Structural Order and Disorder Dictate Sequence And Functional Evolution of the Papillomavirus E7 Protein
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 . The globular C-‐terminal domain (E7C) mediates zinc binding and homodimeriza+on . 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) . 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  show that E7N is as conserved as E7C in spite of the lack of a stable structure (figure A). Thehighly conserved E7N motifs are separated by variable regions. The CR1 region shows high conservation at the The sequence logo  for the C-terminal domain displays (1) four cysteine residues involved in zinc binding (positionshelix-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 nuclearconservation 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 arerestriction 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  reveals two pairs of coevolving amino acidof residues 25 and 29  (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 siteThe phylogenetic analysis suggests that the LxCxE motif, the acidic stretch and the CKII sites have changed severaltimes 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 artiodactylpapillomaviruses, 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 betweenthem. Gamma papillomaviruses often harbor an LxSxE motif. (Figure adapted from ) 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 . NMR measurements indicate that theREFERENCES interaction is mediated by certain residues from each monomers exposed surface (upper panel, left). The proposed Chemes LB et al. Intrinsic disorder in the human papollomavirus E7 protein. In: Flexible Viruses, Uversky DN and Longhi S. Eds. In press. 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 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. 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  (lower panel, right), pointing to a Marino Buslje C et al. Correction for phylogeny, small number of observations and data redundancy improves the identification of coevolvingamino 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 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  suggests that the 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). Radusky L et al. Discovery of functional protein linear motifs using a greedy algorithm and information theory. POSTER.