Identification of an
Helical Motif Sufficient for
Association with Papillomavirus E6*
Jason J.
Chen
§,
Yihui
Hong
,
Edward
Rustamzadeh¶,
James D.
Baleja¶, and
Elliot J.
Androphy
**
From the
Department of Dermatology, New England
Medical Center and Tufts University School of Medicine,
¶ Department of Biochemistry, Tufts University School of Medicine,
and
Department of Molecular Biology and Microbiology, Tufts
University School of Medicine, Boston, Massachusetts 02111
 |
ABSTRACT |
We recently identified a cellular protein named
E6BP or ERC-55 that binds cancer-related papillomavirus E6 proteins
(Chen, J. J., Reid, C. E., Band, V., and Androphy, E. J. (1995) Science 269, 529-531). By construction of a series
of deletion mutants, the region of E6BP that is necessary and
sufficient for complex formation with human papillomavirus type 16 E6
has been mapped to a 25-amino acid domain. The corresponding peptide
was synthesized and found by nuclear magnetic resonance spectroscopy to
bind calcium and fold into a classical helix-loop-helix EF-hand
conformation. Additional deletion mutagenesis showed that 13 amino
acids that form the second
helix mediated E6 association. Alanine
replacement mutagenesis indicated that amino acids of this helix were
most important for E6 binding. Alignment of this
helical E6 binding peptide with the 18-amino acid E6 binding region of E6AP (Huibregtse, J. M., Scheffner, M., and Howley, P. M. (1993) Mol.
Cell. Biol. 13, 4918-4927) and the first LD repeat of another
E6-binding protein, paxillin (Tong, X., and Howley, P. M. (1997)
J. Biol. Chem. 272, 33373-33376), revealed
substantial similarities among these E6 binding domains. The extent of
homology and the mutational data define the peptide as an E6 binding
motif.
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INTRODUCTION |
Papillomaviruses (PV)1
are small DNA viruses that infect various epithelial tissues, including
the epidermis and the epithelial linings of the anogenital tract. Human
papillomaviruses (HPVs) that infect the anogenital tract can be
classified as either high or low risk. The high risk HPV types, of
which HPV type 16 (HPV-16) is the prototype, are strongly associated
with the potential for development of cervical carcinoma (for review,
see Ref. 4). Low risk types are also found in genital and cervical
papillomavirus infections but rarely progress to cancer.
The transforming properties of HPVs reside in two genes, E6
and E7. The E6 and E7 genes are
consistently expressed in HPV-positive cervical cancers and derived
cell lines (5-7). They cooperate to immortalize primary human
keratinocytes (8-13). HPV-16 E6 also cooperates with activated Ras in
the transformation and immortalization of baby mouse kidney cells and
baby rat kidney cells (14, 15). Independently of E7 or ras,
HPV-16 E6 can transform NIH 3T3 cells (16), immortalize human mammary
epithelial cells (17), and induce keratinocyte resistance to calcium
and serum-induced differentiation (18). The activity of E6 in different
biological assays implies it may influence diverse cellular
pathways.
The ability of E6 protein to associate with the cellular tumor
suppressor p53 has been suggested as the mechanism by which the viral
protein promotes cell growth and proliferation (19). Although binding
of high risk HPV E6s with p53 appears to be mediated by another
cellular protein, E6AP (20), direct in vitro association of
E6 with p53 has also been observed (21, 22). The complex of E6 and E6AP
functions as an ubiquitin-protein ligase that results in the specific
ubiquitination and subsequent degradation of p53 (23). Accumulating
evidence suggests that E6 has functions independent of inactivating p53
in cellular transformation (24-36).
We have recently identified a cDNA encoding a cellular protein that
binds papillomavirus E6 (E6BP or ERC-55) (1). E6BP was identified as a
calcium-binding protein of the endoplasmic reticulum (37). The
localization of E6BP is consistent with the localization of E6 to
nonnuclear membranes (38). In vitro binding experiments
demonstrated that E6BP interacted specifically with E6 proteins from
cancer-related HPV types and the bovine papillomavirus type 1 (BPV-1).
