Abstract
E5 is the major transforming oncoprotein of bovine papillomavirus, which activates the platelet-derived growth factor receptor β in a highly specific manner. The short transmembrane protein E5 with only 44 residues interacts directly with the transmembrane segments of the receptor, but structural details are not available. Biophysical investigations are challenging, because the hydrophobic E5 protein tends to aggregate and get cross-linked non-specifically via two Cys residues near its C-terminus. Here, we demonstrate that a truncation by 10 amino acids creates a more manageable protein that can be conveniently used for structure analysis. Synchrotron radiation circular dichroism and solid-state 15N- and 31P-nuclear magnetic resonance spectroscopy show that this E5 variant serves as a representative model for the wild-type protein. The helical conformation of the transmembrane segment, its orientation in the lipid bilayer, and the ability to form homodimers in the membrane are not affected by the C-terminal truncation.
Acknowledgments
We acknowledge the Synchrotron Light Source ANKA for provision of beamtime at the UV-CD12 beamline, and we would like to thank Siegmar Roth and Bianca Posselt for technical assistance in using the beamline and the sample preparation lab. We thank Christian Ried and Prof Dieter Langosch for the pToxRV plasmid and for their support with the ToxR assay. We also thank Dr Sergii Afonin for mass spectrometry of the E5 proteins.
References
Aberle, D., Muhle-Goll, C., Bürck, J., Wolf, M., Reißer, S., Luy, B., Wenzel, W., Ulrich, A.S., and Meyers, G. (2014). Structure of the membrane anchor of pestivirus glycoprotein E(rns), a long tilted amphipathic helix. PLoS Pathog. 10, e1003973.Search in Google Scholar
Brosig, B. and Langosch, D. (1998). The dimerization motif of the glycophorin A transmembrane segment in membranes: importance of glycine residues. Protein Sci. 7, 1052–1056.Search in Google Scholar
Bürck, J., Roth, S., Wadhwani, P., Afonin, S., Kanithasen, N., Strandberg, E., and Ulrich, A.S. (2008). Conformation and membrane orientation of amphiphilic helical peptides by oriented circular dichroism. Biophys. J. 95, 3872–3881.Search in Google Scholar
Burkhardt, A., DiMaio, D., and Schlegel, R. (1987). Genetic and biochemical definition of the bovine papillomavirus E5 transforming protein. EMBO J. 6, 2381–2385.Search in Google Scholar
Burkhardt, A., Willingham, M., Gay, C., Jeang, K.T., and Schlegel, R. (1989). The E5 oncoprotein of bovine papillomavirus is oriented asymmetrically in Golgi and plasma membranes. Virology 170, 334–339.Search in Google Scholar
Clarke, D.T. and Jones, G. (2004). CD12: a new high-flux beamline for ultraviolet and vacuum-ultraviolet circular dichroism on the SRS, Daresbury. J. Synchrotron. Radiat. 11, 142–149.Search in Google Scholar
Fanghänel, S., Wadhwani, P., Strandberg, E., Verdurmen, W.P.R., Bürck, J., Ehni, S., Mykhailiuk, P., Afonin, S., Gerthsen, D., Komarov, I.V., et al. (2014). Structure analysis and conformational transitions of the cell penetrating Peptide transportan 10 in the membrane-bound state. PLoS One 9, e99653.Search in Google Scholar
Freeman-Cook, L.L., Dixon, A.M., Frank, J.B., Xia, Y., Ely, L., Gerstein, M., Engelman, D.M., and DiMaio, D. (2004). Selection and characterization of small random transmembrane proteins that bind and activate the platelet-derived growth factor beta receptor. J. Mol. Biol. 338, 907–920.Search in Google Scholar
Goldstein, D.J. and Schlegel, R. (1990). The E5 oncoprotein of bovine papillomavirus binds to a 16 kd cellular protein. EMBO J. 9, 137–145.Search in Google Scholar
