Antibodies are molecules made by the immune system that protect us from infections. They were discovered over 100 years ago, and for most of that time scientists thought they only worked in the bloodstream. Yet recent research showed that when a virus infects our cells it also carries antibodies in with it. Once inside the cell, a protein called TRIM21 recognises the antibody-covered virus and – together with other proteins called ubiquitin enzymes – targets it for destruction via the cell’s waste disposal system. At the same time TRIM21 sends a signal to the cell’s nucleus to activate certain genes that protect cells across the body from subsequent infection.

The genes activated by TRIM21 have potent antiviral activity. Yet they can also damage the body’s own tissues if they are switched on at the wrong time, which may lead to autoimmune diseases like rheumatoid arthritis and multiple sclerosis. It is thus critical that TRIM21 is carefully regulated and only activated during an infection, but it was not clear how this is achieved.

Dickson, Fletcher et al. now show that although TRIM21 is made all the time and is always ready to detect an incoming virus it is made in an inactive state. This is because part of TRIM21, called a B-Box, inhibits the protein’s own activity. This was an unexpected discovery because, although the B-Box domain is found in around 100 other human proteins, it was unclear what it did. Dickson, Fletcher et al. then combined structural biology and biochemical approaches to show that the B-Box mimics specific enzymes that work with TRIM21, and blocks them from binding to it. This keeps TRIM21 in an inactive state.

Next, Dickson, Fletcher et al. discovered that TRIM21 becomes active when a chemical tag, specifically a phosphate group, is added to the protein. This modification displaces the B-Box, allowing ubiquitin enzymes to bind to TRIM21 and switch on its activity. Further experiments then showed that this process helps regulate TRIM21 during infections with different viruses, including rhinovirus – the virus behind the common cold – and adenovirus – a common cause of respiratory infection.

Antibodies are now used to treat many medical conditions, but present technologies are based on our understanding of how antibodies work outside cells. By revealing the basis of antibody immunity inside cells, these new findings may lead to new treatments for a range of conditions. Future studies could also explore how failures in the TRIM21 system contribute to autoimmune diseases.

Antibodies are an essential component of protective immunity and their induction is a major aim of vaccination. They mediate a sufficiently rapid inflammatory response upon pathogen re-exposure that infection is halted before host colonization can occur. However, the consequences of inappropriate antibody-induced inflammation are severe, including lethal cytokine storms, autoimmunity and chronic inflammatory disease (Suntharalingam et al., 2006). It is therefore essential that antibody immunity is precisely regulated and tightly controlled. Antibody Fc receptors (FcRs) are essential for the diverse effector responses mediated by antibodies (Woof and Burton, 2004). Classical FcRs are expressed on the surface of immune cells. Their signalling induces phagocytosis, antigen presentation, antibody dependent cellular cytotoxicity (ADCC), degranulation and pro-inflammatory cytokine production (Guilliams et al., 2014). Regulating FcR signalling controls the antibody response. FcR immune signalling is influenced at multiple levels by; varying cell type expression and abundance, balancing the activity of inhibitory vs activatory receptors, requiring receptor crosslinking, setting minimal activation thresholds and by synergising with other pattern recognition receptors (PRRs) (Vogelpoel et al., 2015). These diverse mechanisms, deciphered over many decades, operate individually and synergistically to precisely regulate extracellular antibody immunity (Bruhns and Jönsson, 2015).

Recent work from our lab has shown that, in addition to classical FcR immunity mediated at the cell-surface, antibodies exert potent immune function from inside cells via a unique cytosolic IgG receptor called TRIM21 (James, 2014; Mallery et al., 2010; McEwan et al., 2013). TRIM21 is structurally, functionally and evolutionarily distinct from surface FcRs and binds antibodies with a significantly higher affinity (James et al., 2007). TRIM21 is also distinct in being constitutively and ubiquitously expressed in most cell types and tissues. TRIM21 protects cells from pathogen infection by stimulating a dual effector and sensor response. Upon infection, antibody-coated pathogens are detected in the cytosol by TRIM21. TRIM21 recruits cellular degradation machinery, including the AAA ATPase VCP and the proteasome, resulting in the destruction of viral particles and neutralization of infection. TRIM21 simultaneously activates immune transcription pathways, including NF-κB, leading to potent upregulation of pro-inflammatory cytokines including TNF, CXCL10, IL-6 and IFN (McEwan et al., 2013). TRIM21 activation results in cytokine upregulation in mice within hours of infection and is a significant component of the inflammatory response induced by protective antibodies (Watkinson et al., 2015). Deletion of TRIM21 compromises the efficiency of protective antibody immunity and causes virus-induced mortality (Vaysburd et al., 2013).

In contrast to cell-surface FcRs, and despite its capacity for such rapid and potent inflammatory signalling, it is unclear exactly how TRIM21 is regulated. The regulatory mechanisms that exist for classical FcRs are not applicable to TRIM21. TRIM21 is not expressed at the cell surface and therefore cannot be activated by clustering in lipid rafts or employ this as a mechanism to discriminate monomeric IgG from immune complex. Furthermore, as TRIM21 is dimeric, it is capable of binding both heavy chains of a single IgG simultaneously and forming a 1:1 complex. This is in contrast to other FcRs, which bind asymmetrically to a single heavy chain. There is also no known complementary inhibitory receptor to TRIM21 as exist for surface FcRs. Finally, TRIM21 lacks the ITAM and ITIM motifs that drives the function of other FcRs, and whose signalling can be precisely regulated by recruitment of Src family kinases and inositol-lipid phosphatases (Vogelpoel et al., 2015).

