Fig 1.
Overall structure of tbICL in complex with succinate and itaconate.
(A) Reversible catalytic reaction of tbICL, involving isocitrate, glyoxylate, and succinate. (B) Crystal structure of tbICL in the asymmetric unit. The two molecules (subunits A and B) are represented graphically from two different viewpoints. The gray spheres, yellow stick (subunit A), and pink stick indicate Mg2+ ions, succinate, and itaconate, respectively. (C) SEC-MALS profile of tbICl. Measured values using SEC-MALS (red) are plotted as SEC elution volume (x-axis) versus absolute molecular mass (y-axis) distributions on the SEC chromatogram (black) at 280 nm. (D) Tetrameric structure of tbICL. Two 2fold symmetric neighboring molecules (magenta and orange) were identified and modeled with the dimeric structure in the asymmetric unit. (E) The monomeric structure of tbICl (subunit B). (F) Superimposition of subunit B onto subunit A of tbICL.
Table 1.
Data collection and refinement statistics for tbICL in complex with succinate and itaconate.
Fig 2.
Active site structure of tbICL in complex with succinate and itaconate.
(A) Cavities including active sites are colored blue. The dashed ovals indicate active sites. (B) Surface electrostatic potential of tbICL. The scale bar ranges from −5 kT/e (red) to 5 kT/e (blue). The dashed circle indicates the active site in subunit B. Omit maps of succinate (C) and itaconate (D). The omit maps (FO − FC) of succinate and itaconate are colored blue and contoured at the 2.0 sigma and 3.0 sigma level, respectively. Diagrams of succinate (E) and itaconate (F) interactions in the active sites. They interact with water molecules (blue circles) and neighboring residues.
Fig 3.
Structural comparison with other conformers.
Structural comparison of the L5 loop and the C-terminal tail. Subunit A of our tbICL structure (green) is superimposed onto those of the native form (A; orange), glyoxylate and succinate complex (B; magenta), and pyruvate complex (C; wheat) structures. Subunits B are colored gray except for our structure (cyan). All views are restricted to the active site. Succinate from our structure (yellow), glyoxylate (limon), succinate (blue; PDB code: 1F8I), pyruvate (black), and Mg2+ ions (gray) are represented by sticks and spheres. The curved arrows indicate conformational changes of the L5 loop and the C-terminal tail. (D) Subunit B of our tbICL structure (cyan) is superimposed onto those of the structures shown in panel (C). Subunit B, except for our structure, is colored gray for clarity. The pink stick represents itaconate. (E) Magnified view of the active site. Stick symbols and color codes are the same as in panels (A)–(D). (F) Magnified view of the active site in subunit B of our structure, the succinate/glyoxylate-complex structure, and another itaconate (wheat) complex structure (PDB code: 6XPP). Stick symbols and color codes are the same as in panels (E).
Fig 4.
Biophysical properties around the active site.
(A) B-factor distribution of tbICL in complex with succinate and itaconate. The dimeric structure is depicted in putty representation and colored from red (high) to violet (low) in B-factor value order. The dashed circles indicate the L5 loops. (B) Electric field generation of tbICL in complex with succinate and itaconate. Surface electrostatic potential is represented by the same method as in Fig 2B. The potential gradient map of the electric field is contoured at the 0 sigma level. The scale bar for the electric field ranges from −1 kT/e (red) to 1 kT/e (blue). Electric field distributions of our structure (C), the native structure (D: PDB code: 1F61), and the glyoxylate-succinate complex structure (E; PDB code: 1F8I). The arrows indicate active sites.