Goldstein, D.J., Andresson, T., Sparkowski, J.J., and Schlegel, R. (1992). The BPV-1 E5 protein, the 16 kDa membrane pore-forming protein and the PDGF receptor exist in a complex that is dependent on hydrophobic transmembrane interactions. EMBO J. 11, 4851–4859.Search in Google Scholar
Goldstein, D.J., Li, W., Wang, L.M., Heidaran, M.A., Aaronson, S., Shinn, R., Schlegel, R., and Pierce, J.H. (1994). The bovine papillomavirus type 1 E5 transforming protein specifically binds and activates the beta-type receptor for the platelet-derived growth factor but not other related tyrosine kinase-containing receptors to induce cellular transformation. J. Virol. 68, 4432–4441.Search in Google Scholar
Heinzmann, R., Grage, S.L., Schalck, C., Bürck, J., Bánóczi, Z., Toke, O., and Ulrich, A.S. (2011). A kinked antimicrobial peptide from Bombina maxima. II. Behavior in phospholipid bilayers. Eur. Biophys. J. 40, 463–470.Search in Google Scholar
Horwitz, B.H., Burkhardt, A.L., Schlegel, R., and DiMaio, D. (1988). 44-amino-acid E5 transforming protein of bovine papillomavirus requires a hydrophobic core and specific carboxyl-terminal amino acids. Mol. Cell. Biol. 8, 4071–4078.Search in Google Scholar
King, G., Oates, J., Patel, D., van den Berg, H.A., and Dixon, A.M. (2011). Towards a structural understanding of the smallest known oncoprotein: investigation of the bovine papillomavirus E5 protein using solution-state NMR. Biochim. Biophys. Acta 1808, 1493–1501.Search in Google Scholar
Klein, O., Polack, G.W., Surti, T., Kegler-Ebo, D., Smith, S.O., and DiMaio, D. (1998). Role of glutamine 17 of the bovine papillomavirus E5 protein in platelet-derived growth factor beta receptor activation and cell transformation. J. Virol. 72, 8921–8932.Search in Google Scholar
Klein, O., Kegler-Ebo, D., Su, J., Smith, S., and DiMaio, D. (1999). The bovine papillomavirus E5 protein requires a juxtamembrane negative charge for activation of the platelet-derived growth factor beta receptor and transformation of C127 cells. J. Virol. 73, 3264–3272.Search in Google Scholar
Klein, M.J., Grage, S.L., Muhle-Goll, C., Bürck, J., Afonin, S., and Ulrich, A.S. (2012). Structure analysis of the membrane-bound PhoD signal peptide of the Tat translocase shows an N-terminal amphiphilic helix. Biochim. Biophys. Acta 1818, 3025–3031.Search in Google Scholar
Kulke, R., Horwitz, B.H., Zibello, T., and DiMaio, D. (1992). The central hydrophobic domain of the bovine papillomavirus E5 transforming protein can be functionally replaced by many hydrophobic amino acid sequences containing a glutamine. J. Virol. 66, 505–511.Search in Google Scholar
Lange, C., Müller, S.D., Walther, T.H., Bürck, J., and Ulrich, A.S. (2007). Structure analysis of the protein translocating channel TatA in membranes using a multi-construct approach. Biochim. Biophys. Acta 1768, 2627–2634.Search in Google Scholar
Langosch, D., Brosig, B., Kolmar, H., and Fritz, H.J. (1996). Dimerisation of the glycophorin A transmembrane segment in membranes probed with the ToxR transcription activator. J. Mol. Biol. 263, 525–530.Search in Google Scholar
Levitt, M.H., Suter, D., and Ernst, R.R. (1986). Spin dynamics and thermodynamics in solid-state NMR cross polarization. J. Chem. Phys. 84, 4243–4255.Search in Google Scholar
Mattoon, D., Gupta, K., Doyon, J., Loll, P.J., and DiMaio, D. (2001). Identification of the transmembrane dimer interface of the bovine papillomavirus E5 protein. Oncogene 20, 3824–3834.Search in Google Scholar
Meyer, A.N., Xu, Y.F., Webster, M.K., Smith, A.E., and Donoghue, D.J. (1994). Cellular transformation by a transmembrane peptide: structural requirements for the bovine papillomavirus E5 oncoprotein. Proc. Natl. Acad. Sci. USA 91, 4634–4638.Search in Google Scholar
