A quantitative investigation of the interaction mechanism between the HPV16 E7 protein and the RbAB domain in solu-tion revealed that 90% of the binding energy is determined by the LxCxE
Trang 1suppressor by a prototypical viral oncoprotein
Structural modularity, intrinsic disorder and phosphorylation of human papillomavirus E7
Lucı´a B Chemes, Ignacio E Sa´nchez, Clara Smal and Gonzalo de Prat-Gay
Protein Structure-Function and Engineering Laboratory, Fundacio´n Instituto Leloir and IIBBA-CONICET, Buenos Aires, Argentina
Keywords
LxCxE motif; natively unfolded proteins;
phosphorylation; retinoblastoma protein;
viral oncoprotein
Correspondence
Gonzalo de Prat-Gay, Protein
Structure-Function and Engineering Laboratory,
Fundacio´n Instituto Leloir and
IIBBA-CONICET, Av Patricias Argentinas
435, 1405 Buenos Aires, Argentina
Fax: +54 11 5238 7501
Tel: +54 11 5238 7500 ext 3209
E-mail: gpg@leloir.org.ar
(Received 16 November 2009, revised 4
December 2009, accepted 7 December
2009)
doi:10.1111/j.1742-4658.2009.07540.x
DNA tumor viruses ensure genome amplification by hijacking the cellular replication machinery and forcing infected cells to enter the S phase The retinoblastoma (Rb) protein controls the G1⁄ S checkpoint, and is targeted
by several viral oncoproteins, among these the E7 protein from human papillomaviruses (HPVs) A quantitative investigation of the interaction mechanism between the HPV16 E7 protein and the RbAB domain in solu-tion revealed that 90% of the binding energy is determined by the LxCxE motif, with an additional binding determinant (1.0 kcalÆmol)1) located
in the C-terminal domain of E7, establishing a dual-contact mode The stoichiometry and subnanomolar affinity of E7 indicated that it can bind RbAB as a monomer The low-risk HPV11 E7 protein bound 2.0 kcalÆmol)1 more weakly than the high-risk HPV16 and HPV18 type counterparts, but the modularity and binding mode were conserved Phos-phorylation at a conserved casein kinase II site in the natively unfolded N-terminal domain of E7 affected the local conformation by increasing the polyproline II content and stabilizing an extended conformation, which allowed for a tighter interaction with the Rb protein Thus, the E7–RbAB interaction involves multiple motifs within the N-terminal domain of E7 and at least two conserved interaction surfaces in RbAB We discussed a mechanistic model of the interaction of the Rb protein with a viral target
in solution, integrated with structural data and the analysis of other cellu-lar and viral proteins, which provided information about the balance of interactions involving the Rb protein and how these determine the progres-sion into either the normal cell cycle or transformation
Structured digital abstract
l MINT-7383794 , MINT-7383812 , MINT-7383830 , MINT-7383868 , MINT-7383891 ,
MINT-7384056 : E7 (uniprotkb: P03129 ) and Rb (uniprotkb: P06400 ) bind ( MI:0407 ) by fluorescence technologies ( MI:0051 )
l MINT-7383923 : E7 (uniprotkb: P04020 ) and Rb (uniprotkb: P06400 ) bind ( MI:0407 ) by com-petition binding ( MI:0405 )
Abbreviations
AdE1A, adenovirus E1A; BPVN, N-terminal fragment of the BPV1 E7 protein; CKII, casein kinase II; CR1, conserved region 1; CR2,
conserved region 2; CtIP, transcriptional corepressor CtBP-interacting protein; E7(16-40)PP, a synthetic E7(16-40) peptide phosphorylated at serine residues 31 and 32; FITC, fluorescein isothiocyanate; GST, glutathione S-transferase; HDAC, histone deacetylase; HPV, human papillomavirus; IPTG, isopropyl thio-b- D -galactoside; MBP, maltose-binding protein; PII, polyproline type II; Rb, retinoblastoma; SV40LT, SV40 large T antigen; TA, transactivation region; TFE, 1,1,1, trifluoroethanol.
