Sequence rules for a long SPOP-binding degron required for protein ubiquitylation.

The adaptor protein, Speckle-type BTB/POZ protein (SPOP), recruits substrates to the cullin-3-subclass of E3 ligase for selective protein ubiquitylation. The Myddosome protein, Myeloid differentiation primary response 88 (MyD88), is ubiquitylated by the SPOP-based E3 ligase to negatively regulate immune signaling, however, the sequence rules for SPOP-mediated substrate engagement and degradation are not fully understood. Here, we show that MyD88 interacts with SPOP through a long degron that contains the established SPOP-binding consensus and an N-terminal site that we name the Q-motif. Based on sequence similarity to MyD88, we show that additional substrates, including Steroid receptor coactivator-3 (SRC-3), SET domain-containing protein 2 (SETD2) and Caprin1, engage SPOP in this manner. We show that the Q-motif is a critical determinant of these interactions in mammalian cells and determine X-ray crystal structures that show the molecular basis of SPOP associations with these proteins. These studies reveal a new consensus sequence for substrate-binding to SPOP that is necessary for substrate ubiquitylation, thus expanding the sequence rules required for SPOP-mediated E3 ligase substrate recognition.