Figure 4: Crystal structure of the LotA_N OTU domain bound to K6 diUb.

A. 2.8Å crystal structure of LotA_N (1–276, blue) bound to K6 diUb (shades of red). The structure is labeled with visible termini, domain architecture, active site, and OTU variable regions (VR1–3).

B. Close-up view of the K6 diUb-bound LotA_N active site with 2|Fo|-|Fc| electron density overlaid at 1σ for catalytic triad residues. The K6 side chain of Ub^prox and C-terminus of Ub^dist are also shown with overlaid electron density. The LotA_N catalytically inactive C13A variant was used to preserve the diUb linkage.

C. Crystal structures of K6 diUb in isolation (PDB 2XK5) and bound to LotA_N, aligned by their Ub^dist moieties (top). Availability and orientation of common interaction surfaces are shown, with dashed circles indicating the surfaces utilized by either Ub:Ub or LotA_N:Ub interaction.

D. Structural overlay of LotA_N (blue) and LotC (green) bound to their respective distal Ub moieties (red and pink, respectively). The structures are aligned on their core OTU domains, and highlight large differences in Ub orientation, insertion domains, and use of variable regions.

E. Close-up view of the LotA_N S1 site (blue) bound to Ub^dist (red). Interacting residues are shown in ball-and-stick representation, with hydrogen bonds indicated by dashed lines.

F. Cleavage of fluorescent K6 diUb by the indicated LotA_N S1 site variants at 10 nM concentration monitored by fluorescence polarization.

G. Structural overlay of LotA_N (blue) and human USP30 (tan) bound to their respective distal Ub moieties (salmon and purple, respectively). The structures are aligned on their core catalytic domains, and highlight large differences in Ub orientation and features of their S1’ sites.

H. Close-up view of the LotA_N S1’ site (blue) bound to Ub^prox (salmon). Interacting residues are shown in ball-and-stick representation, with hydrogen bonds indicated by dashed lines.

I. Cleavage of fluorescent K6 diUb by the indicated LotA_N S1’ site variants at 10 nM concentration monitored by fluorescence polarization. These data were collected in parallel with those presented in (F), and the WT dataset is shown again for reference.

J. Gel-based LotA_N cleavage assay of K6 diUb variants with indicated Ub^prox mutations. Reactions were quenched at the indicated times and visualized by SDS PAGE with Coomassie staining.

K. Underlying helical domain architecture of the LotA_N (left) and stereotypical (right) adaptive Ub-binding domain (A-UBD), with helices labeled and the Ub-binding α1–2 regions shown in color.

See also Figure S4.