5, GRIP-1, EID-1, and RbDN. Aliquots of RbN were incubated with equivalent amounts of GST-tagged proteins. GST and
GST-p300 Bromo are controls.
(B and B0
) EID-1 interacts independently with RbN and the Rb pocket. Aliquots of EID-1 were incubated with equivalent amounts of GST-tagged
proteins or GST as a negative control. (B0
) Schematic representation; dashed boxes indicate regions of interaction in Rb.
(C) The Arg-rich linker is required for EID-1 binding to RbN. Aliquots of Rb 40–355 WT, Rb 40–355 polyG, Rb 40–355 DEx5, and Rb 40–355 CP2 were
incubated with equivalent amounts of GST-EID-1.
(D) Effect of RbN phophorylation on EID-1 binding. Antibody-FRET-mediated complex detection. GST-RbN was phosphorylated by cyclin A/cdk2
(A/K2) or B/cdk1 (B/K1). Controls omitting enzyme or ATP were run in parallel. Assays used 2.5 nM GST-RbN and 300 nM HisTrx-EID-1. Data shown
are the net of background fluorescence transfer obtained with unfused HisTrx. The net background using HisTrx-EID-1 with GST instead of GST-RbN
is shown. Error bars represent standard deviation over four samples.
(D0
) Anti-phospho-Rb western blots. Rb preparations were analyzed by immunoblot using phosphorylation site-selective and pan-Rb antibodies as
indicated.
(E) p130 N-terminal domain does not interact with EID-1. Aliquots of EID-1 or Rb pocket-FLAG were incubated with equivalent amounts of GST-
tagged proteins, GST as a negative control, or GST-RbN as a positive control.
Molecular Cell

results were obtained for phosphorylation of the Rb pocket
and coordinate phosphorylation of RbN and the Rb pocket
(Figures S15 and S16), neither of which affected the inter-
action of these two parts of Rb with each other.
We also assessed the ability of the Rb pocket to asso-
ciate with RbN in the presence of EID-1 (Figure 6E and Fig-
ure S17). These experiments provided collective evidence
that EID-1 displaces RbN from the Rb pocket, in favor of
binary interactions with RbN and the Rb pocket, respec-
tively (Figure S17). Intriguingly, this displacement could
be recapitulated by the EID-1-derived LxCxE-containing
peptide (Figures 6F and 6F0
), but not by the FxxxV peptide,
which effectively competed for EID-1 binding to RbN (see
Figure 5C).
Figure 5. Separate EID-1 Regions Interact with RbN and the Rb Pocket
(A) A SPOT array of serially overlapping 25-mer EID-1 peptides was probed with RbN WT, RbN polyG, or Rb pocket. Peptide stretches of EID-1
interacting with various Rb constructs are indicated at the top right of individual panels.
(A0
) Identiﬁcation of EID-1 residues critical for interaction with RbN. A SPOT array of 30-mer EID-1 peptides (aa 139–168 [A1-C30] and aa 152–181
[D1-F30]) with serial Ala substitutions as indicated was probed with RbN WT. Single (rows A and D), double (B and E) or triple (C and F) substitutions
were used as indicated.
(B) Summary of EID-1 peptide interactions. Colored residues denote amino acids crucial for interaction with RbN. The EID-1 LxCxE motif is under-
lined. Peptide designs based on the SPOT array results, and used for further study, are indicated.
(C and C0
) Association between GST-RbN (C) and GST-Rb pocket (C0
) with EID-1 in the presence of various EID-1 peptides based on the SPOT assay
results. Afﬁnity precipitation with proteins was performed as indicated. RbN-interacting peptide (aa 144–176, FxxxV), RbN-interacting mutant peptide
(aa 144–176, Mutant), acidic stretch peptide (aa 86–107, Acidic), or LxCxE peptide (aa 172–187, LxCxE) was included in 30-fold molar excess over
GST-Rb and EID-1.
Molecular Cell