The transforming activity of a set of previously characterized BPV-1 E6
mutants correlated well with their E6BP binding ability. These results
suggest that the E6BP interaction plays an important role for BPV-1
E6-induced transformation. More recently, it was reported that BPV-1 E6
associated with paxillin (39) as well as the trans-Golgi
network-specific clathrin adaptor complex AP-1 (40) and that E6
proteins from some high risk HPVs interacts with the human homologue of
the Drosophila discs large tumor suppressor protein hDlg
(41, 42). These observations imply that E6 is capable of interacting
with several cellular factors and participates in several pathways.
In the present study, we mapped the region of the E6BP that is critical
for complex formation with HPV16 E6 and examined the structure of a
peptide corresponding to this region by NMR spectroscopy. The amino
acid sequence of E6BP predicts homology to EF-hand proteins. The
EF-hand coordinates calcium with high specificity. It is named for a
helix-loop-helix motif originally found in the crystal structure of
parvalbumin and is usually present in two to eight copies arranged in
pairs of interacting domains (for review, see Refs. 43 and 44),
although there are some exceptions (45-47). The prototype calcium
binding loop comprises 12 amino acids
(D1X2N3X4D5G6X7X8-11E12),
where a single ligand is contributed to the calcium by each of the side
chains of the first, third, and fifth positions in the sequence, one
ligand is contributed by the backbone carbonyl of the 7th residue, and
another two ligands are provided by the side chain of the 12th residue.
In the NMR spectra of EF-hand-containing proteins, the NH of the
invariant glycine residue at position 6 is observed to be greatly
downfield-shifted (to ~10 ppm) on binding calcium and has been used
as a reporter for the formation of a structured helix-loop-helix
fragment (48). Different EF-hand motifs bind calcium with a wide range
of affinities, with equilibrium dissociation constants from
10
9 M to 10
5 M. The
binding of calcium often causes a conformational change that alters the
interaction with target proteins (43).
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EXPERIMENTAL PROCEDURES |
Plasmids--
The plasmid encoding C terminus 211-amino acid
fusion protein of E6BP and glutathione S-transferase (GST)
(E6BP-211 or GST-E6BP) was described previously (1). Modified pGEX2T
(Amersham Pharmacia Biotech) contains BamHI,
XhoI, ClaI, SpeI, XbaI
sites at the fusion point. Modified pGEX3X (Amersham) contains
BamHI, XhoI, ClaI, SpeI,
XbaI, KpnI, SpeI, and EcoRI
sites at the fusion point. Plasmid pSPBPVE6 encodes BPV-1 E6 in a pSP65
vector. Plasmid pSP16E6 was obtained from Karen Vousden (49).
Mutagenesis--
For GST-E6BP deletions E6BP-N, -N1, -N2, -M,
-dlM1, and -dlM2, restriction endonuclease sites
were used to delete coding sequences from GST-E6BP. E6BP-dlM
was created by polymerase chain reaction amplification of GST-E6BP with
the primers (CGATCGGGATCCGCTAGCATGTCCCCTATACTAGGT) and
(GCGGGATCCTCTTGAATGACAAATTCCG). The fragment was digested with
Msc I and BamHI and then used to replace the
Msc I-BamHI fragment in GST-E6BP. E6BP-C was
constructed by insertion of a BamHI-EcoRI
fragment from GST-E6BP into the BamHI and EcoRI
sites of pGEX1 (Pharmacia). A HindIII-XhoI
fragment of GST-E6BP was inserted into the modified pGEX2T to create
E6BP-C1.
E6BP-EF4 was created by polymerase chain reaction amplification of
E6BP-211 with primers (GCGGGATCCTGACGGAATTTGTCATTCAAG) and
(ATTCTCGAGCTAATTTGCAGTTGGGTCCCACC); E6BP-EF5 was created by polymerase
chain reaction amplification of E6BP-211 with primers (GCGGGATCCTGATACTTGTTGAGAAAGACAG) and (ATTCTCGAGCTAAATGCCCTGATTATTAGG). The fragments were digested with BamHI and XhoI
and inserted into the modified pGEX3X.
