Figure 1.
Candidate residues responsible for pH dependent VDE conformational change.
Superimposition of the VDE central domain structure (VDEcd) at pH 7 and 5. The regions where structure organization is conserved in the two structures are shown in yellow while the ones showing differences are highlighted in red and blue for the inactive (VDEpH7) and active (VDEpH5) structure, respectively. The second monomer of the active dimeric structure is shown in grey to visualize the dimerization interface. Major rearrangements upon activation involve loops L1, L3, L5, L7. Putative residues involved in the pH dependent conformational change (D86, D98, D117, D206, H168) are shown as green sticks. A) Top view of the monomers. B) same as A after x axis rotation of 90°.
Table 1.
pKa of selected VDE residues.
Table 2.
Enzyme activity of VDE variants compared to WT.
Figure 2.
Electrostatic interaction node maps and position of H121-Y214 hydrogen bond.
Electrostatic interaction node maps generated from the results of the multi conformer continuum electrostatic calculations for A) the VDEpH7 and B) the VDEpH5. Acids and bases are shown as red and blue, respectively. The width of the edge is scaled according to the strength of the interaction. Green and red edges indicate stabilizing or de-stabilizing interactions, respectively. Electrostatic interaction node maps of the VDEpH5 show residues of chain B opaque. A contact map of the two subunits is shown in Figure S2. Interaction are only shown if the absolute electrostatic interaction is greater than 0.4 kcal mol−1. The graph densities are 0.059 and 0.029, for VDEpH7 (A) and VDEpH5 (B) node maps, respectively. Interaction cutoff is 0.2 kcal mol−1. Major interaction hubs, representing k-cores of 4 or 7 and 6 in the VDEpH7 (A) or VDEpH5 (B) form, respectively, are highlighted with yellow circles.
Figure 3.
Position of H121-Y214 hydrogen bond in VDEpH7 structure.
A) Top view of the inactive monomer, shown as grey cartoons. H121 and Y214 are shown as red sticks. B) Same as C after x axis rotation of 90° rotation. A part of the β-barrel is not shown for the sake of clear visualization of the H121–Y214 hydrogen bond.
Figure 4.
Results of the VDE structural integrity during 10 ns molecular dynamics simulations.
Representative results of the molecular dynamics simulations of VDE in water show the hydrogen bond, i.e. the distance between ND1-H121 and HH-Y214 (black trace) and the amount of water within the central hydrophobic barrel of VDE (grey trace, solvent contacts within 0.5 nm of Y214-HH). Opening of the barrel structure is required for dimerization and activation of VDE. In (A) protonation states of VDE were chosen as calculated using continuum electrostatics, B–F) the five target residues, i.e. with a pKa within the activation range of VDE have been fixed in their protonated state, one at the time: D98 (B), D117 (C), H168 (D), D206 (E), D86 (F). G) same analysis was performed with all four candidate residues for pH dependent conformational change while in their protonated state.
Figure 5.
Sequence alignment of VDE from different plant and diatom species.
ClustalW alignment of VDE sequences from plants (At, Arabidopsis thaliana, Nt, Nicotiana tabacum, Os, Oryza sativa) and diatoms (Pt, Phaeodactylum tricornutum, Tp, Thalassiosira pseudonana). Only the region corresponding to VDEcd, for which structural data is available is shown. Residues numbering refers to Arabidopsis mature protein. Residues discussed in the text are shown in black, while the others are in grey. Consensus sequence (100% identity) and conservation percentage are also shown.
Figure 6.
A model of VDE activation according to presented data is shown. The first step is the transition from the closed barrel structure (experimental inactive VDE) to a more open structure, where the cavity is more accessible to water and where the H121–Y214 hydrogen bond is destabilized. In a second step the open monomer associates to the membrane and successively dimerizes (thanks to interactions involving D114 and R138), leading to the final active conformation (active VDE). Here Y198 and D177 are available to catalyze the enzymatic reaction. In the absence of D98, D117, H168 and D206, the formation of active dimer is less efficient but possible since steps 2 and 3 are dependent from other residues.