DISCUSSION
Previous structural work has concentrated on the Rb
pocket domain, which provides binding sites for two of
Rb’s best characterized protein ligands, the E2F-1 tran-
scription factor and the papillomavirus E7 oncoprotein
(Lee et al., 1998, 2002; Xiao et al., 2003). No experimental
analysis of the N terminus has previously been reported.
The structure presented here offers experimental insight
into the fold and surface properties of this part of the
retinoblastoma protein and provides a firm structural ba-
sis for dissecting its mode of action and biological sig-
nificance.
Contraryto bioinformaticsanalyses suggesting thepres-
ence of a BRCT domain (Bork et al., 1997; Yamane et al.,
2000), the crystal structure of RbN reveals an architecture
built around tandem cyclin folds, closely resembling that
of the Rb pocket domain. Initially identified in cyclin A
(Brown et al., 1995) and subsequently in the basal tran-
scription factor TFIIB (Nikolov et al., 1995), the cyclin fold
is a known scaffold for protein-protein interactions. How-
ever, whereas cyclinsandTFIIBcontain onetandem repeat
of this fold, Rb unusually contains not one but two tandem
cyclin repeats. Threading analysis (data not shown) shows
that this architecture is shared by the Rb-like pocket pro-
teins p107 and p130.
As in other characterized cyclin fold proteins, the tandem
RbN cyclin folds have diverged substantially. However,
structural alignment of the ‘‘B’’ folds from RbN and the
Rb pocket reveals a degree of identity between equivalent
residues (17.75%) comparable to that between the cyclin
folds within RbN itself (15.25%), cyclin A (15.63%), or TFIIB
(17%) (Noble et al., 1997) (Figure S2C). The identity be-
tween equivalent residues in the ‘‘A’’ folds of RbN and the
Rb pocket is much lower (6.78%), suggesting that conser-
vation in B folds does not just reflect common ancestry, but
indicates a common selective pressure to retain structural
and/or surface features. While most conserved residues in
theRbNB foldare inthecore,somearesurface accessible,
including several whose pocket equivalents participate in
binding LxCxE peptide, arguing for functional retention of
the corresponding surface in RbN.
As in other cyclin fold proteins, multiple binding sites for
ligands can be identified in the RbN structure. Two of
these are involved in lattice contacts in the crystal and
might play a role in oligomerization of Rb (Hensey et al.,
1994), although our own biochemical work with purified
recombinant RbN has provided no evidence for this.
Conservation and interactions with other proteins have
suggested the importance of RbN, while its biological
role has remained ambiguous. Previous efforts to assign
specific function to this region that employed scanning
deletion and insertion mutagenesis (Qian et al., 1992; Riley
et al., 1997) must be re-evaluated in light of the RbN crys-
tal structure, as mutants with loss of function in those
studies invariably disrupted the overall structure of RbN.
Our finding that structural disruption of RbN in vivo radi-
cally impacts on the stability of the entire protein resolves
the conundrum whereby mutation in RbN results in greater
loss of function than total ablation (Qian et al., 1992; Riley
et al., 1997; Yang et al., 2002).
The Arg-rich linker connecting cyclin fold helices 3 and 4
of the RbN B lobe is found to be essential for interaction
with several ligands, including the EID-1 inhibitor of
histone acetylation and differentiation and the previously
recognized Rb interacting proteins p84N5 and GRIP-1.
The requirement of this linker for binding different protein
ligands identifies it as a key structural element, and, to our
knowledge, the first in the RbN domain to which protein-
binding activity can be assigned. Its functional signifi-
cance is further supported by the presence of two cdk
phosphorylation sites within it, and we have shown that
phosphorylation of these sites abrogates these inter-
actions, providing mechanistic insight as to how phos-
phorylation within RbN may contribute to its functional
inactivation.
We have also identified an interaction between the RbN
and pocket regions, which together with the central posi-
tioning of the C terminus of RbN suggests a compact ar-
rangement of the Rb holoprotein with RbN and pocket in
direct contact. This differs from current models, in which
RbN and pocket are considered as essentially separate
structural and functional entities, loosely connected like
beads on a string. The precise topology of the interaction
and the relative juxtaposition of the RbN and pocket
regions is still unclear, although our data exclude involve-
ment of the Arg-rich linker. Disruption of the interaction by
the LxCxE-containing peptide of EID-1 is intriguing, as
binding of LxCxE ligands does not alter the conformation
of the Rb pocket (Lee et al., 1998), and suggests that
EID-1 acts by steric interference, implicating the LxCxE-
binding cleft, or a site nearby, in facilitating the interaction
with RbN. No LxCxE motif is present in RbN itself, exclud-
ing a canonical LxCxE engagement with the Rb pocket.
However, other types of interactions occur near or within
the LxCxE docking cleft, such as the phosphorylated Rb
C terminus, which is thought to engage via a cluster of ba-
sic residues lining the rim of the LxCxE-binding cleft (Lee
et al., 1998; Rubin et al., 2005), and the APC component
cdh1, whose binding is perturbed by mutations that also
disrupt LxCxE binding (Binne et al., 2007).
Our results indicate that the interaction between RbN
and the Rb pocket is not static, but can be modulated
by binding of protein ligands (see model; Figure 7). Indeed
Rb may exist in two opposing states: a ‘‘closed’’ confor-
mation in which RbN and the Rb pocket interact in close
proximity (Figure 7A) and an ‘‘open’’ conformation in
which this interaction is absent and proximity is lost (Fig-
ures 7B and 7C). Our data suggest that the switch from
closed to open is facilitated by recruitment of EID-1 to
the pocket, where it disrupts the RbN-Rb pocket inter-
action, probably by interfacing with the LxCxE-binding
surface (Figure 7C).
Although not addressed here, this raises the possibility
that other ligands might elicit similar conformational re-
sponses. These include LxCxE-containing ligands more
Molecular Cell

