Pathogen sensing by the intracellular antibody receptor, TRIM21, is regulated by B-box autoinhibition and ring phosphorylation

Dickson CF1,2, Fletcher AJ1, Vaysburd M1, Yang JC1, Mallery DL1, Mclaughlin SH1, Chin JW1, Neuhaus D1 and James LC1

  1. MRC Laboratory of Molecular Biology, Cambridge, UK.
  2. SMS, School of Medical Sciences, UNSW, Australia.

TRIM21 is a cytosolic antibody receptor that performs pathogen sensing and antibody-dependant intracellular neutralisation of viruses. Both signalling and effector functions require E3 ubiquitin ligase activity, which is provided by an N-terminal RING domain. TRIM21 induces a potent antiviral immune response, however, it does not constitutively signal, suggesting that the enzymatic activity of the RING is tightly regulated. We have used structural studies together with in vitro ubiquitination assays to show that a domain of unknown function, the B-box domain, represses TRIM21 activity by competing with E2 ubiquitin conjugating enzymes for binding to the RING. We further demonstrated that cellular TRIM21 is phosphorylated by IKKβ and TBK1 at serine 80, located at the RING-B-box interface. By introducing an phosphomimetic or a phosphoserine at position 80 in a recombinant RING-B-box protein we showed that phosphorylation of the interface relieves B-box mediated autoinhibition of the RING by promoting E2 binding and restoring enzymatic activity to the uninhibited state. Cells expressing TRIM21 carrying an S80E mutation constitutively activate NF-κB and produce a more robust cytokine response upon infection with DNA or RNA viruses, while an S80A mutation attenuated cytokine production. In contrast, neutralisation activity was unaffected by mutation of S80, consistent with a different threshold for these two activities. This work identifies several layers of regulation that fine tune the signalling activity of TRIM21 and highlights the importance of controlling inflammation, which unchecked has the potential to cause tissue damage and autoimmune disease.