Trang 2The retinoblastoma tumor suppressor gene (RB1) was
first identified as the causative agent whose loss
resulted in retinoblastoma, a heritable disease of
pedi-atric relevance [1] To date, over 500 distinct
muta-tions in the RB1 gene have been identified in
retinoblastoma tumors, 50 of which are missense
mutations [2,3] The tumor suppressor function of the
Rb protein is underscored by its mutation in a broad
range of human tumors [4] The most extensively
studied function of the Rb protein is in the control
of cell cycle progression at the G1⁄ S boundary,
medi-ated through its interaction with the E2F family of
transcription factors [5] The Rb protein also plays
important roles in chromatin remodeling,
develop-ment, differentiation and apoptosis [6] These multiple
functions are mediated by over 100 interactions with
different protein partners that are dependent on the
cell type, and on the developmental and cell cycle
stages [7]
The Rb protein has a molecular mass of 105 kDa
and is composed of three domains Both the N-terminal
and the AB (RbAB) domains consist of a double cyclin
fold [8,9], while the C-terminal domain (RbC) appears
to be natively unfolded [10] The function of the
N-ter-minal domain is still poorly defined The RbAB domain
mediates transcriptional repression and, together with
the C-terminal domain (RbC), promotes growth arrest
[11,12] Most interacting partners contact more than
one structural domain in the Rb protein [13–15] For
example, the ‘transactivation’ domain of E2F (E2F-TA)
binds to RbAB, whereas the ‘marked box’ domain
(E2F-MB) binds to RbC [10,16] Moreover, there are at
least two distinct highly conserved ligand-binding sites
within the RbAB domain [8] (Figs 1 and S1) Cellular
proteins containing an LxCxE motif interact with a site
(Fig 1A,B) The E2F-TA domains bind to a site
located at the cleft between the A and B subdomains on
the opposite side of RbAB [16,18] (Fig 1C,D)
Early evidence for the tumor suppressor role of the
Rb protein came from the mechanism of action of the
human papillomavirus (HPV) E7 major transforming
protein [19] The interaction between E7 and the Rb protein is required for the induction and maintenance
of the transformed state of human keratinocytes [20] Deregulation of E7 expression upon integration of the HPV genome is believed to play a role in HPV-medi-ated oncogenesis The DNA tumor virus proteins SV40 large T antigen (SV40LT) and adenovirus E1A (AdE1A) also target the Rb protein and share sequence and functional conservation with the HPV E7 protein [21,22] E7, AdE1A and SV40LT each con-tain several functional and structural domains, each of which mediates interactions with different cellular tar-gets The three transforming proteins share conserved region 2 (CR2); E7 and AdE1A also share conserved region 1 (CR1)
E7 is a small ( 100 amino acids) protein composed
of two structural domains We have previously deter-mined that the N-terminal domain (E7N) is natively unfolded [23,24], includes CR1 and CR2, and contains dynamic elements of helical and polyproline type II (PII) secondary structure [23] The globular C-terminal domain (E7C) constitutes conserved region 3 (CR3) and is responsible for protein dimerization and zinc binding [24,25] (Fig 2A) While the CR1 and CR2 domains are required for Rb protein degradation, all conserved E7 regions participate in transformation [26,27] E7 can also oligomerize in vitro and in vivo [28–30] The conformational diversity of E7 may be an evolved trait that allows for multiple modes of pro-tein–protein interaction [31,32]
E7 binds to two structural domains in the Rb protein, namely the RbAB and RbC domains Binding
to both domains is required for E2F displacement [33] The LxCxE motif within the CR2 region of E7 mediates high-affinity binding to the RbAB domain [8,34] (Fig 1A), while the isolated E7C binds to the RbC domain with micromolar affinity [25,35] The crystal structure of the LxCxE–RbAB complex reveals that the motif binds to a conserved shallow groove of the B subdomain in an extended conformation (Fig 1A) The LxCxE motif is followed in E7 CR2 by two conserved serine residues (S31 and S32) and by a
l MINT-7383777 , MINT-7384078 , MINT-7383848 , MINT-7384113 , MINT-7384096 : Rb (uni-protkb: P06400 ) and E7 (uniprotkb: P03129 ) bind ( MI:0407 ) by competition binding ( MI:0405 )
l MINT-7383963 : Rb (uniprotkb: P06400 ) and E7 (uniprotkb: P06788 ) bind ( MI:0407 ) by com-petition binding ( MI:0405 )
l MINT-7384022 , MINT-7384040 : E7 (uniprotkb: P03129 ) and Rb (uniprotkb: P06400 ) bind ( MI:0407 ) by comigration in non denaturing gel electrophoresis ( MI:0404 )
l MINT-7384004 , MINT-7383984 : Rb (uniprotkb: P06400 ) binds ( MI:0407 ) to E7 (uni-protkb: P03129 ) by pull down ( MI:0096 )
Trang 3stretch of acidic amino acids, and HPV16 E7 is phosphorylated at S31 and S32 by casein kinase II (CKII) in vitro and in vivo [36,37] Phosphorylation is required for E7 function, and cell culture assays have suggested that phosphorylation modulates the strength
of the E7–RbAB interaction, but this proposal remains
a matter of debate [37–40]
Indirect evidence suggests that other regions in E7 may contribute to binding to the RbAB domain For example, mutagenesis of a conserved surface patch in the A subdomain of the RbAB domain (Fig 1A,B, right) produces a protein capable of arresting the cell cycle of HeLa cells, implying that this protein was resis-tant to E7 inactivation [41] It is currently unclear whether E7 interacts directly with this surface Similarly,
