Figure 3: Crystal structure of the LotA_N OTU domain.

A. 1.5Å crystal structure of LotA (1–294) labeled with visible termini, domain architecture, active site, and OTU variable regions (VR1–3) described previously³².

B. Close-up view of the LotA_N active site with 2|Fo|-|Fc| electron density overlaid at 1σ for catalytic triad residues. Alternate conformations of the C13 side chain (a and b) are observed, whereas density for the H237 imidazole ring is absent.

C. Cleavage of fluorescent K6 diUb by the indicated LotA_N active site variants at 10 nM concentration monitored by fluorescence polarization.

D. Structural overlay of LotA_N (blue), LotA_M (PDB 7F9X, orange), LotB (PDB 6KS5, yellow), and LotC (PDB 7BU0, green). Structures were aligned by their core OTU domains to demonstrate variability in the insertion domains.

E. Close-up views of the LotA_M, LotB, LotC (PDB 6YK8), and Ub-bound LotC (PDB 7BU0) active sites. Catalytic triad residues are shown in ball-and-stick representation, with hydrogen bonds indicated by dashed lines.

See also Figure S3.