TRIM21 is a member of a large family of ~100 proteins with a common ‘tripartite motif’ architecture, comprising N-terminal RING, B-Box and coiled-coil domains. Instead of recruiting Src and Syk kinases, TRIM21 stimulates immune signaling via the activity of its RING domain, which catalyses the synthesis of polyubiquitin chains. TRIM21 first recruits the E2 Ube2W to modify itself with an N-terminal monoubiquitin (Fletcher and James, 2016). This acts as a primer for the synthesis of an anchored K63-chain catalysed by the heterodimer Ube2N/2V2. TRIM21 is further modified with K48-ubiquitin chains, culminating in proteasomal recruitment. The 19S-resident proteasomal deubiquitinase Poh1 subsequently liberates K63 chains from TRIM21 (Fletcher and James, 2016), and these then activate signal transduction pathways via TBK1, TAB/TAK and NEMO. We therefore hypothesized that TRIM21 immune signaling must be regulated at the fundamental level of its ubiquitination activity.

Here we show that initiating ubiquitination is a key step in regulating TRIM21 activity and that a novel mechanism of autoinhibition and kinase-induced E2 enzyme recruitment controls its potent pro-inflammatory activity.

To investigate the regulation of TRIM21 we tested whether over-expression in the absence of antibody stimuli triggers NFκB. As a positive control, we compared its activity to that of TRIM5α, a related antiviral TRIM protein whose over-expression is sufficient to drive spontaneous NFκB signalling (Pertel et al., 2011). While transfection of full-length TRIM5α caused dose-dependent activation of NFκB, full-length TRIM21 failed to activate NFκB at any dose (Figure 1A). When activated, both TRIM21 and TRIM5α autoubiquitinate, thereby making themselves a degradative substrate for the proteasome. Consistent with the NFκB result, while endogenous TRIM5α underwent rapid recycling we found little evidence of TRIM21 turnover even after several hours (Figure 1B and Figure 1—figure supplement 1). Together these observations suggest either that TRIM21 is constitutively inactive and its ubiquitination activity tightly regulated or it is a less active E3 ligase than TRIM5α. However, comparing the ubiquitination activity of TRIM5α and TRIM21 isolated RING domains (T5-R or T21-R) in vitro revealed that TRIM21 was the more active E3, capable of forming ubiquitin chains within minutes (Figure 1C). RING dimerization via higher order assembly is the proposed mechanism by which TRIMs autoregulate (Wagner et al., 2016). TRIM5α self-assembles and mutations that disrupt RING dimerization inhibit catalytic ability (Yudina et al., 2015). We therefore introduced structurally corresponding mutations into TRIM21 (Figure 1D and Figure 1—figure supplement 2) to test whether RING dimerisation is required for activity. SEC MALS and AUC demonstrated that while the wild-type RING is in monomer-dimer equilibrium, mutants M10E and M10E/M72E do not undergo detectable dimerization (Figure 1E and F). However, both mutants remained active and could catalyse unanchored K63 chain synthesis, Ube2W-primed anchored K63 chain extension and ubiquitin discharge (Figure 1G–I), albeit less efficiently than wild-type. These results demonstrate that a pre-formed TRIM21 RING dimer is not a pre-requisite for ubiquitination activity.

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RING dimerization is not required for E2 enzyme binding, as all interactions occur within a single monomer (Yudina et al., 2015). Instead, dimerization of RING E3s is thought to be needed for additional contacts with the E2-charged ubiquitin. Mutations in the partner RING at this interface reduce TRIM25 catalytic activity in vitro (Figure 2A)(Sanchez et al., 2016). Equivalent mutations in TRIM21 had no impact on ubiquitination, confirming that RING dimerization is not an intrinsic requirement for TRIM21 activity (Figure 2B–D). The mutation E10R reduces ubiquitination by TRIM25, possibly by disrupting a hydrogen bond with ubiquitin (Koliopoulos et al., 2016). We hypothesised that the corresponding glutamate (E13) in TRIM21 may allow a monomeric RING to contact both E2 and bound ubiquitin simultaneously, obviating the requirement for dimerization. Consistent with this hypothesis, E13R reduced TRIM21 catalysis (Figure 2D). Taken together these data show that the monomeric TRIM21 RING is a highly active E3. Thus, the lack of constitutive TRIM21 NFκB activity must be the result of tight regulation, independent of higher order oligomerisation.

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The next domain in the tripartite motif of TRIM21 after the RING is the B-Box2 (hereafter referred to as ‘B-Box’), a domain largely unique to TRIM proteins but whose function (besides an ability to oligomerise in TRIM5α) is unknown. Comparing the activities of RING, RING-Box and MiniTRIM21 proteins (based on a monovalent MiniTRIM5 that preserves the Box-coiled coil interface (Wagner et al., 2016)), revealed that the presence of the B-Box significantly reduces ubiquitination (Figure 3A). This is due to direct inhibition of catalytic activity, as the B-Box prevented ubiquitin discharge from either Ube2D1 or Ube2N (Figure 3B). To understand how the B-Box modulates TRIM21 catalysis we solved a 2 Å crystal structure of the RING-Box protein (Supplementary file 1). Surprisingly, this revealed that the B-Box occupies the E2 binding site on the RING domain (Figure 3C). Comparison to the TRIM5α RING:E2 complex shows that the B-Box is located where helix 1 of the E2 would normally be positioned during RING binding (Figure 3D). Crucially, the B-Box is able to occupy the E2 binding site on the RING by acting as an E2 mimic, forming similar electrostatic interactions. The RING surface contains a negatively charged patch, which is complementary to a strongly positively charged patch on both the E2 and the B-Box (Figure 3E). In particular, Box-RING interaction is stabilized through a salt bridge between residues R118 and E12, which is analogous to the interaction between E2 residue R14 and RING residue E11 in the TRIM5:Ube2N structure (Figure 3D). To test directly whether the B-Box prevents E2 binding we assigned ¹⁵N HSQC spectra of monomeric (M10E) RING and RING-Box constructs and measured chemical shift perturbations (CSPs) upon titration with Ube2N (Figure 4 and Figure 4—figure supplement 1). Titration of Ube2N into the RING resulted in significant CSPs in E2 interface residues (denoted by the green shaded regions in Figure 4A). In contrast, no evidence of Ube2N binding to the RING-Box was observed. Taken together, these data suggest that the B-Box may be an autoinhibitory domain whose function is to regulate RING activity by preventing E2 recruitment.

