(a) Thermodynamic cycle of OTUB1 binding independently to either an E2 or K48 diUbiquitin which in turns favors binding of the other. Each side of the cycle is designated by a letter along with the binding affinities determined in this paper. (b) When comparing each side of the thermodynamic cycle with equilibrium dissociation constants, A × B must equal C × D. Therefore, the constants can be arranged so that Kd be calculated. (c) Binding affinities previously determined, A B and C, and those calculated, D. D is determined using the KM values, representing the affinity of OTUB1 for K48 diUb in the presence of an E2, since Kds were only measured for a select number of E2s.