an E7 construct, encompassing the CR2 and CR3 domains of E7, bound to the RbAB domain more tightly than a CR2 construct and was able to debilitate the E2F–RbAB interaction [16] Finally, E7 CR1 has been shown to contribute to E2F displacement in com-bination with CR2 [27] This E7 region shares a high degree of sequence similarity to the AdE1A CR1 region and can functionally complement it [42] The AdE1A CR1 region binds to the RbAB domain at a site that overlaps with the E2F-TA-binding site [43] (Fig 1D), leading to disruption of the E2F–Rb complex, but an interaction between E7 CR1 and the RbAB domain has not been demonstrated to date
Mechanistic aspects and structure–function relation-ships for the Rb protein remain ill defined [17], in con-trast to those for other well-known tumor suppressors
or oncogenes, such as p53 [44] or Ras [45] A complete understanding of the Rb protein function requires the dissection of all functional surfaces, along with their partners and the strength and mechanism of interac-tion [46] We have dissected individual contact sites and their energetic contribution to the E7–RbAB com-plex, using solution-based measurements of binding affinity at equilibrium This mechanistic and thermody-namic picture of the complex formed by RbAB and E7 paves the way for a better understanding of the Rb cellular complexes that control the cell cycle through-out eukaryotes and their deregulation in HPV infection and oncogenesis
Results
Quantitative dissection of the E7–RbAB interaction in solution
The minimal region required for the interaction between the HPV16 E7 protein and the RbAB pocket has previously been mapped to residues 21-29 of E7,
A
B
C
D
Fig 1 Conserved surface features of the RbAB domain
Conserva-tion scores were calculated using the alignment of the RbAB domain
from 46 vertebrate species and CONSURF [74], and figures were
gen-erated using PYMOL [75] Structures correspond to the following
com-plexes: (A) RbAB ⁄ E7 (PDB ID: 1GUX); (B) RbAB ⁄ SV40-LT (PDB ID:
1GH6); (C) RbAB ⁄ E2F-TA (PDB ID: 1N4M); and (D) RbAB ⁄ E1A-CR1
(PDB ID: 2R7G) Asterisk: H549Y missense mutation [54] Arrows
indicate the rotation of the molecule along the x-axis between two
consecutive images The color scale indicates the residue
conserva-tion score, as calculated using the CONSURF algorithm.
Trang 4containing the LxCxE motif [8] The dissociation
con-stant for this interaction was shown, by isothermal
titration calorimetry, to be 190 nm [8,34], but the
con-tribution of other regions of the E7 protein to the
affinity of the E7–RbAB complex has not been
explored in detail In order to address this issue, we
developed a solution-based assay that allowed us to
perform quantitative and accurate determinations of
stoichiometry and binding affinity at equilibrium, by
measuring the fluorescence anisotropy change upon the
binding of fluorescein isothiocyanate (FITC)-labeled
E7 fragments to RbAB This assay was used to
mea-sure the binding of different fragments of E7
(corre-sponding to well-defined structural and functional
domains and to highly conserved sequence motifs) to
RbAB Figure 2A shows the E7 regions tested
A representative example of the assay is presented in
Fig 2B,C, which show the association of E7N
[E7(1-40)] with RbAB First, the stoichiometry of the
reac-tions was determined by performing titrareac-tions at a
high peptide concentration (Figs 2B and S2) The
anisotropy signal increased linearly up to a 1 : 1 molar
ratio, where it reached a constant value indicating the
saturation of all binding sites This implies that there
is one binding site for the E7(1-40) peptide per RbAB monomer and that the stoichiometry of the E7(1-40)– RbAB interaction is 1 : 1 Far-UV CD spectra of the complexes formed by binding of the RbAB domain to full-length E7, and to E7(1-40) and E7(40-98) peptides, revealed that formation of the complex does not induce significant structural changes in the secondary structure of the interacting proteins (data not shown) Figure 2C shows one representative binding curve per-formed at substoichiometric concentrations, and the residuals of the fit from which the KDvalue was calcu-lated We tested the association of the RbAB domain with a 43-residue N-terminal fragment of the BPV1 E7 protein (BPVN), which does not contain an LxCxE motif This interaction had marginal affinity, which was approximately 106 times lower than that of the full-length E7 protein (Table 1) Figure 2D summarizes all binding curves and shows the dynamic range of the assay, which allowed us to accurately determine subn-anomolar to micromolar dissociation constants The E7(21-29) peptide, comprising the minimal LxCxE motif (DLYCYEQLN) [8], associated with RbAB with a KD of 4.7 ± 1.7 nm (Table 1), and the free energy of binding for this interaction was DG =
A
Fig 2 Interaction of different E7 fragments with the RbAB domain (A) Scheme of HPV16 E7 The positions of conserved regions 1, 2 and
3 (CR1, CR2 and CR3) and the E7 fragments used in this study are shown; the LxCxE motif is underlined Boxes denote the regions con-tained in each fragment: black, LxCxE motif; dark grey, CKII ⁄ PEST motif; light grey, CR1 helix-forming residues Circles denote the position
of FITC moieties (B) Association of E7(1-40) and RbAB at 200 n M E7(1-40) (C) Association of E7(1-40) and the RbAB domain at 5 n M E7(1-40) A fit to a 1 : 1 binding model and residuals are shown The anisotropy value of the free peptide was 0.054 ± 0.001 and the anisotropy
of the complex was 0.124 ± 0.001, indicating that no oligomerization occurred in this binding regime [76] (D) Representative normalized binding curves for the different E7 fragments (symbols are as shown in panel A).