Muhle-Goll, C., Hoffmann, S., Afonin, S., Grage, S.L., Polyansky, A.A., Windisch, D., Zeitler, M., Bürck, J., and Ulrich, A.S. (2012). Hydrophobic matching controls the tilt and stability of the dimeric platelet-derived growth factor receptor (PDGFR) β transmembrane segment. J. Biol. Chem. 287, 26178–26186.Search in Google Scholar
Nilson, L.A. and DiMaio, D. (1993). Platelet-derived growth factor receptor can mediate tumorigenic transformation by the bovine papillomavirus E5 protein. Mol. Cell. Biol. 13, 4137–4145.Search in Google Scholar
Nolandt, O.V., Walther, T.H., Roth, S., Bürck, J., and Ulrich, A.S. (2009). Structure analysis of the membrane protein TatC(d) from the Tat system of B. subtilis by circular dichroism. Biochim. Biophys. Acta 1788, 2238–2244.Search in Google Scholar
Oates, J., Hicks, M., Dafforn, T.R., DiMaio, D., and Dixon, A.M. (2008). In vitro dimerization of the bovine papillomavirus E5 protein transmembrane domain. Biochemistry 47, 8985–8992.Search in Google Scholar
Pace, C.N., Vajdos, F., Fee, L., Grimsley, G., and Gray, T. (1995). How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 4, 2411–2423.Search in Google Scholar
Petti, L. and DiMaio, D. (1992). Stable association between the bovine papillomavirus E5 transforming protein and activated platelet-derived growth factor receptor in transformed mouse cells. Proc. Natl. Acad. Sci. USA 89, 6736–6740.Search in Google Scholar
Petti, L., Nilson, L.A., and DiMaio, D. (1991). Activation of the platelet-derived growth factor receptor by the bovine papillomavirus E5 transforming protein. EMBO J. 10, 845–855.Search in Google Scholar
Petti, L.M., Reddy, V., Smith, S.O., and DiMaio, D. (1997). Identification of amino acids in the transmembrane and juxtamembrane domains of the platelet-derived growth factor receptor required for productive interaction with the bovine papillomavirus E5 protein. J. Virol. 71, 7318–7327.Search in Google Scholar
Russ, W.P. and Engelman, D.M. (1999). TOXCAT: a measure of transmembrane helix association in a biological membrane. Proc. Natl. Acad. Sci. USA 96, 863–868.Search in Google Scholar
Schlegel, R., Wade-Glass, M., Rabson, M.S., and Yang, Y.C. (1986). The E5 transforming gene of bovine papillomavirus encodes a small, hydrophobic polypeptide. Science 233, 464–467.Search in Google Scholar
Settleman, J., Fazeli, A., Malicki, J., Horwitz, B.H., and DiMaio, D. (1989). Genetic evidence that acute morphologic transformation, induction of cellular DNA synthesis, and focus formation are mediated by a single activity of the bovine papillomavirus E5 protein. Mol. Cell. Biol. 9, 5563–5572.Search in Google Scholar
Sparkowski, J., Unders, J., and Schlegel, R. (1995). E5 oncoprotein retained in the endoplasmic reticulum/cis Golgi still induces PDGF receptor autophosphorylation but does not transform cells. EMBO J. 14, 3055–3063.Search in Google Scholar
Sreerama, N., Venyaminov, S.Y., and Woody, R.W. (1999). Estimation of the number of alpha-helical and beta-strand segments in proteins using circular dichroism spectroscopy. Protein Sci. 8, 370–380.Search in Google Scholar
Staebler, A., Pierce, J.H., Brazinski, S., Heidaran, M.A., Li, W., Schlegel, R., and Goldstein, D.J. (1995). Mutational analysis of the beta-type platelet-derived growth factor receptor defines the site of interaction with the bovine papillomavirus type 1 E5 transforming protein. J. Virol. 69, 6507–6517.Search in Google Scholar
Steinbrecher, T., Prock, S., Reichert, J., Wadhwani, P., Zimpfer, B., Bürck, J., Berditsch, M., Elstner, M., and Ulrich, A.S. (2012). Peptide-lipid interactions of the stress-response peptide TisB that induces bacterial persistence. Biophys. J. 103, 1460–1469.Search in Google Scholar
