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J. Biol. Chem., Vol. 282, Issue 1, 353-363, January 5, 2007

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Structural Characterization and Oligomerization of PB1-F2, a Proapoptotic Influenza A Virus Protein^*

Karsten Bruns^¶1, Nicole Studtrucker¹, Alok Sharma^¶, Torgils Fossen^¶||, David Mitzner, André Eissmann, Uwe Tessmer, René Röder^**, Peter Henklein^**, Victor Wray^¶, and Ulrich Schubert²

From the Institute of Clinical and Molecular Virology, University of Erlangen-Nürnberg, Erlangen D-91054, Germany, Heinrich-Pette-Institute, Hamburg D-20251, Germany, the ^¶Department of Structural Biology, Helmholtz Centre for Infection Research, Braunschweig D-38124, Germany, the ^||Department of Chemistry, University of Bergen, N-5007 Bergen, Norway, and the ^**Institute of Biochemistry, Humboldt University, Berlin D-10115, Germany

Recently, a novel 87-amino acid influenza A virus protein with proapoptotic properties, PB1-F2, has been reported that originates from an alternative reading frame in the PB1 polymerase gene and is encoded in most known human influenza A virus isolates. Here we characterize the molecular structure of a biologically active synthetic version of the protein (sPB1-F2). Western blot analysis, chemical cross-linking, and NMR spectroscopy afforded direct evidence of the inherent tendency of sPB1-F2 to undergo oligomerization mediated by two distinct domains located in the N and C termini, respectively. CD and ¹H NMR spectroscopic analyses indicate that the stability of structured regions in the molecule clearly depends upon the hydrophobicity of the solvent. In aqueous solutions, the behavior of sPB1-F2 is typical of a largely random coil peptide that, however, adopts -helical structure upon the addition of membrane mimetics. ¹H NMR analysis of three overlapping peptides afforded, for the first time, direct experimental evidence of the presence of a C-terminal region with strong -helical propensity comprising amino acid residues Ile⁵⁵-Lys⁸⁵ connected via an essentially random coil structure to a much weaker helix-like region, located in the N terminus between residues Trp⁹ and Lys²⁰. The C-terminal helix is not a true amphipathic helix and is more compact than previously predicted. It corresponds to a positively charged region previously shown to include the mitochondrial targeting sequence of PB1-F2. The consequences of the strong oligomerization and helical propensities of the molecule are discussed and used to formulate a hypothetical model of its interaction with the mitochondrial membrane.

Received for publication, July 7, 2006 , and in revised form, October 11, 2006.

The atomic coordinates and structure factors (code 2HN8) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by a grant from the research network FORINGEN, funded by the State of Bavaria, Germany, by German Human Genome Research Project Grant IE-S08T06, and by German Research Council Grants SFB 466-A11, SFB 643-A1, and GRKK 1071. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1 and Table S1.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: Institute for Clinical and Molecular Virology, University of Erlangen-Nürnberg, Schlossgarten 4, D-91054 Erlangen, Germany. Tel.: 49-9131-85-26478; Fax: 49-9131-85-26182; E-mail: ulrich.schubert{at}viro.med.uni-erlangen.de.

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