Protein Expression and Purification--
GST fusion proteins
were expressed in Escherichia coli strain DH5
. One liter
of LB media was inoculated with 100 ml of stationary culture and grown
for 1 h before induction with 0.2 mM
isopropyl-1-thio-
-D-galactopyranoside for 3 h.
Cells were harvested by centrifugation, re-suspended in 50 ml of low
salt association buffer (LSAB, 100 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1% Nonidet P-40, and 1 mM
phenylmethylsulfonyl fluoride) plus 0.03% SDS and 2 mM
dithiothreitol, and lysed by sonication. After centrifugation at
10,000 × g for 10 min, the supernatant was collected
and mixed with glutathione-Sepharose beads (Amersham). The mixture was
subjected to rotary shaking for 2 h at 4 °C. The beads were
then collected by centrifugation at 1000 × g for 2 min, washed three times with 20 volumes of LSAB, and stored at
4 °C. In vitro translated E6 proteins were prepared by
using the rabbit reticulocyte lysate translation system (Promega) and
35S-labeled cysteine (ICN, Irvine, California).
In Vitro Association Experiment--
For in vitro
binding, 30 µl of glutathione-Sepharose containing approximately 4 µg of GST fusion proteins were combined with 1-10 µl of
35S-labeled in vitro translated proteins in LSAB
in a total volume of 250 µl. The mixtures were subjected to rotary
shaking for 3 h at 4 °C. The mixtures were then washed six
times with LSAB, boiled in SDS-gel loading buffer, and electrophoresed
on SDS-polyacrylamide gels. Gels were fixed and scanned by Molecular
Imager (Bio-Rad).
Peptide Synthesis--
A 31-amino acid residue peptide was
synthesized using Fmoc (N-(9-fluorenyl)methoxycarbonyl)
chemistry. The crude deprotected peptide was purified by
phenyl-Sepharose chromatography (50) using a linear gradient of
0-100% B (Buffer A: 10 mM CaCl2, 50 mM Tris, 500 mM NaCl, pH 8; Buffer B: 50 mM Tris, pH 8) and followed by high performance liquid
chromatography on a preparative reverse phase C18 column.
The peptide was greater than 98% pure by analytical HPLC and by
matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)
mass spectral analysis (observed 3689 ± 4, theoretical 3687).
NMR Spectroscopy--
Samples of E6bp peptide were prepared at a
concentrations between 1 and 4 mM (monomer) in 10 and 100%
D2O buffers containing 20 mM NaCl, pH 6.1 ± 0.1, and 0-10% trifluoroethanol (TFE). Spectra were recorded at 30 and 35 °C on a Bruker AMX-500 spectrometer with a proton resonance
frequency of 500.14 MHz. The carrier frequency was set on the water
resonance, which was suppressed using presaturation. One-dimensional
NMR spectra were recorded with 128 summed scans, 2048 real points, and
a spectral width of approximately 7000 Hz. The spectra were processed
using sinebell window functions shifted by 45°, and the residual
water resonance was removed with a gaussian convolution function.
Two-dimensional NOESY and TOCSY spectra were recorded with mixing times
of 75 or 125 and 40 ms, respectively (51). Sequence-specific resonance
assignments were made by identification of intraresidue spin systems
using the 1H-1H through-bond connectivities
found in the TOCSY spectrum followed by sequential assignment of
residues on the basis of sequential d
N,
d
N, and dNN NOE cross-peaks using standard
methods (52).
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RESULTS |
The E6 Binding Domain of E6BP Is a Specific EF-hand
Motif--
E6BP is a 317-amino acid protein that contains six putative
EF-hands (37). The original yeast two hybrid isolate of E6BP that
interacts in vivo with HPV 16 E6 encoded the C-terminal 211 amino acids of E6BP (1). This protein was shown to bind high risk HPV
E6 and the transforming BPV-1 E6 in vitro. To determine the
region of E6BP that interacts with E6, N-terminal, C-terminal, and
internal in-frame deletions were introduced into the C-terminal 211 amino acids of E6BP (Fig. 1A).