Figure 3

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Next, we investigated what relieves B-Box inhibition and promotes TRIM21 ubiquitination activity. Recently it was shown that unrelated innate immune adaptors including MAVS, STING and TRIF all contain a short pLxIS motif (where p is hydrophilic, x is non-aromatic and S is the target serine), which recruits kinases IKKβ or TBK1 resulting in serine phosphorylation and signal potentiation (Liu et al., 2015). Remarkably, TRIM21 contains a very similar motif at the end of its RING domain. Moreover, the target serine in this motif is located at the centre of the B-Box:RING interface (Figure 5A) and undergoes phosphorylation in cells (Figure 5B). We hypothesized that this could provide a mechanism to activate TRIM21 during an immune response. To investigate this further, we raised specific antisera against the phosphoserine peptide ⁶⁷RQLANMVNNLKEISQ⁸¹ (Figure 5C). Using this anti-pS80 serum, we detected cellular phosphorylation of TRIM21 but not an S80A mutant, upon IKKβ overexpression (Figure 5D). To ensure that IKKβ was acting directly rather than inducing the expression of a second kinase via NFκB, we repeated the experiment in the presence of proteasome inhibitor Bortezomib, which inhibits the degradation of IκBα and blocks NFκB activation. Efficient IKKβ phosphorylation of TRIM21 was observed even under conditions where NFκB signaling was abolished (Figure 5E). To confirm that IKKβ directly phosphorylates TRIM21, we incubated recombinant RING-Box and IKKβ proteins in vitro, observing ATP-dependent IKKβ phosphorylation of TRIM21 that was lost upon mutation of motif residues or addition of an IKKβ inhibitor (Figure 5F–H). Robust IKKβ-mediated phosphorylation was also observed using full-length TRIM21 protein (Figure 5I). Finally, knock-out of IKKβ by CRISPR/Cas9 abolished modification of overexpressed TRIM21-His (Figure 5J), suggesting that endogenous kinase phosphorylates TRIM21.

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Previously, it has been shown that while MAVS is phosphorylated by IKKβ and TBK1, STING and TRIF are only phosphorylated by TBK1 suggesting that there is differential kinase dependence even though signaling adapters share the consensus pLxIS motif (Liu et al., 2015). To test whether TRIM21 is a substrate for TBK1 phosphorylation, we incubated the recombinant full-length protein with the kinase in the presence of ATP. TBK1 was capable of phosphorylating TRIM21 in vitro comparably with IKKβ (Figure 6A). Overexpressed TBK1 was also capable of strongly phosphorylating cellular TRIM21 and causing significant stabilization of the protein (Figure 6B). To overcome limitations of kinase overexpression we sought to stimulate TRIM21 phosphorylation by activing the upstream pathway. First, we stimulated the TBK1 pathway by expressing one of its upstream adaptors, MAVS. MAVS expression significantly increased phosphorylation of TRIM21 (Figure 6C). Second, we stimulated cells with the MDA5/RIG-I ligand poly(I:C) (Kato et al., 2008). We observed a rapid increase in TRIM21 phosphorylation upon poly(I:C) stimulation, consistent with TBK1 activation (Figure 6D). Finally, we monitored changes in TRIM21 phosphorylation following infection with antibody-coated adenovirus, under conditions where we have previously demonstrated TRIM21-dependent antiviral activity (McEwan et al., 2013). The data show that in equivalent levels of immunoprecipitated TRIM21 there is a significant increase in the proportion of phosphorylated TRIM21 (Figure 6E).

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We next investigated whether serine phosphorylation can act as a switch to relieve B-Box inhibition and activate TRIM21. To test this, we introduced the phosphomimetic mutation S80E into the RING-Box and measured its ability to catalyse ubiquitin discharge. Remarkably, introduction of S80E into the RING-Box was sufficient to completely restore catalytic activity to the same level as the RING alone (Figure 7A–B). While glutamate is a routinely used phosphomimetic it does not fully recapitulate a phosphoserine. We therefore utilized an evolved orthogonal aminoacyl-tRNA synthetase/tRNA_CUA pair (Rogerson et al., 2015) to incorporate phosphoserine co-translationally at position 80 in MiniTRIM21 protein. Comparison with wild-type protein revealed that S80 phosphorylation potentiates TRIM21 ubiquitination activity (Figure 7C). Phosphorylation-dependence was confirmed by treating the phosphoprotein with phosphatase, which dephosphorylated S80 and restored autoinhibition (Figure 7C). Taken together, this show that modification of S80 is sufficient to control ubiquitination activity of TRIM21 in the presence of the B-Box. Moreover, mutant S80E can be used as a functional mimic of serine phosphorylation at position 80.

Figure 7

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The above data suggest that TRIM21 is kept in a constitutively inactive state by the B-Box domain, which prevents E2 binding and whose autoinhibition is released upon phosphorylation. To test this, we compared NFκB activation by TRIM21 mutants S80E or S80A with TRIM5α. Neither S80A nor wild-type TRIM21 triggered NFκB, but S80E conferred robust activation (Figure 8A). In contrast, mutation of residue S80 did not alter virus neutralization (Figure 8B). This is consistent with previous data that TRIM21 signalling has an activation threshold whereas neutralization does not (Foss et al., 2016). To demonstrate the importance of TRIM21 phosphorylation in immune sensing during infection, we challenged cells expressing different TRIM21 mutants with human adenovirus (Adv) ± human serum IgG and measured TNFA transcription after 4 hr. Significant TNFA induction was only observed during infection in the presence of antibody, and not in TRIM21 knockout cells (K21, Figure 8C and Figure 8—figure supplement 1). Normal TNFA induction was restored in K21 cells by TRIM21 overexpression, confirming TRIM21-dependence. Importantly, expression of S80A failed to rescue TNFA induction, while S80E enabled TNFA induction beyond wild-type levels. A change in TRIM21 phosphorylation upon infection could not be detected but this may be because only a fraction of cellular TRIM21 is recruited and modified during the response. This would be consistent with previous data showing that TRIM21 ubiquitination and degradation is also undetectable, despite being required for activity(Mallery et al., 2010). To confirm the importance of S80 and B-Box inhibition in regulating TRIM21 immune signaling, we repeated our infection experiments using an unrelated RNA virus, human rhinovirus 14 (HRV). As with AdV, expression of wild-type TRIM21 in knockout cells increased TNFA induction upon infection with HRV + antibody (Figure 8D). Mutant S80E greatly potentiated TNFA transcription while S80A failed to reconstitute TRIM21 activity. Similar results were obtained for cytokines CXCL10 and IL6 and the interferon-stimulated gene IFIT1.

