Fig. 4
From: Growth inhibition of Saccharomyces cerevisiae by SUMO-specific nanobodies
VHH1SMT3 and VHH2SMT3 inhibit cleavage of Smt3p-tagged proteins by Ulp1p. (A,B) X-ray crystal structure (PDB: 1EUV) of SUMO protease (Ulp1p) in complex with Smt3p. Residues perturbed upon binding of VHH1SMT3 (A) and VHH2SMT3 (B) as determined by NMR are indicated in the color code of Fig. 3. (C–E) Cleavage of Smt3p-tagged proteins by Ulp1p in the presence and absence of a twofold molar excess of VHH over Smt3p at the indicated time points for: Smt3p-SBP-His6 (C), His6- Smt3p-Ube2G2 (D), and Smt3p-GFP-SBP-His6 (E). Quantification of cleavage, based on 3 independent experiments, is shown on the left. A representative SDS PAGE gel of the reaction is shown on the right. Major protein species are identified to the right. (F) Overlay of plots shown in (C–E) highlight the distinction in amount of cleaved product for the indicated substrates.
