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.