Trang 5)11.2 ± 0.2 kcalÆmol)1 The E7(16-31) and E7(16-40)
peptides, which contain the LxCxE motif plus
addi-tional neighboring sequences from the CR2 region,
and the E7(1-40) peptide, which comprises both CR1
and CR2, had the same affinity for the RbAB domain
as the E7(21-29) peptide (Table 1) The full-length
HPV16 E7 protein bound to the RbAB domain with a
ten-fold increased affinity when compared with the
E7(21-29) peptide (KD= 0.6 ± 0.3 nm), and the free
energy of binding for the interaction between
full-length E7 and the RbAB domain was DG =)12.4 ±
0.3 kcalÆmol)1 (Table 1) Therefore, our data show
that the LxCxE motif contributes about 90% of the
total binding energy for the HPV16 E7–RbAB
interac-tion, providing quantitative support to previous results
[47] The CR1 region does not appear to contribute to
RbAB binding within the context of an E7N
mono-mer, as shown by the fact that the E7(16-40) and
E7(1-40) peptides have the same affinity for the RbAB
domain (Table 1) Finally, we showed that the E7
C-terminal domain contributes 1.0 ± 0.4 kcalÆmol)1to
the total free energy of binding, enhancing the affinity
of the E7–RbAB complex by ten-fold
Previous semiquantitative assays have established
that E7 proteins from HPV types highly associated
with the development of cervical cancer (HPV16 and
HPV18) bind to the full-length Rb protein more
strongly than E7 proteins from HPV types associated
with benign lesions (HPV11 and HPV6) [48] In order
to explore whether similar regions determine the
affinity for RbAB in E7 proteins from high-risk and
low-risk HPV types, we used a competition assay to
measure the association between the RbAB domain
and the E7 proteins from HPV types 16, 18 and 11
We assembled a stoichiometric complex of RbAB and FITC-labeled HPV16 E7 or E7(16-31) and displaced labeled E7 with each of the different full-length pro-teins or N-terminal domains (Fig 3A and Table 2) The HPV11 E7 protein associated with the RbAB domain 2.0 kcalÆmol)1 more weakly than the high-risk HPV16 and HPV18 type counterparts, providing quan-titative support to previous reports [48] The N-termi-nal domain was the main contributor to the binding affinity of E7 from HPV11, HPV16 and HPV18 for the RbAB domain (Fig 3B), pointing to a conserved mode of interaction
Phosphorylation of the conserved CKII sites within the E7 CR2 region increases affinity for RbAB
The sequences C-terminal to the LxCxE motif in HPV16 E7 contain two serine residues, S31 and S32, which are phosphorylated in vitro and in vivo by CKII [36,37] These serine residues are followed by a stretch
of acidic amino acids that constitute an S⁄ TxxD ⁄ E CKII consensus site The PESTfind algorithm suggests
Table 1 Determination of binding affinities for the E7–RbAB
complex The K D was calculated by fitting three to five independent
binding curves to a 1 : 1 binding model, as described in the Materials
and methods.
E7(16-40)PP (CR2PP) b 1.8 ± 0.4 )11.7 ± 0.1
a DG was calculated as DG = )RT *ln(K D ), with RT = 0.582
kcalÆmol)1. bThe stoichiometry for these complexes was
deter-mined to be 1 : 1 by titrations performed at peptide concentrations
at least 10 times greater than the determined KD.
A
B
Fig 3 The LxCxE motif is the main determinant of binding affinity
in HPV-E7 proteins (A) Competition experiments with full-length E7 proteins and a preformed complex of 5 n M RbAB and 5 n M
FITC-HPV16-E7 protein Competitor proteins were: BPV-Nter (s); HPV11-E7 ( ); HPV18-E7 ( ); and HPV16-E7 (d) (B) Comparison
of DG values for different E7 full-length proteins (solid bars) and N-terminal domains (hatched bars) Data are from Table 2.