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To obtain direct evidence that S80 phosphorylation activates TRIM21 by displacing the B-Box domain, we compared the E2 binding of wild-type and S80E RING-Box proteins by NMR. In contrast to wild-type, we detected significant CSPs in canonical E2 binding site residues upon titration of Ube2N into S80E (Figure 9A). There were also changes in residues at the base of helix 2, within the linker and on B-Box residues that contact the RING. This is consistent with restoration of E2 binding and the stabilization of an active conformation in which the B-Box domain is displaced. To further demonstrate B-Box displacement, we compared the dynamics of wild-type and S80E proteins. The {¹H}¹⁵N NOE ratios reveal that the most dynamic region is the linker (residues 83–102) between the RING and B-Box (Figure 9B). This is in agreement with the higher temperature factors observed for these residues in the crystal structure (Figure 9—figure supplement 1). Importantly, mutant S80E has markedly lower enhancement factors both in the linker region and in helix 1 (residues 1–17), against which the B-Box packs (Figure 9B). This increase in local motion is consistent with reduced interaction between RING and B-Box in the S80E mutant. To demonstrate that B-Box displacement promotes access to the E2 binding site on the RING, we carried out paramagnetic relaxation enhancement experiments (solvent PREs). In these experiments, addition of the paramagnetic metal complex Gd(DTPA–BMA) selectively attenuates signals from solvent-exposed surfaces (Pintacuda and Otting, 2002). The S80E mutation caused a dramatic change in surface accessibility precisely localized to the RING-Box interface (Figure 9C and Supplementary file 2). These data confirm that phosphomimetic mutation at serine 80 disrupts the RING:Box interaction and uncovers the E2 binding site.

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Our results suggest intracellular antibody immune signalling is kept in check via tight regulation of the cytosolic antibody receptor TRIM21. TRIM21 activates immune transduction pathways upon sensing an antibody-coated pathogen by synthesizing K63-ubiquitin chains. Controlling synthesis of these chains allows TRIM21 signalling to be regulated at the fundamental level. Here we have shown that this is accomplished by keeping TRIM21 in a constitutively silent state through an autoinhibitory mechanism that blocks E2 enzyme recruitment. Autoinhibition is mediated by the B-Box, providing a function for this ubiquitous but hitherto enigmatic TRIM protein domain. The B-Box inhibits E2 recruitment by acting as an E2 mimic and competing for RING binding. We also show that TRIM21 contains a variant of an IKKβ and TBK1 phosphorylation motif and is phosphorylated at residue S80. Phosphorylation of S80 or its replacement by a phosphomimetic displaces the B-Box, allowing E2 recruitment and potentiating TRIM21 ubiquitination activity, NF-κB signalling and cytokine transcription upon infection with DNA or RNA viruses.

Although TRIM21 regulation is seemingly distinct from that of cell-surface FcRs, parallels exist. For instance, signalling by both types of antibody receptor involves regulation by kinases. In the case of TRIM21, this involves a pLxxIS motif (where p is hydrophilic) and the kinases IKKβ and TBK1 while in other FcRs this involves an YxxL/I motif and recruitment of Src kinases. Importantly, it is not clear for either receptor type exactly how kinase activation occurs. For surface FcRs, this is thought to involve receptor clustering via cross-linking but why this causes Src recruitment and activation is unclear (Huang et al., 1992). TRIM21 may also undergo clustering, via cross-linking or higher order assembly, when it is recruited to opsonized pathogens. This may provide a basal activity that is amplified by kinase phosphorylation. The co-option of IKKβ and TBK1 as amplifying kinases suggests that TRIM21 could participate in its own positive-feedback loop. Again there are parallels to surface FcRs, where tyrosine phosphorylation has been shown to promote receptor clustering, which promotes phosphorylation and so on (Sobota et al., 2005).

Both types of Fc receptor also synergise with other PRRs and immune signalling pathways to amplify and diversify the resulting inflammatory response. Surface FcRs are known to exhibit cross-talk with Toll-like receptors (TLRs), greatly amplifying the production of cytokines such as TNFα during phagocytosis of opsonized bacteria (den Dunnen et al., 2012; Vogelpoel et al., 2014). Similarly, TRIM21 has been shown to synergise with cytosolic nucleic acid receptors such as RIG-I and cGAS, resulting in pathogen-specific responses and multiple signalling waves (Watkinson et al., 2015). Whether there is direct collaboration between TRIM21 and surface FcRs is currently unknown and will be important to determine in future work. Both immune signalling and antigen processing functions of TRIM21 are well placed to synergise with FcR immune complex capture and sorting activities, for instance to promote class I presentation. Precedence exists in the form of collaboration between FcRn and surface FcRs to drive antigen processing by dendritic cells (DCs)(Baker et al., 2011; Qiao et al., 2008).

