1. STRUCTURAL
ORDER
AND
DISORDER
DICTATE
SEQUENCE
AND
FUNCTIONAL
EVOLUTION
OF
THE
PAPILLOMAVIRUS
E7
PROTEIN
LUCIA
B.
CHEMES¶,
JULIANA
GLAVINA§,
CRISTINA
MARINO-­‐BUSLJE¶,
GONZALO
DE
PRAT-­‐GAY¶
AND
IGNACIO
E.
SANCHEZ§
¶PROTEIN
STRUCTURE,
FUNCTION
AND
ENGINEERING
LABORATORY,
FUNDACION
INSTITUTO
LELOIR
AND
IIBBA-­‐CONICET,
BUENOS
AIRES,
ARGENTINA.
§PROTEIN
PHYSIOLOGY
LABORATORY,
DEPARTAMENTO
DE
QUIMICA
BIOLOGICA,
FACULTAD
DE
CIENCIAS
EXACTAS
Y
NATURALES-­‐UNIVERSIDAD
DE
BUENOS
AIRES,
ARGENTINA
INTRODUCTION
E7
is
the
main
transforming
protein
in
papillomaviruses
and
plays
an
important
role
in
oncogenesis
[1].
The
globular
C-­‐terminal
domain
(E7C)
mediates
zinc
binding
and
homodimeriza+on
[2].
The
intrinsically
disordered
N-­‐terminal
domain
(E7N)
harbors
several
linear
mo+fs
that
mediate
interac+on
with
cellular
targets,
including
the
high
affinity
LxCxE
binding
site
for
the
Re+noblastoma
protein
(Rb)
[3].
We
have
analyzed
sequence
and
func+onal
evolu+on
of
E7
using
210
natural
sequences.
E7N DOMAIN CONSERVATION! E7C DOMAIN CONSERVATION!
RbAB (E2F SITE) RbAB (LxCxE SITE) RbAB (LxCxE SITE)
p600 p300 RbC, p21CIP, p27KIP, TBP, AP1, Mi2B, IGFBP3, S4 proteasome, MPP2
P107 p130 p21CIP TBP F-Actin
IRF-1 Cullin-2 FHL2 p300 IRF-1 FHL2 CKII HPV-E2C IRF-1, h-TID, pCAF, Cullin2-UBC, E2F1, FHL2, NuMA, DNMT1
CR1-Helix! LxCxE! CKII-PEST! NES!
* Coevolving residue pairs" * **
* * A
* * *
* B
*
CONSERVATION! CO-EVOLUTION!
A INFORMATION CONTENT! B CKII-PEST REGION!
Zn-binding cysteines" Surface residues" D61/T72"
Monomer interface" Dimer interface" C45/Q56"
The sequence logos [4] show that E7N is as conserved as E7C in spite of the lack of a stable structure (figure A). The
highly conserved E7N motifs are separated by variable regions. The CR1 region shows high conservation at the The sequence logo [4] for the C-terminal domain displays (1) four cysteine residues involved in zinc binding (positions
helix-forming Rb-targeting residues 6-13 and at uncharacterized residues 1-3. The LxCxE motif also shows 44, 47, 77 and 80, displayed in red in figure A), (2) a highly conserved leucine-rich region that acts as a nuclear
conservation at residues that are outside the canonical motif (pos. 19, 23, 25 and 26). One third of the E7 sequences export signal, (3) six conserved positions (displayed in blue in figure A) that form the core of each monomer, and (4)
lack a CKII phosphorylation site, while only 2.5% of them lack a stretch of acidic residues (n>3) (figure B). The tight six conserved positions (displayed in cyan in figure A) that stabilize the dimer interface. Four conserved residues are
restriction in sequence separation between the LxCxE motif and the CKII/PEST region together with the coevolution surface exposed (yellow in figure A). A mutual information analysis [5] reveals two pairs of coevolving amino acid
of residues 25 and 29 [5] (black asterisks), suggests that the two motifs form an evolutionary and functional unit. positions that form contacts across the dimerization interface (figure B).
EVOLUTION OF E7N LINEAR MOTIFS! E7C CYSTEINE CLUSTERS!