Trang 6that this site overlaps with a PEST degradation motif
[49] Figure 4A shows the sequence of the HPV16 E7
CR2 region, indicating the relative positions of the
LxCxE motif, the phosphorylatable serine residues and
the CKII⁄ PEST region within CR2 Aligned below this
sequence is a sequence logo created from the alignment
of all 56 E7 proteins from genital HPV types (Fig S3)
The sequence logo clearly shows that serine residues
are nearly as conserved as the LxCxE motif Inspection
of individual sequences revealed that all 56 E7 proteins
present at least one CKII consensus site between positions 30 and 34 This region also contained a high proportion of negatively charged amino acids (D⁄ E), with 97% of sequences presenting a net charge that was equal to or lower than -6
The striking conservation of sequence features within the CR2 region of E7 underscores the impor-tance of this region for E7-mediated transformation The CKII⁄ PEST region of E7 and its phosphorylation have been postulated to play a role in the E7–Rb protein interaction Here, we directly tested this hypothesis by comparing binding to the RbAB domain for E7(16-40) and for a synthetic E7(16-40) peptide phosphorylated at serine residues 31 and 32 [E7(16-40)PP] Phosphorylation increased the affinity four-fold (Table 1) The difference in free energy of binding
of both peptides, DDG =)0.7 ± 0.3 kcalÆmol)1, was significant across repeated assays We further validated the data by carrying out competition experiments, where a stoichiometric complex of FITC-labeled E7(16-31) and the RbAB domain was titrated with increasing amounts of unlabeled 40) or E7(16-40)PP peptides Competition experiments (Fig 4B,C) confirmed a positive contribution of phosphorylation
to RbAB-binding affinity The difference in free
A
Fig 4 Phosphorylation of the E7 CR2 region increases the affinity for the RbAB domain (A) Conservation of sequence features within E7 CR2 Upper panel: sequence of the HPV16 E7(16-40) peptide The LxCxE motif is underlined, and the position of phosphoryl serine residues and the CKII ⁄ PEST consensus are marked Lower panel: sequence logo of the CR2 region from genital E7 proteins The height of the stack
of letters at each position denotes the level of conservation (the maximum value is 4.32), while the relative proportions of each residue rep-resents the relative abundance (B) Competition experiments with CR2 peptides and a preformed complex of 25 n M RbAB and the 25 n M
FITC–E7(16-31) peptide Competitor peptides were: E7(16-31) (d), E7(16-40) (.), E7(16-40)PP (s) and BPVN ()) (C) Comparison of DG val-ues for the E7(16-40) and E7(16-40)PP peptides with those for the E7(16-31) peptide Data are from Table 1 and from panel B.
Table 2 The LxCxE motif determines binding affinity in distantly
related HPV E7 proteins.
Fragment K D (n M ) DGa(kcalÆmol)1) DDGb(kcalÆmol)1)
Full-length protein
HPV18 E7 7.8 ± 0.5 )10.9 ± 0.04 0.7 ± 0.06
N-terminal domain
HPV18 E7 12.2 ± 0.8 )10.6 ± 0.04 0.2 ± 0.1
HPV11 E7 366 ± 25 )8.6 ± 0.04 2.2 ± 0.1
a DG = )RT*ln(K D ), with RT = 0.582 kcalÆmol)1 b DDG was
calcu-lated as DDG = DG ) DG E716
Trang 7energy of binding from competition experiments was
DDG =)1.4 ± 0.2 kcalÆmol)1, in agreement with
the direct binding data Our data demonstrated that
phosphorylation of the CKII⁄ PEST region contributes
significantly to the RbAB–E7 interaction, enhancing
the affinity by fourfold to 10-fold
Structural correlates of E7 phosphorylation at the
CKII sites
We have previously shown that E7N is an extended bona
fidestructural domain, with regions of dynamic residual
secondary structure in solution Far-UV CD analyses
showed that HPV16 E7(1-40) displayed an extended PII
structure, which was stabilized by phosphorylation of
serine residues S31 and S32 [23] We tested the E7 CR2
region for PII content by measuring the far-UV CD
spectra of the E7(16-40) and the E7(16-40)PP peptides
at 5C Both peptides presented a CD spectrum
charac-teristic for a disordered polypeptide with a positive band
at 218 nm, which is characteristic of the PII
conforma-tion (Fig 5A) PII conformaconforma-tions are sensitive to
tem-perature, with higher temperatures decreasing the
intensity of the 218 and 198 nm peaks Increasing the
temperature to 85C decreased the intensity of both
peaks for both peptides, characteristic for the disruption
of the PII structure (Fig 5A) The difference spectra (5–
85C) clearly showed the induction at 5 C of the
218 nm peak (Fig 5A, inset) The denaturant GdmCl is
known to stabilize PII structures [50] We have
previ-ously shown that the stability of PII conformations can
be estimated from GdmCl titrations, by validating
changes in the CD spectra with NMR measurements of