TRIM21 belongs to a large family of structurally related proteins, many of which have known roles in cell signalling. They include other proteins with roles in immune signalling, such as the antiretroviral TRIM5(14) and RIG-I activator TRIM25 (Gack et al., 2007). While progress has been made in understanding the structure and function of TRIMs, it is still unclear how TRIM proteins are activated and regulated. Existing models of activation were solely based on substrate-induced aggregation or higher order assembly. This was thought to be necessary for dimerization of the RING domains located at either end of the molecule, which was assumed to be a pre-requisite for ubiquitination activity (Koliopoulos et al., 2016). However, our data show that this is not the case for TRIM21, and so cannot be assumed for other members of the TRIM family. Furthermore, higher order assembly alone does not explain the discrepancy between in vitro and cellular TRIM ubiquitination activity. TRIM RINGs, including those of TRIM5, 25 and 32, are active in vitro at concentrations where they are monomeric (Yudina et al., 2015; Sanchez et al., 2016; Koliopoulos et al., 2016). Given that TRIMs are not constitutively active in cells, there must be another level of regulation. It has been proposed that binding of a ubiquitin-conjugated E2 may drive RING equilibrium towards the dimeric form, explaining the in vitro activity. However, this would not explain the absence of activity in cells where the same phenomenon would occur. Moreover, if E2-Ub can drive TRIM RING dimerization then a pre-existing dimer is by definition no longer a pre-requisite for function and higher order assembly cannot be the sole regulator. It seems likely that the activation and regulation of TRIMs is a complex process that is regulated at multiple levels. This is exemplified by TRIM21 whose neutralization and signalling functions are both mediated by ubiquitination, but only signalling requires release of B-Box inhibition. Previously we have observed that TRIM21 signalling has a distinct threshold whereas neutralization takes place at very low antibody occupancy (<2 antibodies) (McEwan et al., 2012) and under conditions of sub-optimal TRIM21:antibody and antibody:antigen kinetics (Foss et al., 2016; Bottermann et al., 2016). Therefore, basal activity via higher order assembly may be sufficient for neutralization but not signalling. Strict regulation of signalling is intuitive, as it ensures a proportional response and avoids inappropriate triggering. Setting activation thresholds and regulating signalling may mitigate against any endosomal leakage of antibodies into the cytosol. The activation of TRIM21 by leaked immune complexes in lysosome-maturation-defective macrophages of lupus-prone mice illustrates the consequences of such an event (Monteith et al., 2016). However, while the requirement for tight regulation of TRIM21 signalling is intuitive the mechanism behind this has remained elusive. Our data suggest that it is achieved by a novel autoinhibitory mechanism in which the B-Box domain prevents ubiquitination by acting as an E2 mimic and the kinases IKKβ and TBK1 promote ubiquitination by phosphorylating the RING to relieve B-Box inhibition. Use of innate immune kinases intimately associates TRIM21 with the antiviral state not only at the level of gene expression as an ISG but also post translationally and in a similar manner to key immune adaptors MAVS, STING and TRIF (Liu et al., 2015).

1. 1. Adams PD
   2. Afonine PV
   3. Bunkóczi G
   4. Chen VB
   5. Davis IW
   6. Echols N
   7. Headd JJ
   8. Hung LW
   9. Kapral GJ
   10. Grosse-Kunstleve RW
   11. McCoy AJ
   12. Moriarty NW
   13. Oeffner R
   14. Read RJ
   15. Richardson DC
   16. Richardson JS
   17. Terwilliger TC
   18. Zwart PH

   (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution

   Acta Crystallographica Section D Biological Crystallography 66:213–221.

   https://doi.org/10.1107/S0907444909052925

   • PubMed
   • Google Scholar
2. 1. Baker K
   2. Qiao SW
   3. Kuo TT
   4. Aveson VG
   5. Platzer B
   6. Andersen JT
   7. Sandlie I
   8. Chen Z
   9. de Haar C
   10. Lencer WI
   11. Fiebiger E
   12. Blumberg RS

   (2011) Neonatal Fc receptor for IgG (FcRn) regulates cross-presentation of IgG immune complexes by CD8-CD11b+ dendritic cells

   PNAS 108:9927–9932.

   https://doi.org/10.1073/pnas.1019037108

   • PubMed
   • Google Scholar
3. 1. Bottermann M
   2. Lode HE
   3. Watkinson RE
   4. Foss S
   5. Sandlie I
   6. Andersen JT
   7. James LC

   (2016) Antibody-antigen kinetics constrain intracellular humoral immunity

   Scientific Reports 6:37457.

   https://doi.org/10.1038/srep37457

   • PubMed
   • Google Scholar
4. 1. Bruhns P
   2. Jönsson F

   (2015) Mouse and human FcR effector functions

   Immunological Reviews 268:25–51.

   https://doi.org/10.1111/imr.12350

   • PubMed
   • Google Scholar
5. 1. Chen VB
   2. Arendall WB
   3. Headd JJ
   4. Keedy DA
   5. Immormino RM
   6. Kapral GJ
   7. Murray LW
   8. Richardson JS
   9. Richardson DC

   (2010) MolProbity: all-atom structure validation for macromolecular crystallography

   Acta Crystallographica Section D Biological Crystallography 66:12–21.

   https://doi.org/10.1107/S0907444909042073

   • PubMed
   • Google Scholar
6. 1. den Dunnen J
   2. Vogelpoel LT
   3. Wypych T
   4. Muller FJ
   5. de Boer L
   6. Kuijpers TW
   7. Zaat SA
   8. Kapsenberg ML
   9. de Jong EC

   (2012) IgG opsonization of bacteria promotes Th17 responses via synergy between TLRs and FcγRIIa in human dendritic cells

   Blood 120:112–121.

   https://doi.org/10.1182/blood-2011-12-399931

   • PubMed
   • Google Scholar
7. 1. Emsley P
   2. Cowtan K

   (2004) Coot: model-building tools for molecular graphics

   Acta Crystallographica Section D Biological Crystallography 60:2126–2132.

   https://doi.org/10.1107/S0907444904019158

   • PubMed
   • Google Scholar
8. 1. Fletcher AJ
   2. Christensen DE
   3. Nelson C
   4. Tan CP
   5. Schaller T
   6. Lehner PJ
   7. Sundquist WI
   8. Towers GJ

