Model of molecular contributors to FARV mono- and poly-Ub activity.
(A) FARV vOTU (reddish orange) in a Coot-calculated secondary structure overlay with CCHFV vOTU (gray) bound to Ub (PDB ID 3PRP). The selectivity pocket of FARV vOTU is shown in Panel I, with other elements potentially diminishing FARV vOTU Ub activity in Panels II and III. Black dashes show hydrogen bond interactions. Inset shows the Ub-AMC activity relative to WT of FARV and TAGV vOTU mutants. Error bars represent the standard deviation of two independent experiments. (B) Enzymatic activity of FARV vOTUΔ79–107 compared to WT for Ub-AMC and K48/K63 FRET-TAMRA (Panel I), gel cleavage assays of K48/K63 di-Ub with FARV vOTUΔ79–107 (Panel II), and gel cleavage assays of K48/K63 tri-Ub with WT FARV vOTU and FARV vOTUΔ79–107 (Panel III). (C) Model of tri-Ub binding with FARV vOTU. The proximal Ub of K6 linked (PDB ID 5OHP), K11 linked (PDB ID 5LRV), K48 linked (PDB ID 5E6J), and K63 linked (PDB ID 2JF5) di-Ub was anchored to bound mono-Ub based on a secondary structure alignment in Coot. The filled circle indicates the common space that would likely be occupied by the Ub interacting with the second site of interaction of FARV vOTU.