PII structure [51] GdmCl increased the 218 nm band in
the E7(16-40)PP peptide, but not in the E7(16-40)
pep-tide (Fig 5B), suggesting that the E7(16-40)PP peppep-tide
has a higher propensity for PII structure The titration
of the E7(16-40)PP peptide with GdmCl is shown in Fig 5C, along with a fit of the data to a two-state coil-PII model The calculated free energy for the coil-coil-PII equilibrium in 0 m GdmCl is 1.7 ± 0.7 kcalÆmol)1, which corresponds to 4.6 ± 6% of the PII population
in the absence of denaturant Although the model used
is a crude estimate of the true conformational equilibria
of the peptide, and the estimated parameters have high errors as a result of noise in the measurements, the GdmCl titration data clearly show that the E7(16-40)PP peptide is in equilibrium between coil and PII conforma-tions Overall, our data indicate that both peptides from the HPV16 E7 CR2 region present residual PII structure
in equilibrium with disordered conformations GdmCl titrations strongly suggest that phosphorylation modu-lates the coil–PII equilibrium, increasing the PII propen-sity of the E7 CR2 region
The E7 C-terminal domain binds independently
to RbAB The increased affinity of the full-length E7 protein compared with the E7 N-terminal domain suggested that additional regions within the E7 C-terminal domain contribute to association with the RbAB domain In order to test for a direct interaction between E7C and RbAB, we performed a pull-down assay with recombinant purified proteins by forming a stoichiometric complex of His-tagged RbAB with E7 and E7C (Fig 6A) Most of the full-length E7 protein (96%), and a fraction of the E7 C-terminal domain (23%), bound to RbAB at a concentration of 10 lm These results confirmed a direct association of the RbAB domain with both E7 and E7C, and suggested that the E7C–RbAB interaction was weaker than the
Fig 5 Phosphorylation increases the PII content of the E7 CR2 region (A) Far-UV CD spectra of the 40) (solid line) and the E7(16-40)PP (dashed line) peptides, performed at 5 C and 85 C Inset: difference spectra (5–85 C) for the 40) (solid line) and the E7(16-40)PP (dashed line) peptides (B) CD spectra of E7(16-40) and E7(16-E7(16-40)PP between 0 and 6 M GdmCl Titration points graphed are [GdmCl] = 0, 1.2, 1.9, 2.4, 3.2, 3.7, 4.9 and 5.9 M The curves corresponding to 0 and 5.9 M GdmCl are shown in bold (C) GdmCl titration of the E7(16-40)PP peptide Data were fit to a two-state coil-PII equilibrium (DG H2O
E7(16-40)PP = 1.7 ± 0.7 kcalÆmol)1; m = 0.44 ± 0.22 kcal mol)1Æ M )1).
Trang 8interaction of the full-length E7 protein with the
RbAB domain Direct titration showed that the E7C–
RbAB complex had a dissociation constant of
2.7 ± 0.6 lm (Table 1, Fig 6B) Titration with BPVN
E7 yielded a dissociation constant of 400 lm or higher,
supporting the specificity of the E7C–RbAB
interac-tion A peptide containing the CR2 region of E7 did
not compete with E7C binding, indicating that E7C
does not bind to the RbAB domain at the
LxCxE-binding cleft (Table 1, Fig 6B)
The E7 CR1 region can form an alpha helix and
binds independently to RbAB
The CR1 region from E1A binds to the RbAB domain
with micromolar affinity (KD= 1 lm) [43] at the
interface between the A and B subdomains, which is also the binding site for E2F-TA (Fig 1C,D) The fact that the E7 and AdE1A CR1 regions have similar functional properties [42] suggests that E7 CR1 might also bind to the RbAB domain at the E2F-TA-binding site
E1A CR1 and E2F-TA form a six-residue helix in the bound conformation (Fig 1C, residues boxed in Fig 7A) [16,18,43] Four AdE1A residues that estab-lish intermolecular contacts with the RbAB domain (P41, L43, H44 and L49), and two residues that stabi-lize the helix by an intramolecular hydrogen bond (T42 and E45) [43], are conserved in E7 CR1 (E7 residues 6-10 and 15; Fig 7A) Furthermore, the AGADIR algorithm [52] suggests that E7 residues 6 to
15 have local helical propensity (data not shown) We tested whether the E7 CR1 region could form an a-helix in solution by measuring the far-UV CD spec-trum of E7(1-20) in the presence of 1,1,1, trifluoroetha-nol (TFE), which is known to stabilize helical conformations in peptides [53] The addition of 60% TFE induced an a-helix structure in E7(1-20) (Fig 7B and inset) A fit of the TFE titration data to a two-state coil-helix model yielded a free energy for a-helix formation in 0% TFE of 1.3 ± 0.2 kcalÆmol)1, corre-sponding to a residual a-helix population of 10 ± 4%
in the absence of cosolvent These results show that the E7 and E1A CR1 regions have similar conforma-tional properties