REGION 2" Zn-binding cysteines"
REGION 1" Zinc ion"
Two E7C sequence regions show high frequencies of Cysteine (6 to 21%), with most E7 proteins having at least one
extra cysteine in addition to the two canonical CxxC motifs. One cysteine-rich region (blue) is close in space and
sequence to the first CxxC motif and the zinc ion. The second region (green) is close in space to the zinc ion
coordinated by the opposite monomer. The additional cysteines may stabilize alternative conformations of the domain
through non-native coordination of the zinc ion.
CONSERVED PEPTIDE-BINDING SITE IN E7C!
HPV45 E7C (PDB 2B9D) PYGO1_MOUSE (PDB 2YYR)
P21-binding site Information content H3-binding site
The phylogenetic analysis suggests that the LxCxE motif, the acidic stretch and the CKII sites have changed several
times during papillomavirus evolution. Changes in sequence of the LxCxE motif are coupled to changes in phenotype
(delta, gamma and alpha 2 sp.), pointing to adaptive evolution events. In reptilian, avian and some artiodactyl
papillomaviruses, E7N is substituted by a domain with no sequence similarity to canonical E7N sequences.
Whenever the LxCxE motif is present, the acidic stretch follows, further supporting the functional association between
them. Gamma papillomaviruses often harbor an LxSxE motif. (Figure adapted from [6])
CONCLUDING
REMARKS
•
Sequence
evolu+on
in
the
disordered
E7N
domain
shows
that
some
of
its
short
func+onal
mo+fs
evolve
in
a
coordinate
manner
and
that
the
domain
has
been
subject
to
several
episodes
of
adap+ve
evolu+on.
The
high
func+onal
density
within
PUTATIVE E7C BINDING MOTIF E7C/PYGO1
E7N
could
explain
the
large
number
of
targets
found
for
this
small
protein.
•
Evolu+on
of
the
E7C
domain
is
dictated
by
dimeriza+on,
canonical
zinc
binding
by
the
two
CxxC
mo+fs
and
likely
also
by
zinc
binding
by
unpaired
cysteines
and
binding
of
short
Ser/Pro-­‐rich
sequences
within
host
proteins.
HPV45 E7C binds to a short peptide from the host cellular protein p21 [2]. NMR measurements indicate that the
REFERENCES
interaction is mediated by certain residues from each monomer's exposed surface (upper panel, left). The proposed
[1] Chemes LB et al. Intrinsic disorder in the human papollomavirus E7 protein. In: Flexible Viruses, Uversky DN and Longhi S. Eds. In press.
[2] Ohlenschlager O et al. Solution sturcture of the partially folded high-risk human papilloma virus 45 oncoprotein E7. Oncogene 2006, 25:5953-9.
site overlaps well with a conserved surface patch in E7C (upper panel, middle), suggesting that peptide binding is a
[3] Lee JO et al. Structure of the retinoblastoma tumour suppressor pocket domain bound to a peptide from HPV E7. Nature 1998, 361:859-65. property shared by most E7C domains.
[4] Schneider TD, Stephens RM. Sequence logos: a new way to display consensus sequences. Nucleic Acids Res. 1990, 18:6097-100. The structures of the E7C domain and host phd domains superimpose well [7] (lower panel, right), pointing to a
[5] Marino Buslje C et al. Correction for phylogeny, small number of observations and data redundancy improves the identification of coevolving
amino acid pairs using mutual information. Bioinformatics 2009, 25:1125-1131
plausible evolutionary origin for E7C. Many phd domains bind peptides at a site that corresponds to the proposed
[6] Bravo IG et al. The clinical importance of understanding the evolution of papillomaviruses. Trends in Microbiology 2010, 18:432-438. functional surface of E7C (upper panel, right). A motif search in the sequences of E7C targets [8] suggests that the
[7] Suhrer S et al.. COPS-a novel workbench for explorations in fold space. Nucleic Acids Res. 2009, 37(Web Server issue):W539-544. domain binds peptides rich in proline and serine residues (lower panel, left).
[8] Radusky L et al. Discovery of functional protein linear motifs using a greedy algorithm and information theory. POSTER.