Ordered assembly of the cytosolic RNA-sensing MDA5-MAVS signaling complex via binding to unanchored K63-linked poly-ubiquitin chains

Abstract

The RNA sensor MDA5 recruits the signaling adaptor MAVS to initiate type I interferon signaling and downstream antiviral responses, a process that requires K63-linked polyubiquitin chains. Here, we examined the mechanisms whereby K63-polyUb chain regulate MDA5 activation. Only long unanchored K63-polyUb_n (n ≥ 8) could mediate tetramerization of the caspase activation and recruitment domains of MDA5 (^MDA5CARDs). Cryoelectron microscopy structures of a polyUb₁₃-bound ^MDA5CARDs tetramer and a polyUb₁₁-bound ^MDA5CARDs-^MAVSCARD assembly revealed a tower-like formation, wherein eight Ubs tethered along the outer rim of the helical shell, bridging ^MDA5CARDs and ^MAVSCARD tetramers into proximity. ATP binding and hydrolysis promoted the stabilization of RNA-bound MDA5 prior to MAVS activation via allosteric effects on CARDs-polyUb complex. Abundant ATP prevented basal activation of apo MDA5. Our findings reveal the ordered assembly of a MDA5 signaling complex competent to recruit and activate MAVS and highlight differences with RIG-I in terms of CARD orientation and Ub sensing that suggest different abilities to induce antiviral responses.

Keywords: HDX-MS; K63-polyUb chain; MAVS; MDA5; cryo-EM.