We tested for the association between E7 CR1 and the RbAB domain using three different approaches First, we used nondenaturing PAGE and FITC-labeled E7 peptides to test for complex formation (Fig 7C)
As a positive control, we tested the association
domain, and as a test for the specificity of the interac-tion, we used ovalbumin in place of RbAB Both E7(1-40) and E7(1-20) formed a complex with RbAB but not with the control protein ovalbumin, confirming the specificity of the interactions (Fig 7C) A pull-down assay, similar to that performed with E7C, did not show significant interaction (data not shown), sug-gesting that the E7(1-20)–RbAB complex has a lower affinity than the E7C–RbAB complex Fluorescence titration gives a dissociation constant of 19 ± 1 lm
Table 1) Titration with BPVN yielded a dissociation constant of 400 lm or higher The 20-fold higher affin-ity for the E7(1-20)–RbAB complex supports the speci-ficity of the interaction Peptides containing the CR2 region did not compete for the E7(1-20)–RbAB inter-action (Fig 7C), which indicates that E7(1-20) does not bind RbAB at the LxCxE-binding site
A
B
Fig 6 E7C binds independently to the RbAB domain (A)
Pull-down assay for the RbAB–E7C interaction His-RbAB was
incu-bated with E7 (lanes 3-4) or with E7C (lanes 7-8) Lanes 1-2 and
5-6: control experiments excluding His-RbAB The labels to the left
of the gel indicate the position of each protein % E7: percentage
of E7 or E7C protein in the bound (B) and unbound (U) fractions, as
quantified by densitometry (see the Materials and methods) (B)
Binding of E7C to the RbAB domain in solution Titrations were
per-formed at 1 l M FITC-E7C; the titrant was RbAB (d, KD= 4.8 ± 0.5
l M ), RbAB-E7(16-40) (s, K D = 6.4 ± 0.9 l M ) A control experiment
was performed using 5 l M FITC-BPVN (4, K D > 400 l M ).
Trang 9Despite its vast importance as the guardian of the cell cycle and its clinical relevance in human cancers, struc-tural and thermodynamic understanding of the mecha-nisms of action of the Rb protein is far behind that of p53, the keeper of the genome, mutated in most can-cers and targeted by the same DNA tumor viruses that target the Rb protein [44] In this work, we set out to investigate the interaction mechanism of the RbAB pocket domain with one of the paradigmatic viral oncoproteins, HPV E7, which targets it for degrada-tion Precise quantitative assessment of Rb protein interactions is fundamental for understanding viral-mediated subversion of cell cycle control and allows novel shared features of viral and cellular Rb protein interaction partners to be uncovered
We measured the contribution of the LxCxE motif
of E7 to be 90% of the total binding free-energy, and showed that this motif is also the main determinant of binding for E7 proteins from three prototypical HPV types (Figs 2 and 3) The free energy of binding for full-length HPV16 E7 was 1.0 kcalÆmol)1 higher than that of the E7N domain, revealing that the E7C domain contributes a 10-fold increase in affinity through a dual-contact mode of interaction Careful examination of conserved surface patches in the RbAB domain suggests a putative binding site for E7C, located in the RbA domain close to the AB cleft (Rb residues E492, F514, P515, K548 and H549; Fig 1A,B, right) This site is nearly as conserved as the LxCxE cleft, the lysine-rich patch and the E2F-binding site [8], and a tumorigenic missense mutation, H549Y, has been described at this surface (Fig 1A,B, asterisk) [3,54], which strongly suggests that this is an important functional surface in the RbAB domain for which cellular binding partners are likely to be described in the future [7] Mutations in this region affect cell cycle regulation by E7 [41], suggesting that E7 may bind at this interaction site and displace Rb protein cellular targets
The viral transforming proteins AdE1A and SV40LT, in addition to nine cellular protein targets of
Rb [17] [histone deacetylase (HDAC)1, HDAC2, tran-scriptional corepressor CtBP-interacting protein (CtIP), 95kDa retinoblastoma-associated protein (RBP95), ETS-related transcription factor 1 (Elf1), HMG Box transcription factor 1 (HBP1), kinetochore protein Hec1 (Hec1), RBP1 and replication factor C subunit 1 (RFC1)], present a putative serine-phosphorylation site following the LxCxE motif (Fig 8) In addition, in vivo phosphorylation of the AdE1A, SV40-LT and HDAC sites has functional consequences [55–57] In HPV E7,
A
B
C
D
Fig 7 The E7 CR1 region forms an a-helix and interacts with the
RbAB domain (A) Alignment of the E7 CR1 region with the E1A CR1
and E2F1-TA RbAB-binding sites Bold: residues in AdE1A involved in
complex formation with RbAB and conserved in E7 Asterisks:
resi-dues of E1A and E2F1 involved in the RbAB-binding a-helix [16,43].
