Abstract
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 (1)H 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 alpha-helical structure upon the addition of membrane mimetics. (1)H NMR analysis of three overlapping peptides afforded, for the first time, direct experimental evidence of the presence of a C-terminal region with strong alpha-helical propensity comprising amino acid residues Ile(55)-Lys(85) connected via an essentially random coil structure to a much weaker helix-like region, located in the N terminus between residues Trp(9) and Lys(20). 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.
Highlights
The genome of Influenza A virus (IAV), a representative of the orthomyxoviruses, consists of eight separate linear segments of negative sense RNA and was thought to encode 10 gene products
With the goal of understanding the molecular mechanism involved in the biological function of the regulatory IAV protein PB1-F2, we describe here the first structural characterization of the molecule derived from the IAVPR8 isolate
The sequence of the sPB1-F2 molecule used in the present investigation corresponds to the strain IAVPR8 (4, 11)
Summary
Peptide Synthesis and Purification—The synthesis, purification, and molecular characterization of sPB1-F2 and the three related fragments PB-(1– 40), PB-(30 –70), and PB-(50 – 87) derived from influenza A virus strain A/Puerto Rico/8/34(H1N1) (isolate IAVPR8) (4) are described in detail elsewhere (11). CD Spectroscopy—CD spectra of the protein and related peptides were recorded at room temperature and a concentration of 0.2 mg/ml in 0.5-mm cuvettes on a Jasco J-810 spectropolarimeter in a wavelength range from 260 to 180 nm at various TFE concentrations as described previously (8). Final structures were displayed and manipulated on an Silicon Graphics OCTANE-work station using the program BRAGI (16). After 30 min, the cross-linking reaction was terminated by the addition of glycine and 10 l of SDS sample buffer (2% SDS, 1% -mercaptoethanol, 10% glycerol in 65 mM Tris/HCl, pH 6.8). To follow the oligomerization of sPB1-F2 by Coomassie staining, the peptide was incubated at room temperature in PBS to a final protein concentration of 250 g/ml. After Coomassie staining (0.5% Coomassie Brilliant Blue G-250, 10% acetic acid, 25% isopropyl alcohol), the gel was destained (25% methanol, 10% acetic acid), and processed for imaging
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