   (2015) TRIM5α requires Ube2W to anchor Lys63-linked ubiquitin chains and restrict reverse transcription

   The EMBO Journal 34:2078–2095.

   https://doi.org/10.15252/embj.201490361

   • PubMed
   • Google Scholar
9. 1. Fletcher AJ
   2. James LC

   (2016) Coordinated neutralization and immune activation by the cytosolic antibody receptor TRIM21

   Journal of Virology 90:4856–4859.

   https://doi.org/10.1128/JVI.00050-16

   • PubMed
   • Google Scholar
10. 1. Foss S
    2. Watkinson RE
    3. Grevys A
    4. McAdam MB
    5. Bern M
    6. Høydahl LS
    7. Dalhus B
    8. Michaelsen TE
    9. Sandlie I
    10. James LC
    11. Andersen JT

    (2016) TRIM21 immune signaling is more sensitive to antibody affinity than its neutralization activity

    The Journal of Immunology 196:3452–3459.

    https://doi.org/10.4049/jimmunol.1502601

    • PubMed
    • Google Scholar
11. 1. Gack MU
    2. Shin YC
    3. Joo CH
    4. Urano T
    5. Liang C
    6. Sun L
    7. Takeuchi O
    8. Akira S
    9. Chen Z
    10. Inoue S
    11. Jung JU

    (2007) TRIM25 RING-finger E3 ubiquitin ligase is essential for RIG-I-mediated antiviral activity

    Nature 446:916–920.

    https://doi.org/10.1038/nature05732

    • PubMed
    • Google Scholar
12. 1. Gibson DG
    2. Young L
    3. Chuang RY
    4. Venter JC
    5. Hutchison CA
    6. Smith HO

    (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases

    Nature Methods 6:343–345.

    https://doi.org/10.1038/nmeth.1318

    • PubMed
    • Google Scholar
13. 1. Guilliams M
    2. Bruhns P
    3. Saeys Y
    4. Hammad H
    5. Lambrecht BN

    (2014) The function of Fcγ receptors in dendritic cells and macrophages

    Nature Reviews Immunology 14:94–108.

    https://doi.org/10.1038/nri3582

    • PubMed
    • Google Scholar
14. 1. Huang MM
    2. Indik Z
    3. Brass LF
    4. Hoxie JA
    5. Schreiber AD
    6. Brugge JS

    (1992)

    Activation of fc gamma rii induces tyrosine phosphorylation of multiple proteins including fc gamma RII

    The Journal of Biological Chemistry 267:5467–5473.

    • PubMed
    • Google Scholar
15. 1. James LC
    2. Keeble AH
    3. Khan Z
    4. Rhodes DA
    5. Trowsdale J

    (2007) Structural basis for PRYSPRY-mediated tripartite motif (TRIM) protein function

    PNAS 104:6200–6205.

    https://doi.org/10.1073/pnas.0609174104

    • PubMed
    • Google Scholar
16. 1. James LC

    (2014) Intracellular antibody immunity and the cytosolic Fc receptor TRIM21

    Current Topics in Microbiology and Immunology 382:51–66.

    https://doi.org/10.1007/978-3-319-07911-0_3

    • PubMed
    • Google Scholar
17. 1. Jinek M
    2. Chylinski K
    3. Fonfara I
    4. Hauer M
    5. Doudna JA
    6. Charpentier E

    (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity

    Science 337:816–821.

    https://doi.org/10.1126/science.1225829

    • PubMed
    • Google Scholar
18. 1. Kato H
    2. Takeuchi O
    3. Mikamo-Satoh E
    4. Hirai R
    5. Kawai T
    6. Matsushita K
    7. Hiiragi A
    8. Dermody TS
    9. Fujita T
    10. Akira S

    (2008) Length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-I and melanoma differentiation-associated gene 5

    The Journal of Experimental Medicine 205:1601–1610.

    https://doi.org/10.1084/jem.20080091

    • PubMed
    • Google Scholar
19. 1. Koliopoulos MG
    2. Esposito D
    3. Christodoulou E
    4. Taylor IA
    5. Rittinger K

    (2016) Functional role of TRIM E3 ligase oligomerization and regulation of catalytic activity

    The EMBO Journal 35:1204–1218.

    https://doi.org/10.15252/embj.201593741

    • PubMed
    • Google Scholar
20. 1. Liu H
    2. Naismith JH

    (2008) An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol

    BMC Biotechnology 8:91.

    https://doi.org/10.1186/1472-6750-8-91

    • PubMed
    • Google Scholar
21. 1. Liu S
    2. Cai X
    3. Wu J
    4. Cong Q
    5. Chen X
    6. Li T
    7. Du F
    8. Ren J
    9. Wu YT
    10. Grishin NV
    11. Chen ZJ

    (2015) Phosphorylation of innate immune adaptor proteins MAVS, STING, and TRIF induces IRF3 activation

    Science 347:aaa2630.

    https://doi.org/10.1126/science.aaa2630

    • PubMed
    • Google Scholar
22. 1. Mallery DL
    2. McEwan WA
    3. Bidgood SR
    4. Towers GJ
    5. Johnson CM
    6. James LC

    (2010) Antibodies mediate intracellular immunity through tripartite motif-containing 21 (TRIM21)

    PNAS 107:19985–19990.

    https://doi.org/10.1073/pnas.1014074107

    • PubMed
    • Google Scholar
23. 1. McEwan WA
    2. Hauler F
    3. Williams CR
    4. Bidgood SR
    5. Mallery DL
    6. Crowther RA
    7. James LC

    (2012) Regulation of virus neutralization and the persistent fraction by TRIM21

    Journal of Virology 86:8482–8491.

    https://doi.org/10.1128/JVI.00728-12

    • PubMed
    • Google Scholar
24. 1. McEwan WA
    2. Tam JC
    3. Watkinson RE
    4. Bidgood SR
    5. Mallery DL
    6. James LC