(B) TFE titration of the E7(1-20) peptide Data were fit to a
two-state helix-coil transition model (DGH2O= 1.3 ± 0.2 kcalÆmol)1;
m = 25 ± 3 kcal mol)1Æ M )1) Inset: difference spectrum (60–0% TFE)
showing the conformation induced by TFE addition (C) Interaction
between E7(1-20) and the RbAB domain, determined using
nondena-turing PAGE Arrows mark the position of peptides⁄ complexes:
1 = free peptide, 2 = E7(1-40)–RbAB complex, 3 = E7(1-20)–RbAB
complex (D) Interaction between E7(1-20) and RbAB in solution.
Experiments were performed at 5 l M E7(1-20) The titrants
were RbAB (d, KD= 19 ± 1 l M ; Table 1), RbAB–E7(16-40) ( , KD=
26 ± 1 l M ) and RbAB–E7(1-40) (h, K D = 30 ± 2 l M ) A control
exper-iment was performed using 5 l M FITC-BPVN (4, K D > 400 l M ).
Trang 10phosphorylation is essential for S-phase re-entry of
dif-ferentiating keratinocytes in organotypic raft models
[39,40] and contributes to E7-mediated transformation
[37] Our results offer the first molecular insight into the functional role of E7 phosphorylation, by provid-ing direct evidence that phosphorylation of serines 31 and 32 of HPV16 E7 increases affinity for the RbAB domain (Fig 4) The E7 region surrounding these resi-dues is natively unfolded [23] and presents a high den-sity of negative charge, which may interact with a conserved lysine-rich patch contiguous to the LxCxE cleft [41,58] We showed that phosphorylation affects the local conformation of the E7(16-40) fragment, increasing the PII content of this region and stabilizing
an extended conformation that optimizes binding to the LxCxE cleft (Fig 5) PII-coil transitions induced
by phosphorylation in a similar natively unfolded PEST region can modulate the stability of a protein to intracellular degradation [51], which could also be the case for the E7 oncoprotein
The isolated E7 CR1 region is able to bind to the RbAB domain in vitro with measurable affinity, pos-sibly undergoing a coil-to-helix transition (Fig 7) In the AdE1A protein, the 70-residue spacer between the CR1 and CR2 regions allows for the simulta-neous binding of both motifs at opposite sides of the same RbAB molecule (Fig 1A,D) [59] Our data clearly show that the E7(16-40) and E7(1-40) pep-tides have the same affinity for the RbAB domain (Table 1) This result implies that the HPV E7 CR1 region does not contribute to binding when CR2 is present, which is probably because of the short eight-residue spacer separating both binding motifs
In a complex between the Rb protein and the weak E7 dimer [29], the CR2 region of one E7 molecule may bind to the LxCxE cleft, while the CR1 region
of the other E7 molecule binds to the E2F site of an RbAB monomer This mode of interaction may cooperate in the displacement of E2F, as previously suggested [25,27]
Our results highlight the modular nature of E7 and its interaction with the RbAB domain (Fig 8, top) It has long been recognized that AdE1A and SV40LT also present multiple interaction modules that bind to different Rb protein domains [21,22,59] This is also a feature of prototypical Rb protein interacting part-ners, such as E2F1, HDAC, CtIP and EP300 interact-ing inhibitor of differentiation 1 (EID-1) (Fig 8, bottom) The three secondary sites in E7 (E7C, CR1 and the CKII⁄ PEST region) contribute far less than expected from their binding energy in isolation (this work), which suggests that their main role is to finely tune affinity and to target multiple interaction sur-faces of the RbAB domain It will be interesting to investigate how the action of these modules is inte-grated with other known E7 interaction sites within
Fig 8 Interaction modules and affinities of viral and cellular Rb
protein targets Proteins and affinities reported are from: HPV-E7
[8,25,34,62] and, from this work, AdE1A [15,43,57], SV40LT
[22,34,56], E2F1 [10,16,18], HDAC [14,34,55], CtIP [13,77] and
EID-1 [9,78,79] The interaction sites in each protein are marked as
boxes Linear motifs are marked in color: red (LxCxE motif), dark
blue (CKII site), light blue [cyclin-dependent kinase phosphorylation
(Cdk) site], orange (phosphorylatable serine residues), green (helix
motif), violet (PENF motif) and yellow (FxxxV motif) Dark grey,
interactions mediated by globular domains; light grey, interactions
at unknown sites Structural domains are indicated above each
car-toon, and the Rb domains targeted, and the affinities, are indicated
below each site When known, the affinities of the full-length
pro-teins and the effects of phosphorylation are indicated.