Legionella pneumophila regulates the activity of UBE2N by deamidase-mediated deubiquitination (2019)
Figure 3

Fig. 3 Structural analysis of the mechanism of substrate recognition and catalysis by MvcA.

A. Domains present in the MvcA-UBE2N-Ub complex. Top panel: A linear model of the domains in the region used for structural determination in which the tail domain (blue), core domain (green) and the insertion domain (yellow) are shown. Lower panel: Ribbon representation of the domains of MvcA and its substrate observed in the crystals. The tail domain contains three helices (α2, α3 and α15). The linkers between the tail domain and the core domain are termed loop 1, 2 and 5 (red) and those that connect the insertion domain and the core domain are termed loop 3 and 4 (red). Ubiquitin (Ub) is shown in magenta and UBE2N in cyan. The view on right is generated by rotating the structure shown on the left by 180 degrees around the indicated axis.

B. The movement of the tail domain and the insertion domain in MvcA upon substrate binding. Note the shift (arrows) of the tail domain (right) and the insertion domain between Apo MvcA(marine) and MvcA in the complex (green). The left box highlights the rotation suffered by the insertion domain upon complex formation, and the right box shows the rotation observed in the tail domain.

C. The structure of UBE2N (cyan)-Ub (magenta) in the complex. Lys92 of UBE2N and Gln40 of ubiquitin linked by an isopeptide bond produced from MavC-catalyzed transglutamination are shown as sticks. The conformational change of the 310 helix of UBE2N is indicated by a box. The structure of apo-UBE2N (PDB code: 1JAT) not conjugate to ubiquitin is shown in grey for comparison.

D. Key residues involved in the formation of the catalytic center from MvcA (core domain: green, insertion domain: yellow, tail domain: blue) and their interactions with UBE2N-Ub (UBE2N, cyan; Ub, magenta). Left panel: the detail view of the interactions. Right panel: the 2Fo-Fc map of the left panel (it was contoured at 1.2 σ). Residues in the catalytic triad (Ala(Cys)83-His244-Q265, yellow), Ser82, Arg140, Gly242, Ser243, and Phe268 of MvcA, Lys92 and Asp93 of UBE2N, as well as Asp39 and Gln40 of Ub are shown in sticks. Hydrogen bonds are shown as dashed lines.

E-G. Detailed views of UBE2N contact patches A (E), contact B (F) and contact C (G). Key residues involved in interactions between MvcA (core domain: green, insertion domain: yellow, tail domain: blue) and UBE2N (cyan) are labeled and shown as sticks. Leu330

of MvcA is labeled in red stick in F and yellow stick in the bottom right panel, which shows it interacting with a nonpolar pocket of UBE2N (shown in electrostatic surface). Hydrogen bonds are shown as dashed lines.

H. Mutational analysis of residues important for substrate recognition by MvcA predicted by structural analysis. For deubiquitination reactions, the indicated amounts of MvcA or its mutants were incubated with 2.0 μg UBE2N for 5 min in 25 μl reactions. For ubiquitin deamidase activity, the indicated amounts of MvcA or its mutants were incubated with 10 μg of ubiquitin for 60 min in 25 μl reactions. The ratio of cleavage was analyzed by SDS-PAGE and then densitometry. Each bar represents the ratio of cleavage or deamidation obtained from three independent experiments. Bars and error bars: means and standard error of the mean (SEM).