Structural characterization of the mechanosensitive channel candidate MCA2 from Arabidopsis thaliana

Abstract

Mechanosensing in plants is thought to be governed by sensory complexes containing a Ca²⁺-permeable, mechanosensitive channel. The plasma membrane protein MCA1 and its paralog MCA2 from Arabidopsis thaliana are involved in mechanical stress-induced Ca²⁺ influx and are thus considered as candidates for such channels or their regulators. Both MCA1 and MCA2 were functionally expressed in Sf9 cells using a baculovirus system in order to elucidate their molecular natures. Because of the abundance of protein in these cells, MCA2 was chosen for purification. Purified MCA2 in a detergent-solubilized state formed a tetramer, which was confirmed by chemical cross-linking. Single-particle analysis of cryo-electron microscope images was performed to depict the overall shape of the purified protein. The three-dimensional structure of MCA2 was reconstructed at a resolution of 26 Å from 5,500 particles and appears to comprise a small transmembrane region and large cytoplasmic region.

Figure 1. Carboxyl-terminally tagged MCA1 and MCA2 retained their activities.

(A) Ca²⁺ accumulation in yeast mid1 mutant cells carrying various plasmids. Cells were grown to the exponential phase in SD.Ca100 medium and incubated for an additional 2 h at 30°C with 185 kBq/ml of ⁴⁵CaCl₂ (1.81 kBq/nmol). The radioactivity that accumulated in these cells was measured as described in the Materials and Methods. The mean for at least three independent experiments (± SD) is shown for each yeast strain. The plasmids carried by the mid1 mutant are as follows: YEpT-MCA1-6H (for MCA1-6H), YEpT-MCA2-6H (MCA2-6H), and YEpTDHXho (control). *, p<0.001 versus control. (B) Ca²⁺ accumulation in insect Sf9 cells expressing MCA1-6H or MCA2-6H. Cells were infected with a recombinant baculovirus carrying MCA1-6H or MCA2-6H cDNA at a MOI of 1.0 and incubated for 2 days at 25°C. Infected cells were harvested, washed, and resuspended in uptake solution as described in the Materials and Methods. The suspension was incubated for 30 min with 11.1 kBq/ml of ⁴⁵CaCl₂ (0.444 kBq/nmol). The radioactivity that accumulated in the cells was measured as described above. The mean for three independent experiments (± SD) is shown for Sf9 cells expressing MCA1-6H, MCA2-6H, or β-glucuronidase (control). **, p<0.05 versus control.

Figure 2. Expression profiles in Sf9 cells infected with a recombinant baculovirus.

Western blot analysis of the expression of the MCA1-6H (A, C) and MCA2-6H (B, C) proteins using anti-MCA1 (A), anti-MCA2 (B), and anti-6xHis tag (C) antibodies. Differences in the post-infection times and MOI are shown above the panels.

Figure 3. Size-exclusion chromatography and stoichiometry analysis.

(A) Size-exclusion chromatogram of affinity-purified MCA2-6H. The retention volumes of each molecular weight standard are shown with molecular weights above the panel. The inset shows the results of SDS-PAGE visualized by CBB staining of the first and second elution (E1 and E2) from the affinity column. (B) SDS-PAGE under non-reducing conditions of purified MCA2-6H followed by CBB staining. Arrowheads indicate the possible monomer, dimer, trimer, and tetramer of MCA2-6H. The bottom arrowhead shows an additional band, which may correspond to a monomer with an internal disulfide bond of MCA2-6H. (C) SDS-PAGE under reducing conditions of cross-linked MCA2-6H followed by CBB staining. Arrowheads represent the positions of the monomer, dimer, trimer, and tetramer of MCA2-6H. The time (min) of crosslinking is shown under the bottom of each lane.

Figure 4. Cryo-EM micrographs of (A) ZPC cryo-EM and (B) defocus phase contrast cryo-EM.

Those two images were taken at the same area under (A) in-focus and (B) 5 µm underfocused conditions. (C) Comparison of the rotationally averaged power spectrum of the ZPC cryo-EM micrograph before and after filtering. The dashed line represents an original ZPC cryo-EM image and the continuous line represents a filtered ZPC cryo-EM image. (D) (E) Examples of particles picked from original ZPC cryo-EM images and filtered ZPC cryo-EM images, respectively. The contrast of each particle stack has been inverted.

Figure 5. Cryo-EM structure reconstruction (A) Class-averages from the reference-free two-dimensional classification.

Of the 200 total classes, 14 are shown. (B) Surface representation of the initial model that was used for projection-matching refinement. (C) Fourier shell correlation (FSC) between reconstructions from even and odd halves of the data set, plotted against spatial frequency. The value fell to the criterion level of 0.5 at a resolution of 26 Å. (D) Projection angle distribution. Each particle image represents a projection of the three-dimensional channel particle, with the projection direction defined by the Euler angles β and γ. The angle γ represents a rotation about the symmetry axis, and β is a rotation normal to the axis. Because of the four-fold symmetry, unique projection directions are described by values of both β and γ in the range 0 to 90°. The intensities of the dots in the figure represents the number of particle images assigned to each pair of β and γ values, and shows that the angle distribution was well sampled as required for good 3D reconstructions. (E) Comparison between reference-free 2D classification (top raw), classes for final reconstruction (middle raw), and reprojections of 3D reconstruction (bottom raw).

Figure 6. Surface representation of MCA2-6H viewed from four different angles.

Surface representations of MCA2-6H corresponding to a molecular size of 200 kDa. (A) Top view, (B) bottom view, (C) side view, and (D) side view turned 45° in the z-axis, which is superposed with the crystal structure of the transmembrane segment of the M2 proton channel protein from influenza A virus (PDB #3C9J) and schematic illustration of the bilayer lipid membrane to propose membrane topology.