Second site of FARV vOTU interaction with di-Ub.

(A) Model of FARV vOTU (reddish orange) bound to K63-linked di-Ub (purple; PDB ID 2JF5). FARV vOTU was overlaid with CCHFV vOTU bound to Ub (PDB ID 3PRP; not rendered) based on secondary structure alignment of the vOTUs. The distal Ub was anchored to the bound mono-Ub by aligning the secondary structure in Coot, followed by manual bond rotations within Lys63 of the proximal Ub in PyMol to model a plausible fit with minimal clashes based on the CCHFV vOTU active site and protease surface. The predicted region of FARV vOTU engagement with the proximal Ub is indicated by a black box. (B) Closeup view of the predicted region, with the residues selected for mutation shown as sticks. Activity of the mutants relative to WT is shown for Ub-AMC, Ub-Rh110, K48 di-Ub FRET-TAMRA, and K63 di-Ub FRET-TAMRA (right).