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

• Published in: The Journal of biological chemistry 2007-01, Vol.282 (1), p.353-363
• Main Authors: Bruns, Karsten, Studtrucker, Nicole, Sharma, Alok, Fossen, Torgils, Mitzner, David, Eissmann, André, Tessmer, Uwe, Röder, René, Henklein, Peter, Wray, Victor, Schubert, Ulrich
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Circular Dichroism; Influenza A virus; Influenza A virus - metabolism; Lysine - chemistry; Magnetic Resonance Spectroscopy; Models, Biological; Models, Molecular; Molecular Sequence Data; Protein Binding; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Solvents - chemistry; Tryptophan - chemistry; Viral Proteins - chemistry; Viral Proteins - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M606494200

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 α-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 α-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.