Table 1.
Data and Refinement Statistics.
Figure 1.
X-ray Crystal Structures of WT-HiDapE and HiDapET.
A) Dimer architecture based on the structure of WT-HiDapE. B) Close-up view of the catalytic domain of WT-HiDapE. Ribbon diagram showing the active site formed by six loops (LI–LVI); five of them coordinate the Zn ions (LI–IV & VI). C) Superimposition of the structure of WT-HiDapE (black) over HiDapET (magenta). Active site Zn(II) ions are shown as black and magenta spheres for WT-HiDapE and HiDapET, respectively. Regions where differences are most prominent are labeled Region I (yellow) and Region II (orange). Yellow and magenta circles highlight two disordered loop regions in the HiDapET structure. The red dots marked disordered loop that contains conserved His residue.
Table 2.
Dynamic light scattering data.
Figure 2.
X-ray Crystal Structures of VcDapET.
A) Superimposition of apo-VcDapET (blue) over [ZnZn(VcDapET)] (cyan), showing the identical nature of the catalytic domains. Zinc atoms for VcDapET are shown as black spheres. B) Comparison of [ZnZn(VcDapET)] (cyan) and [ZnZn(HiDapET)] (magenta). Regions I (yellow) and II (orange) identify the areas where the most significant differences between the two structures exist. Six loops (LI–LVI) forming the active site are labeled. Zinc atoms for VcDapET are shown as black spheres while the residues coordinating the metal ions are shown as lines. C) Close-up view of the active site environment of the [ZnZn(VcDapET)] with the 2Fo−Fc electron density map (with the Zn ions and ethylene glycol molecules omitted from the calculation). D) Close-up view of residues from loops I–IV and VI interacting with the Zn(II) ions in the structure of [ZnZn(VcDapET)]. E) The 2Fo−Fc (blue, 1 σ) and Fo−Fc electron-density maps (red and green at −3σ and 3σ) of the LV loop region in the apo-VcDapET.
Figure 3.
Diagrams Showing Regions of Flexibility in Truncated DapE Proteins.
A) MOLMOL diagram of [ZnZn(VcDapET)] molecular dynamics. B) MOLMOL diagram of [ZnZn(HiDapET)] molecular dynamics (the thickness of the line is proportional to the variation of the protein structure during the simulation). The crystallographic temperature factors indicating that the most dynamic (in red) and the most rigid (in blue) parts of the protein: C) [ZnZn(VcDapET)]. D) [ZnZn(HiDapET)].
Figure 4.
Molecular Dynamic Simulation Showing Regions of Flexibility in Catalytic Domain.
A) [ZnZn(VcDapE)]. B) [ZnZn(HiDapE)]. C) AAP. The thickness of the line is proportional to the variation of the protein structure during the simulation. AS indicates the active site area, LVeq. equivalent of the LV loop in HiDapE).
Figure 5.
The Active Site of WT-HiDapE Showing Loop V.
T325 resides on loop V directly over the dinuclear active site.
Figure 6.
The Role of the Dimerization Domain in the Stabilization of Loop V in WT-HiDapE.
A) Superimposition of the WT-HiDapE (gray) and VcDapET (cyan) structures is shown. Loop V of WT-HiDapE and VcDapET is labeled as HiLV and VcLV, respectively. WT-HiDapE residues interacting with the sulfate ion (stick model) are shown as gray lines. Corresponding residues in VcDapET (except for R258 that is absent in the deletion mutant) are shown as yellow (R179 and R180) and orange (G214) lines. B) Specific orientation of the active site loop V in VcDapET and the corresponding loop in AAP. Overlay of the VcDapET (cyan) and AAP (purple) structures is shown. The AAP loop and VcDapET loop V are labeled as ApLV and VcLV, respectively. Stabilization of loop V in AAP by a disulfide bridge is indicated where Cys223 and Cys227 of AAP and the residues involved in zinc-binding in VcDapET are shown as sticks. Zinc ions of VcDapET are shown as black spheres. Zinc-bound ethylene glycol was omitted for clarity.