These were constructed either with existing restriction sites or by
employing polymerase chain reaction. Mutant proteins were synthesized
in E. coli as GST fusions. Equal amounts of GST-E6BP fusion
proteins were assayed for their abilities to associate with in
vitro translated 35S-labeled HPV16 E6. As shown in
Fig. 1B, all constructs containing amino acid residues
194-218 were capable of binding to E6, whereas truncated forms of E6BP
without this region could not bind. Amino acids outside this region may
contribute to the interaction, as none of the deletion mutants bound as
efficiently as E6BP-211. This E6 binding domain falls within the fourth
EF-hand motif in E6BP and contains all the putative loop and
flanking
-helical sequences.

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Fig. 1.
A specific EF-hand motif of E6BP is both
necessary and sufficient for E6 binding. Glutathione-Sepharose
beads containing GST-E6BP fusion proteins were combined with
35S-labeled in vitro translated proteins HPV-16
E6 in LSAB. After incubation and washes, the bound products were
separated by SDS-polyacrylamide gel electrophoresis. E6 binding was
quantified by phosphoimager analysis. A, construction of
deletion mutations of E6BP as GST-fusion proteins. B,
identification of E6 binding domain in E6BP by in vitro
association experiment. C, construction of additional
GST-E6BP fusion proteins. D, the fourth EF-hand motif of
E6BP binds E6. Mutant numbers indicate the amino acid boundaries in
E6BP constructs. Blackened boxes represent EF-hand motifs.
Input was loaded directly into the well and represents 10% of the
35S-labeled E6 used in each binding reaction.
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To confirm these results, we engineered an additional GST fusion that
incorporated only 25 amino acids (residues 194-218) from the fourth
EF-hand (E6BP-M) and tested it in the in vitro binding assay
(Fig. 1C). We also prepared an internal deletion of the
fourth EF-hand (E6BP-dlM) of the original 211 amino acid isolate of E6BP. As shown in Fig. 1D, E6BP-M efficiently
bound to HPV-16 E6, whereas E6BP-dlM did not. A GST fusion
protein containing the complete 36-amino acid fourth EF-hand of
(E6BP-EF4) bound HPV-16 E6. The control GST fusion protein containing
the 36-amino acid fifth EF-hand (E6BP-EF5) did not bind. These results
demonstrate that the fourth EF-hand in E6BP specifically mediates
association with E6.
Although only amino acids 194-218 were sufficient for binding E6,
comparison to other helix-loop-helix EF-hand domains (43) suggested
that formation of the first helix may require residues N-terminal to
the 25-amino acid E6 binding domain. A peptide was therefore
synthesized as a 31-amino acid fragment (residues 188-218 of E6BP) for
analysis by NMR spectroscopy. For further discussion, this peptide is
renumbered as residues 1 through 31 and named E6bd peptide.
The Peptide-containing E6 Binding Domain Folds into a Classical
Helix-Loop-Helix EF-hand Conformation--
In the absence of metal
ion, the peptide adopted a mostly random-coil conformation, as
evidenced by the lack of dispersion of resonances in the spectrum,
noted particularly in the aromatic and upfield methyl-group regions and
by the lack of downfield-shifted amide and H
resonances (48). Upon
addition of the first three or four equivalents of calcium to the
sample, very few changes were noted in the spectrum. Significant
spectrum changes were observed as the calcium-to-peptide mol ratio was
increased to 20:1, including the downfield migration of the amide
resonance of glycine 17 to a final position around 10 ppm (Fig.
2). No significant spectrum changes were
noted as the concentration of metal ion was increased to a ratio of
80:1. The weak binding of calcium to E6bp, as observed by NMR, is
consistent with results obtained for a similar peptide derived
from other EF-hand-containing proteins, such as troponin C
(48).

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Fig. 2.
