Fig 1.
Mouse IRG1 purification and activity analysis.
(A) Itaconate production catalyzed by IRG1 using cis-aconitate as the substrate. (B) Size-exclusion chromatography profile. The calibrated positions of the size markers are indicated above the profile. (C) MALS profile; the red line indicates the experimental mass size. (D) HPLC profiles. The eluted positions of cis-aconitate (upper panel) and itaconate (lower panel) are indicated by black arrows. (E) HPLC profiles showing IRG1 activity. The peak produced by the enzymatic reaction is indicated by a black arrow.
Table 1.
Data collection and refinement statistics.
Fig 2.
Crystal structure of mouse IRG1.
(A) Cartoon representation of dimeric mouse IRG1. (B) Domain boundary of mouse IRG1. The positions of the helical domain and the lid domain are indicated using a bar diagram. A rainbow-colored cartoon representation of monomeric mouse IRG1 is shown in the lower panel. The chain from the N- to C-terminus is colored blue to red. (C) Cartoon representation showing the two distinct domains of mouse IRG1.
Fig 3.
Structural comparison of mouse IRG1 with the recently reported mouse IRG1 (PDB ID: 6R6T).
(A) Comparison of the dimeric structure of our mouse IRG1 with the 6R6T model by superposition analysis. (B) Superposition of the monomeric structure of our mouse IRG1 and the 6R6T model. The tilt angle of the lid domain of our mouse IRG1 is indicated above the cartoon. (C) Details of the active site of our mouse IRG1 compared with that of the 6R6T model. The two loops around the active site are labeled as A1 loop and A2 loop. (D) The 2Fo-Fc electron density map contoured at the 1.0 σ level around A1 loop. (E) Close-up view of the active site. The unknown Fo-Fc electron density contoured at the 3σ level is indicated with a blue mesh, the residues that form the active site are labeled, and the positions of the A1 and A2 loops are indicated by black arrows. (F) The interactions between A1 loop and A2 loop. Red-dash lines indicate hydrogen bonds. (G) Electrostatic surface representation of our mouse IRG1 and mouse IRG1 (6R6T). Dotted black circles indicate the active sites.