Molecular Cell

generally, but also the phosphorylated C-terminal region
of Rb itself, whose binding near the LxCxE-binding site
could functionally inactivate Rb partly by disrupting com-
posite surfaces formed by association of RbN and the
Rb pocket. Our observations may therefore represent
a general mechanism whereby ligands direct conforma-
tional changes in Rb that cause functional inactivation,
and/or orchestrate the assembly of specific Rb com-
plexes, and may provide a rational explanation for the
prevailing evidence that Rb participates in functionally dis-
tinct multiprotein complexes made from nonoverlapping
sets of Rb ligands.
EXPERIMENTAL PROCEDURES
Crystallization and Structure Determination
Purified RbN (aa 40–355) was partially digested with trypsin (Promega),
purified on a Resource Q, and concentrated to 8 mg/ml. Crystals
grown at 4
C by hanging drop vapor diffusion from mixtures containing
equal volumes of protein and reservoir solutions containing 0.2 M Na
acetate, 25% (w/v) PEG 4000, and 0.1 M Tris (pH 8.0) were flash frozen
in mother liquor made up to 25% (v/v) MPD. Diffraction data of SeMet
crystals were collected on ID29 and ID14 at ESRF and processed
using MOSFLM and SCALA (Leslie, 2006). SAD phases were calcu-
lated in SOLVE/RESOLVE (Terwilliger and Berendzen, 1999), and the
model was refined with REFMAC5 (Murshudov et al., 1997). Model
building used COOT (Emsley and Cowtan, 2004), and images were
Figure 6. The RbN Domain Interacts with the Rb Pocket
In vitro protein-binding assays. Immunoblots were probed with antibodies as indicated.
(A and A0
) RbN interacts with the Rb pocket (aa 379–792) and RbDN (aa 379–928). Aliquots of RbN were incubated with equivalent amounts of GST-
tagged Rb pocket or GST as a negative control. (A0
) Schematic. Dashed boxes indicate regions of interaction in the Rb large pocket.
(B and B0
) RbN interacts with the Rb pocket independently of the pocket spacer region (aa 580–640). (B) Aliquots of RbN were incubated with equiv-
alent amounts of GST-tagged proteins as indicated. (B0
) Aliquots of modified Rb pocket with the spacer removed by thrombin (D aa 589–636;
RbPDSp) were incubated with equivalent amounts of GST-tagged proteins as indicated.
(C) RbN does not use CP2, the projection, or the Arg-rich linker for its interaction with the Rb pocket. Aliquots of Rb 40–355 WT, Rb 40–355 CP2, Rb
40–355 DEx5, and Rb 40–355 polyG were incubated with equivalent amounts of GST-Rb 379–792.
(D) Effect of RbN phosphorylation on pocket binding. Antibody FRET and data presentation were done as for Figure 4 with phosphorylated RbN as
shown but FLAG-tagged Rb pocket in the place of EID-1.
(E–F0
) Association between RbN domain and pocket in the presence of EID-1. Affinity precipitation with proteins was performed as indicated. Full-
length EID-1 (aa 1–187) was added in equimolar amounts and at a 3-fold or 10-fold molar excess (E). EID-1 RbN-interacting peptide (aa 144–176,
FxxxV), EID-1 acidic stretch peptide (aa 86–107, Acidic), or EID-1 LxCxE peptide (aa 172–187, LxCxE) was included at a 30-fold molar excess (F).
LxCxE peptide was added at a 1-fold, 3-fold, 9-fold, or 27-fold molar excess (F0
).
Figure 7. Interaction with EID-1 Causes
Conformational Rearrangement of Rb
Model for ligand-induced conformational
response of Rb. (A) Full-length Rb can exist in
a conformation in which RbN and the pocket
directly interact. (B) Occupancy of the LxCxE-
binding site impairs association between RbN
and the Rb pocket resulting in the protein
adopting an open conformation. (C) Although
not experimentally tested here, phosphoryla-
tion of the Rb C terminus could have a similar
effect.
Molecular Cell

generated using PyMol (DeLano Scientific) and Chimera (Pettersen
et al., 2004). Crystallographic statistics are in Table 1. Details on other
methods can be found in the Supplemental Data.
Supplemental Data
Supplemental Data include Supplemental Experimental Procedures
and 17 figures and can be found at http://www.molecule.org/cgi/
content/full/28/3/371/DC1/.
ACKNOWLEDGMENTS
The work was supported by Cancer Research UK and the Institute of
Cancer Research. FSS was the recipient of a fellowship from Conseje-
rıá de Educacio´ n y Ciencia (Comunidad Valenciana) Spain. We thank
Angela Paul for mass spectrometry analysis, ESRF Grenoble for ac-
cess, Professor Dick Gordon for support and use of laboratory space,
David Komander and Mark Roe for assistance in data collection,
Richard Harris, Roger George, and John Ladbury for assistance in
NMR and CD spectroscopy, Feixia Chu for help with crosslinking
analysis, and Roger Ahern for statistics advice.
Received: August 9, 2006
Revised: May 21, 2007
Accepted: August 27, 2007
Published: November 8, 2007
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Accession Numbers
Coordinates and structure factors have been deposited in the Protein
Data Bank with PDB Code 2QDJ.
Molecular Cell

Crystal Structure of the Retinoblastoma Protein

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