Demonstration of calcium binding by E6
binding peptide by 1H NMR spectroscopy. A, the
metal-free peptide was at 1.0 mM concentration in a
solution containing 20 mM NaCl. B, addition of
80 mM calcium chloride (CaCl2) to the peptide
induces chemical shift dispersion. The downfield-shifted resonance of
glycine 17 is indicated with an arrow and is typical of a
calcium-bound EF-hand. The addition of a small amount of TFE (5-10%)
narrows some resonance line widths. Sample conditions were 1.0 mM peptide in a buffer containing 8% TFE, 20 mM NaCl, pH 6.0.
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In the apo form, the resonance line widths were narrow (about 3 Hz) and
consistent with a monomeric form of the peptide (53). In the
calcium-bound state, resonances for the residues of E6bd peptide
initially were broad (about 10-20 Hz) and more so for residues 17-19
(about 20-50 Hz), indicating oligomerization (data not shown). Several
EF-hand peptides have been noted before to homodimerize and mimic the
heterodimer detected in full-length proteins (43, 44, 48, 54, 55). Our
data were consistent with homodimer formation (see below). In addition
to dimerization, the E6bd peptide visibly aggregated upon binding
calcium, consistent with the exposure of a significant hydrophobic
patch. As has been observed before for other EF-hand-containing
peptides, the addition of a small amount of the organic solvent TFE
improved solubility and sharpened the resonances without appreciably
changing resonance positions (56). Subsequent samples of E6bd peptide
used between 5 and 10% TFE and 60 equivalents of calcium. However,
line widths still remained slightly broader (10-20 Hz) than what would
be expected (4-8 Hz) for a dimer of 7000 Da (53), probably because of
residual aggregation.
The sequential assignment of the 1H NMR spectrum was
carried out using the conventional two-dimensional NMR approach. TOCSY spectra were used to identify through-bond connectivities associated with spin systems of residues, and NOESY spectra were used to establish
interresidue connectivities (52). The chemical shift assignments for
the calcium-bound peptide are presented in Table I. Fig. 3
shows a contour plot of the NH to NH region of a NOESY spectrum and
illustrates the quality of the NMR data. From a qualitative interpretation of the NOESY cross-peak intensities and the chemical shift data, it was clear that the peptide comprised two helices separated by a loop region. Fig. 4
summarizes the NOE data and illustrates stretches of
d
(i, i + 3) and
dNN(i, i + 1), strong
d
N(i, i + 1), and weak
d
N(i, i + 1) connectivities, confirming the presence of helical secondary structure extending approximately from residue 4 to residue 14 and from residue 21 to
residue 27. A short stretch of sequence corresponding to a strand of a
sheet is observed for residues 18-20. The ends of the structural
elements cannot be determined exactly from a qualitative analysis of
this sort and can only be defined more precisely from a complete
structure determination to be presented
elsewhere.2 Our preliminary
analysis of the NOE data showed contacts between residues and is
consistent with an antiparallel dimer similar to the previously solved
structures of EF-hand-containing proteins. A model of the E6bd peptide
was prepared using the calcium-bound EF-hand pair observed in the
2.1-Å resolution structure of turkey troponin C (57). The EF-hands
formed dimer contacts nearly throughout the entire length of the
peptide sequence and was centered about a small
sheet formed using
residues 18 to 20 of each monomer (Fig.
5).

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Fig. 3.
The NH-NH region of the two-dimensional
NOESY spectrum. Resonance assignments were accomplished by tracing
connections between the NH, , and protons of a residue
(i), and the NH of the next residue (i + 1). The
helical nature of this peptide is illustrated by medium and strong NH
to NH NOE cross-peaks between residues adjacent in sequence. Sample
conditions are given in Fig. 3, and the NOE mixing time was 125 ms.
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Fig. 4.
Summary of the conformational data. The
sequence of the peptide, corresponding to residues 289-318 of E6BP, is
renumbered as residues 1-31. The residues that ligate calcium are
boxed. These residues form single ligands to calcium using
their side chains, except for F18, which uses its backbone carbonyl,
and E23, which contributes a double ligand. NOE cross-peaks describing
NH-to-NH contacts, H -NH contacts, H -NH contacts, and H to
H (i + 3) contacts are illustrated with the thickness of
the filled-in bar corresponding to NOE intensity. The
absence of a bar indicates that the NOE was not observed, whereas the
presence of the dotted line indicates that the NOE was
likely but was obscured by resonance overlap. A hollow bar
indicates that the NOE involves a glycine residue and is not
applicable. The chemical shift index (CSI) is positive if
the difference in chemical shift between observed and a random coil
peptide was greater than 0.1 ppm or negative if it was less than 0.1 ppm (61). A positive index indicates sheet, whereas a negative
index indicates helix. A summary for the secondary structure of the
peptide is shown under the conformational data.