Strandberg, E., Kanithasen, N., Tiltak, D., Bürck, J., Wadhwani, P., Zwernemann, O., and Ulrich, A.S. (2008). Solid-state NMR analysis comparing the designer-made antibiotic MSI-103 with its parent peptide PGLa in lipid bilayers. Biochemistry 47, 2601–2616.Search in Google Scholar
Surti, T., Klein, O., Aschheim, K., DiMaio, D., and Smith, S.O. (1998). Structural models of the bovine papillomavirus E5 protein. Proteins 33, 601–612.Search in Google Scholar
Talbert-Slagle, K., Marlatt, S., Barrera, F.N., Khurana, E., Oates, J., Gerstein, M., Engelman, D.M., Dixon, A.M., and DiMaio, D. (2009). Artificial transmembrane oncoproteins smaller than the bovine papillomavirus E5 protein redefine sequence requirements for activation of the platelet-derived growth factor beta receptor. J. Virol. 83, 9773–9785.Search in Google Scholar
van Stokkum, I.H., Spoelder, H.J., Bloemendal, M., van Grondelle, R., and Groen, F.C. (1990). Estimation of protein secondary structure and error analysis from circular dichroism spectra. Anal. Biochem. 191, 110–118.Search in Google Scholar
Wadhwani, P., Bürck, J., Strandberg, E., Mink, C., Afonin, S., and Ulrich, A.S. (2008). Using a sterically restrictive amino acid as a 19F NMR label to monitor and to control peptide aggregation in membranes. J. Am. Chem. Soc. 130, 16515–16517.Search in Google Scholar
Wadhwani, P., Strandberg, E., Heidenreich, N., Bürck, J., Fanghänel, S., and Ulrich, A.S. (2012). Self-assembly of flexible β-strands into immobile amyloid-like β-sheets in membranes as revealed by solid-state 19F NMR. J. Am. Chem. Soc. 134, 6512–6515.Search in Google Scholar
Wadhwani, P., Strandberg, E., van den Berg, J., Mink, C., Bürck, J., Ciriello, R.A., and Ulrich, A.S. (2014). Dynamical structure of the short multifunctional peptide BP100 in membranes. Biochim. Biophys. Acta 1838, 940–949.Search in Google Scholar
Wallace, B.A. and Janes, R.W. (2009). Modern Techniques for Circular Dichroism and Synchrotron Radiation Circular Dichroism Spectroscopy (Amsterdam, NL: IOS Press).Search in Google Scholar
Wallace, B.A. and Mao, D. (1984). Circular dichroism analyses of membrane proteins: an examination of differential light scattering and absorption flattening effects in large membrane vesicles and membrane sheets. Anal. Biochem. 142, 317–328.Search in Google Scholar
Wallace, B.A. and Teeters, C.L. (1987). Differential absorption flattening optical effects are significant in the circular dichroism spectra of large membrane fragments. Biochemistry 26, 65–70.Search in Google Scholar
Walther, T.H. and Ulrich, A.S. (2014). Transmembrane helix assembly and the role of salt bridges. Curr. Opin. Struct. Biol. 27, 63–68.Search in Google Scholar
Wei, P., Liu, X., Hu, M.-H., Zuo, L.-M., Kai, M., Wang, R., and Luo, S.-Z. (2011). The dimerization interface of the glycoprotein Ibβ transmembrane domain corresponds to polar residues within a leucine zipper motif. Protein Sci. 20, 1814–1823.Search in Google Scholar
Whitmore, L. and Wallace, B.A. (2004). DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic Acids Res. 32, W668-73.Search in Google Scholar
Whitmore, L., Woollett, B., Miles, A.J., Klose, D.P., Janes, R.W., and Wallace, B.A. (2011). PCDDB: the Protein Circular Dichroism Data Bank, a repository for circular dichroism spectral and metadata. Nucleic Acids Res. 39, D480-6.Search in Google Scholar
Windisch, D., Hoffmann, S., Afonin, S., Vollmer, S., Benamira, S., Langer, B., Bürck, J., Muhle-Goll, C., and Ulrich, A.S. (2010). Structural role of the conserved cysteines in the dimerization of the viral transmembrane oncoprotein E5. Biophys. J. 99, 1764–1772.Search in Google Scholar
Wu, Y., Huang, H.W., and Olah, G.A. (1990). Method of oriented circular dichroism. Biophys. J. 57, 797–806.Search in Google Scholar
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