    (2013) Intracellular antibody-bound pathogens stimulate immune signaling via the Fc receptor TRIM21

    Nature Immunology 14:327–336.

    https://doi.org/10.1038/ni.2548

    • PubMed
    • Google Scholar
25. 1. McKenna S
    2. Spyracopoulos L
    3. Moraes T
    4. Pastushok L
    5. Ptak C
    6. Xiao W
    7. Ellison MJ

    (2001) Noncovalent interaction between ubiquitin and the human DNA repair protein Mms2 is required for Ubc13-mediated polyubiquitination

    Journal of Biological Chemistry 276:40120–40126.

    https://doi.org/10.1074/jbc.M102858200

    • PubMed
    • Google Scholar
26. 1. Monteith AJ
    2. Kang S
    3. Scott E
    4. Hillman K
    5. Rajfur Z
    6. Jacobson K
    7. Costello MJ
    8. Vilen BJ

    (2016) Defects in lysosomal maturation facilitate the activation of innate sensors in systemic lupus erythematosus

    PNAS 113:E2142–E2151.

    https://doi.org/10.1073/pnas.1513943113

    • PubMed
    • Google Scholar
27. 1. Murshudov GN
    2. Vagin AA
    3. Dodson EJ

    (1997) Refinement of macromolecular structures by the maximum-likelihood method

    Acta Crystallographica Section D Biological Crystallography 53:240–255.

    https://doi.org/10.1107/S0907444996012255

    • PubMed
    • Google Scholar
28. 1. Perkins DN
    2. Pappin DJ
    3. Creasy DM
    4. Cottrell JS

    (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data

    Electrophoresis 20:3551–3567.

    https://doi.org/10.1002/(SICI)1522-2683(19991201)20:18<3551::AID-ELPS3551>3.0.CO;2-2

    • PubMed
    • Google Scholar
29. 1. Pertel T
    2. Hausmann S
    3. Morger D
    4. Züger S
    5. Guerra J
    6. Lascano J
    7. Reinhard C
    8. Santoni FA
    9. Uchil PD
    10. Chatel L
    11. Bisiaux A
    12. Albert ML
    13. Strambio-De-Castillia C
    14. Mothes W
    15. Pizzato M
    16. Grütter MG
    17. Luban J

    (2011) TRIM5 is an innate immune sensor for the retrovirus capsid lattice

    Nature 472:361–365.

    https://doi.org/10.1038/nature09976

    • PubMed
    • Google Scholar
30. 1. Pintacuda G
    2. Otting G

    (2002) Identification of protein surfaces by NMR measurements with a pramagnetic Gd(III) chelate

    Journal of the American Chemical Society 124:372–373.

    https://doi.org/10.1021/ja016985h

    • PubMed
    • Google Scholar
31. 1. Qiao SW
    2. Kobayashi K
    3. Johansen FE
    4. Sollid LM
    5. Andersen JT
    6. Milford E
    7. Roopenian DC
    8. Lencer WI
    9. Blumberg RS

    (2008) Dependence of antibody-mediated presentation of antigen on FcRn

    PNAS 105:9337–9342.

    https://doi.org/10.1073/pnas.0801717105

    • PubMed
    • Google Scholar
32. 1. Rogerson DT
    2. Sachdeva A
    3. Wang K
    4. Haq T
    5. Kazlauskaite A
    6. Hancock SM
    7. Huguenin-Dezot N
    8. Muqit MM
    9. Fry AM
    10. Bayliss R
    11. Chin JW

    (2015) Efficient genetic encoding of phosphoserine and its nonhydrolyzable analog

    Nature Chemical Biology 11:496–503.

    https://doi.org/10.1038/nchembio.1823

    • PubMed
    • Google Scholar
33. 1. Sanchez JG
    2. Chiang JJ
    3. Sparrer KMJ
    4. Alam SL
    5. Chi M
    6. Roganowicz MD
    7. Sankaran B
    8. Gack MU
    9. Pornillos O

    (2016) Mechanism of TRIM25 catalytic activation in the antiviral RIG-I pathway

    Cell Reports 16:1315–1325.

    https://doi.org/10.1016/j.celrep.2016.06.070

    • PubMed
    • Google Scholar
34. 1. Schuck P

    (2003) On the analysis of protein self-association by sedimentation velocity analytical ultracentrifugation

    Analytical Biochemistry 320:104–124.

    https://doi.org/10.1016/S0003-2697(03)00289-6

    • PubMed
    • Google Scholar
35. 1. Skelton NJ
    2. Akke M
    3. Kördel J
    4. Thulin E
    5. Forsén S
    6. Chazin WJ

    (1992) 15N NMR assignments and chemical shift analysis of uniformly labeled 15N calbindin D9k in the apo, (Cd2+)1 and (Ca2+)2 states

    FEBS letters 303:136–140.

    https://doi.org/10.1016/0014-5793(92)80505-B

    • PubMed
    • Google Scholar
36. 1. Sobota A
    2. Strzelecka-Kiliszek A
    3. Gładkowska E
    4. Yoshida K
    5. Mrozińska K
    6. Kwiatkowska K

    (2005) Binding of IgG-opsonized particles to Fc gamma R is an active stage of phagocytosis that involves receptor clustering and phosphorylation

    The Journal of Immunology 175:4450–4457.

    https://doi.org/10.4049/jimmunol.175.7.4450

    • PubMed
    • Google Scholar
37. 1. Suntharalingam G
    2. Perry MR
    3. Ward S
    4. Brett SJ
    5. Castello-Cortes A
    6. Brunner MD
    7. Panoskaltsis N

    (2006) Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412

    New England Journal of Medicine 355:1018–1028.

    https://doi.org/10.1056/NEJMoa063842

    • PubMed
    • Google Scholar
38. 1. Vaysburd M
    2. Watkinson RE
    3. Cooper H
    4. Reed M
    5. O'Connell K
    6. Smith J
    7. Cruickshanks J
    8. James LC