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Fig. 5.
Model for the E6bp peptide. The peptide
was modeled using the x-ray structure of the calcium-bound motif from
troponin C. The helical elements are shown as cylinders. The
side chains and backbone carbonyl groups that ligate the calcium ions
are shown.
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The E6 Binding Domain Is a Short
Helical Peptide That Is
Homologous to E6 Binding Regions Found in Other E6-binding
Proteins--
To further map the domain that is important for E6
binding, additional deletion mutants from the E6 binding domain
(E6BP-M) were constructed and tested for E6 binding (Fig.
6A). Because BPV-1 E6 binds
this domain with higher efficiency than HPV-16 E6, subsequent
quantitative experiments were performed with BPV-1 E6. As shown in Fig.
6B, deletion of the first
helix (E6bd-20) did not affect
binding nor did deletion of two additional amino acids from C-terminal
end of E6bd (E6bd-18). More importantly, a peptide of 13 amino acids
encompassing the second
helix (E6bd-13) retained the ability to
bind E6, although at reduced efficiency. Notably, the majority of the
loop region of the EF-hand motif was not present in this 13-amino acid
peptide. The ability of the second
helix to bind E6 demonstrates
that the interaction of E6BP with E6 is independent of calcium binding,
as the first
helix and loop region from the EF-hand motif are both
required for coordination of calcium.

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Fig. 6.
An helical motif is sufficient for
association with E6. Glutathione-Sepharose beads containing
GST-E6BP fusion proteins were combined with 35S-labeled
in vitro translated BPV-1 E6 proteins in LSAB. After
incubation and washes, the bound products were separated by
SDS-polyacrylamide gel electrophoresis. E6 binding was analyzed by
Molecular Imager. A, construction of deletion and point
mutations of E6bd GST fusion proteins. Numbers for binding
are the percentage of association from each mutant normalized to the
level seen with E6bd-M. The average of three independent experiments,
including standard deviations, is given. B, an helical
motif is important for association with E6. The image of one
representative experiment is shown. Sequences of the E6 binding domain
(E6bd) 7 through 31 are given. Input was loaded directly into the well
and represents 10% of the 35S-labeled E6 used in each
binding reaction.
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Next, alanine replacement mutations in the E6 binding domain (E6bd)
were constructed to define amino acids important for E6 interaction
within this
helix (Fig. 6A). Some mutations were also
made in the surrounding regions. As expected, mutants E6bd-V19A, -S20A,
-Y28A, -R29A, and -W30A, which are adjacent to the
helix, bound E6
at wild-type levels or with modest changes (Fig. 6B). The
mutant E6bd-F18A showed some reduced binding (about 3-fold), probably
reflecting the role of hydrophobic residue interactions between E6 and
E6bd. Although some mutations within the
helix (E6bd-E22A and
-E23A) showed modest changes of E6 binding activity, all other mutants
within the
helical structure demonstrated substantial decreases.
Consistent with the notion that hydrophobic residues play important
roles in E6-E6bd interaction, E6bd-L21A, E6bd-F24A, and E6bd-L25A were
severely impaired for their ability to bind E6. Notably, a change of
leucine to alanine at amino acid 25 (E6bd-L25A) totally abolished
binding. Maintenance of E6 association by the mutant E6bd-E23A
substantiated the prediction that the interaction of E6BP with E6 is
independent of calcium binding, as glutamic acid 23 contributes two
ligands to calcium. Finally, we created a leucine to proline change at
amino acid 25, which was expected to disrupt the
helical structure
of E6bd. The L25P mutation totally abolished E6 binding, indicating
importance of the
helix. In summary, the sequence of amino acids in
E6BP that bind E6 is FVSLEEFLGD, with
the amino acids in bold most critical.
