Covalent modification of the NF-κB essential modulator (NEMO) by a chemical compound can regulate its ubiquitin binding properties in vitro.

Abstract Posttranslational modification by ubiquitin plays important roles in multiple physiological and pathological processes. Ubiquitin-binding proteins play a critical role in recognizing and relaying polyubiquitin-based signaling. NEMO (NF-κB Essential Modulator) is a central player in canonical NF-κB signaling whose major function is to bind to K63- and/or M1 (or linear)-linked polyubiquitin chains generated in response to cell stimulation. Here we show that Withaferin A (WA), a steroidal lactone, causes a change in NEMO′s interaction with specific types of polyubiquitin chains in vitro. WA induces full-length recombinant NEMO to bind to long K48-linked polyubiquitin chains but not tetra-ubiquitin species. Significantly, the UBAN (ubiquitin binding in ABIN and NEMO) domain, essential for NEMO′s ability to bind M1/K63-linked polyubiquitin, is dispensable for the WA-induced gain-of-function activity. Mass spectrometric analysis demonstrated that WA covalently modifies NEMO on a cysteine residue within the C-terminal zinc finger (ZF) domain. Point mutations to the ZF can reverse the WA-induced K48-polyubiquitin binding phenotype. Our study demonstrates the feasibility of directly altering the ubiquitin interaction properties of a ubiquitin-binding protein by a chemical compound, thereby shedding light on a novel drug class to potentially alter polyubiquitin-based cellular processes.