    (2013) Intracellular antibody receptor TRIM21 prevents fatal viral infection

    PNAS 110:12397–12401.

    https://doi.org/10.1073/pnas.1301918110

    • PubMed
    • Google Scholar
39. 1. Vogelpoel LT
    2. Baeten DL
    3. de Jong EC
    4. den Dunnen J

    (2015) Control of cytokine production by human fc gamma receptors: implications for pathogen defense and autoimmunity

    Frontiers in Immunology 6:79.

    https://doi.org/10.3389/fimmu.2015.00079

    • PubMed
    • Google Scholar
40. 1. Vogelpoel LT
    2. Hansen IS
    3. Rispens T
    4. Muller FJ
    5. van Capel TM
    6. Turina MC
    7. Vos JB
    8. Baeten DL
    9. Kapsenberg ML
    10. de Jong EC
    11. den Dunnen J

    (2014) Fc gamma receptor-TLR cross-talk elicits pro-inflammatory cytokine production by human M2 macrophages

    Nature Communications 5:5444.

    https://doi.org/10.1038/ncomms6444

    • PubMed
    • Google Scholar
41. 1. Wagner JM
    2. Roganowicz MD
    3. Skorupka K
    4. Alam SL
    5. Christensen D
    6. Doss G
    7. Wan Y
    8. Frank GA
    9. Ganser-Pornillos BK
    10. Sundquist WI
    11. Pornillos O

    (2016) Mechanism of B-box 2 domain-mediated higher-order assembly of the retroviral restriction factor TRIM5α

    eLife 5:e16309.

    https://doi.org/10.7554/eLife.16309

    • PubMed
    • Google Scholar
42. 1. Watkinson RE
    2. McEwan WA
    3. Tam JC
    4. Vaysburd M
    5. James LC

    (2015) TRIM21 promotes cGAS and RIG-I sensing of viral genomes during infection by antibody-opsonized virus

    PLoS Pathogens 11:e1005253.

    https://doi.org/10.1371/journal.ppat.1005253

    • PubMed
    • Google Scholar
43. 1. Winn MD
    2. Ballard CC
    3. Cowtan KD
    4. Dodson EJ
    5. Emsley P
    6. Evans PR
    7. Keegan RM
    8. Krissinel EB
    9. Leslie AG
    10. McCoy A
    11. McNicholas SJ
    12. Murshudov GN
    13. Pannu NS
    14. Potterton EA
    15. Powell HR
    16. Read RJ
    17. Vagin A
    18. Wilson KS

    (2011) Overview of the CCP4 suite and current developments

    Acta Crystallographica. Section D, Biological Crystallography 67:235–242.

    https://doi.org/10.1107/S0907444910045749

    • PubMed
    • Google Scholar
44. 1. Woof JM
    2. Burton DR

    (2004) Human antibody-Fc receptor interactions illuminated by crystal structures

    Nature Reviews Immunology 4:89–99.

    https://doi.org/10.1038/nri1266

    • PubMed
    • Google Scholar
45. 1. Yudina Z
    2. Roa A
    3. Johnson R
    4. Biris N
    5. de Souza Aranha Vieira DA
    6. Tsiperson V
    7. Reszka N
    8. Taylor AB
    9. Hart PJ
    10. Demeler B
    11. Diaz-Griffero F
    12. Ivanov DN

    (2015) RING dimerization links higher-order assembly of TRIM5α to synthesis of K63-Linked polyubiquitin

    Cell Reports 12:788–797.

    https://doi.org/10.1016/j.celrep.2015.06.072

    • PubMed
    • Google Scholar

Author details

1. Claire Dickson

   Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom

   Contribution

   Data curation, Formal analysis, Investigation, Writing—review and editing, Conceived the project and wrote the manuscript, Performed X-ray crystallography and in vitro ubiquitination assays

   Contributed equally with

   Adam J Fletcher

   Competing interests

   No competing interests declared

2. Adam J Fletcher

   Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom

   Contribution

   Formal analysis, Investigation, Writing—review and editing, Conceived the project and wrote the manuscript, Carried out cellular experiments and kinase assays

   Contributed equally with

   Claire Dickson

   Competing interests

   No competing interests declared

3. Marina Vaysburd

   Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom

   Contribution

   Investigation, Carried out virus-induced cytokine infections

   Competing interests

   No competing interests declared

4. Ji-Chun Yang

   Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom

   Contribution

   Investigation, Performed NMR experiments

   Competing interests

   No competing interests declared

5. Donna L Mallery

   Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom

   Contribution

   Investigation, Assisted with ubiquitination assays

   Competing interests

   No competing interests declared

6. Jingwei Zeng

   Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom

   Contribution

   Investigation, Assisted with cell experiments, Provided new reagents

   Competing interests

   No competing interests declared

7. Christopher M Johnson

   Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom

   Contribution

   Investigation, Performed multi-angle light scattering

   Competing interests

   No competing interests declared

8. Stephen H McLaughlin

   Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom

   Contribution

   Investigation, Performed AUC

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9135-6253

9. Mark Skehel

   Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom

   Contribution

   Supervision

   Competing interests

   No competing interests declared

10. Sarah Maslen

    Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom

    Contribution

    Investigation

    Competing interests

    No competing interests declared

11. James Cruickshank

    Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom

    Contribution

    Supervision, Investigation

    Competing interests

    No competing interests declared

12. Nicolas Huguenin-Dezot

    Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom

    Contribution

    Methodology

    Competing interests

    No competing interests declared

13. Jason W Chin

    Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom

    Contribution

    Supervision, Methodology

    Competing interests

    No competing interests declared

14. David Neuhaus

    Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom

    Contribution

    Data curation, Supervision, Analysed and supervised NMR

    Competing interests

    No competing interests declared

15. Leo C James

    Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom

    Contribution

    Conceptualization, Formal analysis, Supervision, Writing—original draft, Writing—review and editing, Conceived the project and wrote the manuscript

    For correspondence

    lcj@mrc-lmb.cam.ac.uk

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-2131-0334

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