Influenza virus Matrix Protein M1 preserves its conformation with pH, changing multimerization state at the priming stage due to electrostatics

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

Structural Analysis of Influenza A Virus Matrix Protein M1 and Its Self-Assemblies at Low pH

<div><p>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.</p></div

Shape restoration of the NM-domain.

<p>Left panel: experimental SAXS data (1), the transformed from <i>p(r)</i> and extrapolated to zero scattering angle intensity (2), scattering pattern computed from the GASBOR model (3). Insert: distance distribution function <i>p(r)</i> computed by GNOM. Right panel: the model reconstructed by GASBOR (red balls, dummy residues, green balls: dummy water molecules) (a), crystal structure of the NM-domain (PDB code 1AA7) (b).</p

Experimental SAXS patterns from the full length M1 protein (left panel) and the NM-domain (right panel).

<p>The individual curves correspond to the varying solute concentrations; for M1, curves 1 to 4 represent c =  4.5 mg/ml; 3.4 mg/ml, 2.3 mg/ml and 1.7 mg/ml, respectively; for NM-domain, curves 1 to 3 represent c = 3.8 mg/ml, 3.0 mg/ml and 1.5 mg/ml, respectively.</p

AFM topography image of M1 protein structures formed at negatively charged surface.

<p>The inset displays a magnification of the region outlined by white frame.</p

Guinier and Kratky plots for the M1 protein and for the NM-domain.

<p>Left panel: Guinier plots of M1 (1 – experimental data; 2 – Guinier fit) and of the NM-domain: (3 – experimental data; 4 – Guinier fit). Right panel: Kratky plots for M1 (1) and NM-domain (2).</p

The <i>R_g</i> and <i>D_max</i> computed from the solution of the full length M1 protein as a function of the cut-off value at small angles <i>s_min</i>.

<p>The <i>R_g</i> and <i>D_max</i> computed from the solution of the full length M1 protein as a function of the cut-off value at small angles <i>s_min</i>.</p

Flexibility of the C-terminal of M1 analysed by EOM.

<p>Left panel: experimental SAXS data (1) and the scattering from the selected ensemble. Middle and right panels: <i>R_g</i> and <i>D_max</i> distributions, respectively (random pool (1), selected ensemble (2)).</p

Sedimentation velocity experiment on the M1 protein (left) and the NM-domain (right).

<p>Top panels, raw data; absorbance at 280 nm plotted as a function of the radial position, every fifth curve is shown; lower panels, fitted distributions of the sedimentation coefficients.</p