Asymmetry in ubiquitin binding by A20 reveals early recognition features of the ubiquitin code

Linear (Met1‐linked) polyubiquitin chains play essential roles in NF‐κB signaling, with readers such as A20 specifically recognizing these chains via specialized domains. Although structural data exist for the linear diubiquitin‐A20 ZF7 complex, the basis for its strong preference for linear polyubiquitin remains unclear. Here, we investigated the early steps of ubiquitin recognition by A20‐ZF7, identifying that A20‐ZF7 exhibits weak binding to monoubiquitin and distinct exchange kinetics at its two ubiquitin‐binding sites, with slow exchange at the proximal site reflecting stronger binding. Comparison of monoubiquitin and linear diubiquitin binding suggested a multistep mechanism involving a kinetically resolvable intermediate state absent in the monoubiquitin interaction. This intermediate likely facilitates proper ubiquitin chain positioning and contributes to A20‐ZF7's specificity for linear polyubiquitin. Impact statement We reveal transient intermediate states in ubiquitin recognition by A20 ZF7, showing that specificity for linear chains arises from a multistep kinetic pathway. This work highlights the importance of dynamic binding processes in decoding ubiquitin signaling.