Previous studies of E6AP defined a span of 18 amino acids that is
necessary for interaction with E6 (2). Comparison of the amino acid
sequence of the
helical E6 binding domain of E6BP with this
18-amino acid E6 binding region revealed a striking degree of homology
with a common motif, L(E/Q)E(F/L)LG(D/E) (Fig. 7A). Moreover, this motif has
similarity to the LD1 motif in paxillin, which has been reported to be
critical for BPV-1 E6 binding (3). Using a secondary structure analysis
program (58), the E6 binding motif from E6AP and the paxillin LD motifs
are each strongly predicted to form an
helix. We have thus
identified an
helical E6 binding domain that is conserved among
these E6-binding proteins. The consensus sequence is
LhX
Ls
, where
is a hydrophobic residue, h denotes an amino acid that can make multiple hydrogen
bonding interactions with its side chain, s is an amino acid
with a small side chain,
is a negatively charged amino acid, and
X is any amino acid.

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Fig. 7.
An helix is conserved among E6-binding
proteins. A, comparison of E6 binding domains reveals a
conserved motif. The E6 binding peptides of E6bd (residues 18-29), the
18 amino acids (amino acids 391-408) of E6AP (2), and the sequence of
LD1 from paxillin (amino acids 3-15) (3) are aligned. h,
amino acid that can form a double hydrogen bond, i.e. Asp,
Glu, Gln. X, any amino acid. , hydrophobic amino acid.
s, small amino acid such as Gly or Ala. , acidic residue.
B, amino acids in the second helix of the helix-loop-helix
that comprise the EF-hand in a helical wheel representation. In E6BP,
replacement of either Leu-21, Phe-24, Leu-25, or Asp 27 by Ala reduced
binding to E6 by at least 10-fold. Alanine replacements of the Gly-26
results in about a 5-fold loss in binding, whereas replacement of the
other residues did not appreciably affect binding. Left, the
motif for E6BP (EF-hand 4). Center, the motif for E6AP.
Right, an example of a nonbinding E6 motif (the homologous
residues from EF-hand 5 of E6BP).
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DISCUSSION |
The high risk HPV E6 proteins are consistently expressed in all
HPV-positive cervical carcinomas. E6 is a small protein and is not
known to possess intrinsic enzymatic activity. Similar to the
oncoproteins of other tumor viruses, E6 is thought to exert multiple
functions through interaction with cellular factors. Five proteins have
been reported to associate directly with E6: E6AP (1, 20), E6BP (1),
paxillin (39), hDlg (41, 42), and AP-1 (40). Although association with
E6AP is believed to be necessary for E6 to target p53 for degradation,
interaction with the other proteins, including E6BP, represents a
p53-independent property of E6.
In the present study, we first mapped the region of the E6BP that is
necessary and sufficient for complex formation with HPV16 E6 to a
25-amino acid residue domain. A peptide containing this E6 binding
domain was synthesized and shown by NMR spectroscopy to bind calcium
and fold into a classical dimeric helix-loop-helix conformation.
Subsequently we determined that a smaller peptide of 13 amino acids
containing the second
helix of the EF-hand retained the ability to
bind E6. Smaller peptides have not been tested yet, so this may not be
the minimal interaction domain.
Comparison of the
helical E6 binding domain identified in this
study with the E6 binding region of E6AP and paxillin revealed strong
homology. Alanine replacement mutagenesis studies in E6BP clearly
demonstrated the importance of these common amino acids. The greatest
effects on E6 binding resulted from substitutions of the hydrophobic
amino acids and the negatively charged amino acid on one side of the
helix (Fig. 7B). Thus E6 can bind proteins bearing the
sequence LhX
Ls
(see above). Proteins that do
not contain this sequence or ones that are not
helical do not bind E6 (Figs. 1D, and 7B). For example, in EF-hand 5 there is a proline at the s position of the consensus
sequence, which is likely to disrupt the helical structure. In
addition, a tryptophan occupies the negatively charged amino acid
position of the consensus sequence.
