Interaction of Amphipathic Peptide from Influenza Virus M1 Protein with Mitochondrial Cytochrome Oxidase.

The Bile Acid Binding Site (BABS) of cytochrome oxidase (CcO) binds numerous amphipathic ligands. To determine which of the BABS-lining residues are critical for interaction, we used the peptide P4 and its derivatives A1-A4. P4 is composed of two flexibly bound modified α-helices from the M1 protein of the influenza virus, each containing a cholesterol-recognizing CRAC motif. The effect of the peptides on the activity of CcO was studied in solution and in membranes. The secondary structure of the peptides was examined by molecular dynamics, circular dichroism spectroscopy, and testing the ability to form membrane pores. P4 was found to suppress the oxidase but not the peroxidase activity of solubilized CcO. The Ki(app) is linearly dependent on the dodecyl-maltoside (DM) concentration, indicating that DM and P4 compete in a 1:1 ratio. The true Ki is 3 µM. The deoxycholate-induced increase in Ki(app) points to a competition between P4 and deoxycholate. A1 and A4 inhibit solubilized CcO with Ki(app)~20 µM at 1 mM DM. A2 and A3 hardly inhibit CcO either in solution or in membranes. The mitochondrial membrane-bound CcO retains sensitivity to P4 and A4 but acquires resistance to A1. We associate the inhibitory effect of P4 with its binding to BABS and dysfunction of the proton channel K. Trp residue is critical for inhibition. The resistance of the membrane-bound enzyme to inhibition may be due to the disordered secondary structure of the inhibitory peptide.

Amphipathic alpha-helices and putative cholesterol binding domains of the influenza virus matrix M1 protein are crucial for virion structure organisation.

The influenza virus matrix M1 protein is an amphitropic membrane-associated protein, forming the matrix layer immediately beneath the virus raft membrane, thereby ensuring the proper structure of the influenza virion. The objective of this study was to elucidate M1 fine structural characteristics, which determine amphitropic properties and raft membrane activities of the protein, via 3D in silico modelling with subsequent mutational analysis. Computer simulations suggest the amphipathic nature of the M1 α-helices and the existence of putative cholesterol binding (CRAC) motifs on six amphipathic α-helices. Our finding explains for the first time many features of this protein, particularly the amphitropic properties and raft/cholesterol binding potential. To verify these results, we generated mutants of the A/WSN/33 strain via reverse genetics. The M1 mutations included F32Y in the CRAC of α-helix 2, W45Y and W45F in the CRAC of α-helix 3, Y100S in the CRAC of α-helix 6, M128A and M128S in the CRAC of α-helix 8 and a double L103I/L130I mutation in both a putative cholesterol consensus motif and the nuclear localisation signal. All mutations resulted in viruses with unusual filamentous morphology. Previous experimental data regarding the morphology of M1-gene mutant influenza viruses can now be explained in structural terms and are consistent with the pivotal role of the CRAC-domains and amphipathic α-helices in M1-lipid interactions.

Structural analysis of influenza A virus matrix protein M1 and its self-assemblies at low pH.

Influenza A virus matrix protein M1 is one of the most important and abundant proteins in the virus particles broadly involved in essential processes of the viral life cycle. The absence of high-resolution data on the full-length M1 makes the structural investigation of the intact protein particularly important. We employed synchrotron small-angle X-ray scattering (SAXS), analytical ultracentrifugation and atomic force microscopy (AFM) to study the structure of M1 at acidic pH. The low-resolution structural models built from the SAXS data reveal a structurally anisotropic M1 molecule consisting of a compact NM-fragment and an extended and partially flexible C-terminal domain. The M1 monomers co-exist in solution with a small fraction of large clusters that have a layered architecture similar to that observed in the authentic influenza virions. AFM analysis on a lipid-like negatively charged surface reveals that M1 forms ordered stripes correlating well with the clusters observed by SAXS. The free NM-domain is monomeric in acidic solution with the overall structure similar to that observed in previously determined crystal structures. The NM-domain does not spontaneously self assemble supporting the key role of the C-terminus of M1 in the formation of supramolecular structures. Our results suggest that the flexibility of the C-terminus is an essential feature, which may be responsible for the multi-functionality of the entire protein. In particular, this flexibility could allow M1 to structurally organise the viral membrane to maintain the integrity and the shape of the intact influenza virus.

Spatial structure peculiarities of influenza A virus matrix M1 protein in an acidic solution that simulates the internal lysosomal medium.

The structure of the C-terminal domain of the influenza virus A matrix M1 protein, for which X-ray diffraction data were still missing, was studied in acidic solution. Matrix M1 protein was bombarded with thermally-activated tritium atoms, and the resulting intramolecular distribution of the tritium label was analyzed to assess the steric accessibility of the amino acid residues in this protein. This technique revealed that interdomain loops and the C-terminal domain of the protein are the most accessible to labeling with tritium atoms. A model of the spatial arrangement of the C-terminal domain of matrix M1 protein was generated using rosetta software adjusted to the data obtained by tritium planigraphy experiments. This model suggests that the C-terminal domain is an almost flat layer with a three-α-helical structure. To explain the high level of tritium label incorporation into the C-terminal domain of the M1 protein in an acidic solution, we also used independent experimental approaches (CD spectroscopy, limited proteolysis and MALDI-TOF MS analysis of the proteolysis products, dynamic light scattering and analytical ultracentrifugation), as well as multiple computational algorithms, to analyse the intrinsic protein disorder. Taken together, the results obtained in the present study indicate that the C-terminal domain is weakly structured. We hypothesize that the specific 3D structural peculiarities of the M1 protein revealed in acidic pH solution allow the protein greater structural flexibility and enable it to interact effectively with the components of the host cell.

Isolation of influenza virus A hemagglutinin C-terminal domain by hemagglutinin proteolysis in octylglucoside micelles.

A method of isolation of hydrophobic membrane-bound C-terminal domain of influenza virus A hemagglutinin (HA) is suggested. The method is based on the insertion of HA into octylglucoside micelles followed by pepsin or thermolysin hydrolysis. Subsequent treatment of proteolytic digests with chloroform-hexafluoroisopropanol mixture resulted in the extraction of a few hydrophobic peptides into organic phase. Mass-spectrometry (MALDI-TOF) analysis revealed that the peptides with ion masses corresponding to the anchoring C-terminal domain with or without modifications predominated in the organic solution. The data obtained confirmed our speculation on the possibility of the suggested isolation scheme following from the strong interactions of anchoring domains in compact trimeric structure of HA spikes combined with micelle protection effect. Several appropriate peptides presence in the organic phase apparently arises from the presence of a few accessible proteolytic sites in HA transmembrane region.