Deciphering the Mechanism of Interactions between Polyubiquitin Chains and Caspase Activation and Recruitment Domains Using Protein–Polymer-Engineering-Assisted Native Mass Spectrometry

Polyubiquitin (polyUb) chain topology plays important roles in determining Ub chain conformation and therefore directs specific cellular signal transductions. However, little is known about the conformational dynamics of polyUb chains and their complex topology. A major challenge lies in the inherent heterogeneity of native polyUb chains, which often exist as mixtures of different lengths and linkage types, making it difficult to achieve molecular-level characterization. Herein, we synthesized a panel of Lys63(K63)-linkage-specific polyUb chain ladders via E1–E2 cascade reactions as a platform for complexation with K63-linkage-specific binding proteins, including RNA sensors RIG-I, MDA5, and their adaptor MAVS, which govern the antiviral-signaling pathways in innate immunity. Taking advantage of precise stoichiometry control, we used native mass spectrometry to decipher their dynamic signaling mechanisms. The flexible range of polyUb chain lengths allowed polyUb to populate mono-, di-, and trimeric states and the corresponding conformations in solutions. Hexamer and undecamer were identified as the minimum-length polyUb chains adopting conformers enabling stable binding to RIG-I CARDs and MDA5 CARDs at a 1:4 stoichiometry, respectively, demonstrating that a single Ub6 or Ub11 chain can stabilize the tetrameric structures of these antiviral-signaling complexes. Alternatively, polyUb chains enable the flexible accommodation of alternative stoichiometric constraints to regulate the tetramerizing events by RIG-I CARDs and MDA5 CARDs. Further in-depth analysis of RIG-I/MDA5 CARDs– MAVS CARD–K63-polyUb14 + complexes uncovered the assembly of macromolecular complexes featuring over 9 layers of MAVS CARD filaments. These findings elucidate the intricate interplay between the length of K63-polyUb chains and their dynamic binding stoichiometries and topologies during signaling transductions.