Skip to main content
. Author manuscript; available in PMC: 2020 Jan 22.
Published in final edited form as: Nature. 2019 Jul 22;572(7769):387–391. doi: 10.1038/s41586-019-1439-1

Fig. 4. Structural analysis of the mechanism of SidJ-catalyzed glutamylation.

Fig. 4

a. Domain organization of SidJ. SidJ consists of the N-terminal domain (orange), the Central domain (yellow) and the C-terminal domain (green).

b. Ribbon diagram representation of the SidJ-CaM complex. In top panels, the N-terminal domain (orange), Central domain (yellow), C-terminal domain (green) of SidJ and CaM (red) are shown. The view on top right is generated by rotating the structure shown on the top left by 180° around the indicated axis. The N and C termini of SidJ are labeled with letters. The missing residues are shown in dashed lines. Lower panels depict interactions between SidJ and the N-lobe/C-lobe of CaM. Residues important for binding are shown in sticks and hydrogen bonds are indicated by dashed lines.

c. Binding of CaM to SidJ and its mutants. The binding affinity was evaluated using microscale thermophoresis. The dissociation constant (Kd) was calculated by the NanoTemper Analysis 2.2.4 software. Data shown were one representative from three experiments with similar results.

d. Ribbon representation of the SidJ-CaM-AMP complex. Key residues of SidJ involved in AMP binding are shown in yellow sticks, AMP is labeled in magenta sticks. Hydrogen bonds are shown as dashed lines. Electron density of an SA (simulated annealing) Fo-Fc omit map for AMP contoured at 3.0 σ.

e. Mutational analysis of residues important for binding AMP. Each SidJ mutant was incubated with SdeA, ATP, L-glutamate and CaM for 2 h before measuring SdeA’s ubiquitin ligase activity.

f-g. Activation of SidJ by ATP analogs. The indicated compounds were incubated with SdeA, GST-SidJ, L-glutamate and CaM for 2 h at 37°C before monitoring SdeA’s activity in ubiquitinating Rab33b. Note that analogs defective in hydrolysis at the α site cannot activate SidJ.

h. The role of residues important for AMP binding in SidJ self-AMPylation. Each SidJ mutant was incubated with 32P-α-ATP, Mg2+ and CaM for 2 h at 37°C and the incorporation of 32P-α-ATP was detected by autoradiography. In panels c, e-h, data shown were one representative from at least three independent experiments with similar results.