Influenza A virus matrix protein M1 plays an essential role in the virus lifecycle, but its functional and structural properties are not entirely defined. Here we employed small-angle X-ray scattering, atomic force microscopy and zeta-potential measurements to characterize the overall structure and association behavior of the full-length M1 at different pH conditions. We demonstrate that the protein consists of a globular N-terminal domain and a flexible C-terminal extension. The globular N-terminal domain of M1 monomers appears preserved in the range of pH from 4.0 to 6.8, while the C-terminal domain remains flexible and the tendency to form multimers changes dramatically. We found that the protein multimerization process is reversible, whereby the binding between M1 molecules starts to break around pH 6. A predicted electrostatic model of M1 self-assembly at different pH revealed a good agreement with zeta-potential measurements, allowing one to assess the role of M1 domains in M1-M1 and M1-lipid interactions. Together with the protein sequence analysis, these results provide insights into the mechanism of M1 scaffold formation and the major role of the flexible and disordered C-terminal domain in this process.
NoteThe present study partly includes recently published preliminary results [Ksenofontov AL et al. (2011) 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-a-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.
The first attempt has been made to suggest a model of influenza A virus matrix M1 protein spatial structure and molecule orientation within a virion on the basis of tritium planigraphy data and theoretical prediction results. Limited in situ proteolysis of the intact virions with bromelain and surface plasmon resonance spectroscopy study of the M1 protein interaction with lipid coated surfaces were used for independent confirmation of the proposed model.
The method of tritium planigraphy, which provides comprehensive information on the accessible surface of macromolecules, allows an attempt at reconstructing the three-dimensional structure of a protein from the experimental data on residue accessibility for labeling. The semiempirical algorithm proposed for globular proteins involves (i) predicting theoretically the secondary structure elements (SSEs), (ii) experimentally determining the residueaccessibility profile by bombarding the whole protein with a beam of hot tritium atoms, (iii) generating the residueaccessibility profiles for isolated SSEs by computer simulation, (iv) locating the contacts between SSEs by collating the experimental and simulated accessibility profiles, and (v) assembling the SSEs into a compact model via these contact regions in accordance with certain rules. For sperm whale myoglobin, carp and pike parvalbumins, the cro repressor, and hen egg lysozyme, this algorithm yields the most realistic models when SSEs are assembled sequentially from the amino to the carboxyl end of the protein chain.Studies on the protein spatial structure use a panoply of physical, chemical, and biological methods, the foremost of which are certainly x-ray analysis and high-resolution NMR. Notwithstanding their merit, these methods are quite laborious and have a number of inherent limitations as applied to macromolecules. This makes topical a search for alternative means of obtaining structural information, including theoretical approaches (1-4).There are ways to predict the tertiary structure of globular proteins by determining the hypothetically possible sites of contact between secondary structure elements (SSEs) and then arranging the latter into a three-dimensional (3D) complex that should reflect the spatial fold of the macromolecule (5-8). Their weak point is the multiplicity of the admissible models they produce. Thus even for a fairly simple protein, sperm whale myoglobin, there may be several hundred structures. Even if certain restrictions are imposed (6) (their number can be cut to 20), the choice of a single version therefrom remains largely arbitrary. Building a realistic 3D model would be greatly facilitated if data were available on the actually existing contacts. Such information can be obtained experimentally by tritium planigraphy. Our early work with pike parvalbumin III (a globular Ca 2ϩ binding protein), which demonstrated that planigraphic data can be used for spatial modeling (9), was a stepping stone to developing a basically novel concept of protein 3D reconstruction that would combine the conventional SSE prediction algorithms with analysis of the accessibility of amino acid residues for external tritiation. In that pilot study, labeling of each residue in the whole molecule was considered relative to its labeling in a fully exposed state [tripeptide, Gly-Xaa-Gly (10)]; thus, we initially did not distinguish the changes in accessibility (shielding) due to contacts between SSEs. However, this is a point of principal importance...
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