EF-hand-containing proteins interact with their target proteins in
different ways (43). In all cases, association is predominantly mediated through hydrophobic residues and is complemented by acidic side chains from the EF-hands interacting with basic amino acid residues of the target molecule. The consensus binding motif contains several hydrophobic residues and one negatively charged residue. We
showed that the residues Leu-21, Phe-24, Leu-25, and Asp-27 were
critical for E6 binding. The hydrophobic Phe-18 residue, although not
essential, may enhance the interaction between E6 and E6 binding
domain. Alternatively, it may contribute to dimerization of the domain.
We observed that the E6bd domain peptide dimerizes on binding calcium,
but the role of this dimerization on interaction with E6 is not
understood. Since GST is dimeric, it is likely that the GST fusions to
E6BP derivatives used in the binding experiments are also dimeric. The
amino acid sequence of E6 itself predicts a pseudodimeric protein (59),
and it could be that two E6 binding motifs simultaneously interact with
E6. Our future experiments are aimed at examining the conformation of
E6bd peptides while bound to E6.
Despite bearing EF-hand motifs, the mutational data show that E6BP does
not require calcium for binding E6. In addition, we find that the
extent of binding is insensitive to the addition of calcium (data not
shown). Other EF-hand-containing proteins, such as calmodulin and
calcylin, have been observed to interact with some target proteins in
the calcium-free state (reviewed in Ref. 43). The other proteins that
bind E6, E6AP, paxillin, and AP-1 have no known calcium binding motifs
and are unlikely to bind calcium. We hypothesize that the E6·E6BP
complex targets a regulatory protein in a calcium-dependent
manner, although that protein has not yet been discovered.
The fourth protein that binds E6, hDlg, appears to operate by using a
different mechanism (41). The hDlg protein does not contain the
consensus motif. E6 binding has been mapped to interaction between its
third PDZ domain and the C-terminal four-amino acid residues of HPV-16
E6. A PDZ domain is a protein recognition surface that recognizes
peptides bearing the sequence Xaa-Thr/Ser-Xaa-Val-COO
.
Although the C terminus of HPV-16 E6 contains the
XTXL motif, BPV-1 E6 does not. Crystallographic
analysis of a PDZ domain-peptide complex shows that the target peptide
is bound in an extended conformation (60). The most recently identified
E6-binding protein, AP-1, does not contain all elements of the
consensus sequence, although the exact region required for interaction
has not been defined (40). In contrast, based on our observations, we
predict that E6AP, E6BP, and paxillin use an
-helical motif to bind
E6. Identification of a conserved E6 binding motif provides a basis for
structure-based drug design to block HPV-associated malignant transformation.
 |
ACKNOWLEDGEMENT |
We thank Dr. Michael Berne for peptide
synthesis and members of our laboratories for helpful suggestions.
 |
FOOTNOTES |
*
This research was supported by National Institutes of Health
Grants RO1 CA73558 and U01 AI38001 (to E. J. A.), R29 AI34918 (to
J. D. B.), and a Alpha Omega Alpha Student Research Fellowship (to
E. R.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Supported by the Postdoctoral Fellowship from the American Cancer
Society, Massachusetts Division, Inc., and the Dermatology Foundation
Dermik Laboratories, Inc. Career Development Award.
**
To whom correspondence should be addressed: Dept. of Dermatology,
New England Medical Center, Box 166, 750 Washington St., Boston, MA
02111. Tel.: 617-636-1493; Fax: 617-636-6190; E-mail: eandroph{at}opal.tufts.edu.
1
The abbreviations used are: PV, papillomavirus;
HPV, human PV; BPV, bovine PV; GST, glutathione
S-transferase; TFE, trifluoroethanol; LSAB, low salt
association buffer; NMR, nuclear magnetic resonance; NOE, nuclear
Overhauser effect; NOESY, NOE spectroscopy; TOCSY, total correlation
spectroscopy.
2
S. Veeraraghavan, X. Be, E. Androphy, and J. Baleja, unpublished information.
 |
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