Fig. 1. FLS2–BAK1 interface correlation is enhanced upon the flg22 binding. The horizontal axis indicates the interface residues in BAK1, and the vertical axis shows the interface residues in FLS2. (a) Interface correlation of FLS2–BAK1 without flg22; (b) with flg22.

Fig. 2. (a), (c) Pitch length and (b), (d) radius distribution of FLS2 superhelix structure for FLS2, FLS2–flg22, and FLS2–flg22–BAK1 at the start (upper panel) and end (lower panel) episodes of the whole 200 ns MD simulations. From left to right, pitch length and its variance decrease. The radius also fluctuates less and slightly decreases.

Fig. 3. First 3 dominant normal modes of FLS2. (a) Left: mode 1 (side view) shows the bending motion with two ends moving asynchronously; Middle: mode 2 (top view) shows the twisting motion with two ends rotating around the center axis in opposite directions; Right: mode 3 (side view) shows another bending motion with two synchronous ends. (b) The x, y, z components (in normalized scale) of the first three normal modes (NMs).

Fig. 4. Principal component analysis of FLS2 MD trajectory. (a) The x, y, z dimensions of the first three principal components. PC1 and PC2 indicate bending modes and PC3 twisting mode of helix. (b) The FLS2 structure distribution density of the MD trajectories in the 2D space spanned by PC1 and PC2 for FLS2-only (lower left), FLS2–flg22 (lower middle), and FLS2–flg22–BAK1 (lower right).

Fig. 5. (a) Explained variance ratio by the top 10 PCs in PCA. Top 3 components (bending1, bending2, and twisting mode) contribute to around 70% of the variance; (b) Correlation of the top 3 components of PCA and the top 3 modes of ANM. PC1 matches best with NM1 (Pearson correlation coefficient 0.96); PC2 corresponds to NM3 (0.93); PC3 to NM2 (0.86). (c) Top PCs and NMs projection correlation. The projection coefficients of the FLS2 MD structures correlate well for PC1–NM1 (left), PC2–NM3 (center), and PC3–NM2 (right), with the correlation coefficients indicated.

Fig. 6. Top PCs and NMs projection distribution. The distribution of projection coefficients of FLS2 structure in (a) PC1, (b) PC2, and (c) PC3 direction. The FLS2 structure in the three different complexes shows no obvious difference in PC1 and PC2 directions while shows an obvious global shift for FLS2, FLS2–flg22, and FLS2–flg22–BAK1 conditions, with FLS2–flg22 being the intermediate state.

Fig. 7. Proposed interaction mechanism of the FLS2–flg22–BAK1 complex. (a) Interaction of FLS2–flg22–BAK1. N- and C-terminal ends of flg22 interact with At-FLS2 and promote the binding of BAK1. Interaction of FLS2–flg15–BAK1 in (b) Arabidopsis thaliana (At) and (c) Solanum lycopersicum (Sl). The new N-terminus of flg15 fails to bind with At-FLS2, thus resulting in little restriction on the twisting dynamics of FLS2 and, therefore much weaker FLS2–BAK1 association or correspondingly, much weaker downstream immune response. In Sl, the N-terminus of flg15 binds with Sl-FLS2 to induce a tight binding of BAK1.

Fig. 8. Conservation analysis of FLS2–flg22 binding sites. (a) Conservation of flg22. The N-terminus (first 7 residues) is less conserved than the C-terminus. (b) The corresponding binding sites of FLS2 with flg22 N-terminus are also less conserved than those with flg22 C-terminus.