A detailed understanding of the molecular mechanisms whereby ubiquitin (Ub) recognizes enzymes in the Ub proteasome system is crucial for understanding the biological function of Ub. Many structures of Ub complexes have been solved and, in most cases, reveal a large structural epitope on a common face of the Ub molecule. However, owing to the generally weak nature of these interactions, it has been difficult to map in detail the functional contributions of individual Ub side chains to affinity and specificity. Here we took advantage of Ub variants (Ubvs) that bind tightly to particular Ub-specific proteases (USPs) and used phage display and saturation scanning mutagenesis to comprehensively map functional epitopes within the structural epitopes. We found that Ubvs that bind to USP2 or USP21 contain a remarkably similar core functional epitope, or "hot spot," consisting mainly of positions that are conserved as the wild type sequence, but also some positions that prefer mutant sequences. The Ubv core functional epitope contacts residues that are conserved in the human USP family, and thus it is likely important for the interactions of Ub across many family members.
Submitter: Marie Ary
Submission Date: June 14, 2017, 5:50 p.m.
|Number of data points||2049|
|Proteins||Ubiquitin variant Ubv.2.1 ; Ubiquitin-specific protease 2 (USP2) ; Ubiquitin variant Ubv.21.4|
|Assays/Quantities/Protocols||Experimental Assay: kcat ; Experimental Assay: kcat/Km ; Experimental Assay: Km ; Experimental Assay: IC50 for inhibition of USP2 ; Experimental Assay: Binding of Ubv.2.1 variants to USP2 ; Experimental Assay: Binding of Ubv.21.4 variants to USP21 ; Derived Quantity: Fold effect (kcat/Km variant relative to kcat/Km WT) ; Derived Quantity: IC50(K6N;T12H)/IC50 ; Computational Protocol: ΔΔG-bind|
|Libraries||IC50s for inhibition of USP2 by Ub.wt and Ubvs (Table 1) ; ΔΔG-bind from computational saturation scan of Ubv.2.1 point variants with USP2 (Fig. S4C) ; Kinetic values from Ub-AMC cleavage by USP2 variants (Table S1) ; Saturation scan analysis of 25 positions in Ubv.21.4-USP21 interface (Fig. S2B) ; Saturation scan analysis of 25 positions in Ubv.2.1-USP2 interface (Fig. S2A) ; ΔΔG-bind from computational saturation scan of Ubv.21.4 point variants with USP21 (Fig. S4D)|