Structure and catalytic activation of the TRIM23 RING E3 ubiquitin ligase

INTRODUCTION

Tripartite motif (TRIM) proteins comprise a large family of E3 ligases, with about 100 different human proteins identified¹. TRIM proteins regulate many different cellular processes and pathways, and in particular have important roles in antiviral defense, activation of the innate immune response, inflammation, and development of cancer. TRIMs share a common domain organization – called the tripartite or RBCC motif – which consists of an N-terminal RING domain, followed by one or two B-box domains, a coiled-coil domain, and a C-terminal domain, which is variable across the protein family² (Fig. 1A). The RING domain functions as a ubiquitin (Ub) E3 ligase and cooperates with E2 enzymes to generate polyUb chains. Recent studies have shown that despite considerable sequence divergence, the coiled-coil domains of different TRIM proteins share a common quaternary fold^3–6. Two subunits pack in an anti-parallel manner, forming an elongated rod-shaped scaffold with a RING domain on each end; the two RING domains are separated by 17–20 nm and so cannot directly interact with each other. On the other hand, other studies have shown that the isolated RING domains of TRIM5α, TRIM25, and TRIM32 engage E2-Ub conjugates and catalyze Ub conjugation as close-packed dimers^7–9. Taken together, these observations imply that the catalytically active form of TRIMs comprises at least 2 coiled-coil mediated dimers (4 protein molecules). Thus, the polyubiquitination activity of a TRIM protein requires higher-order assembly or oligomerization beyond its basal, coiled-coil mediated dimeric quaternary fold.

The above requirement for RING activation is exploited by the cell to regulate when, where, and how TRIM proteins synthesize polyUb chains. One mechanism is exemplified by TRIM5α, which inhibits replication of HIV-1 and other retroviruses in a ubiquitin-dependent manner¹⁰. TRIM5α assembles into a hexagonal network that surrounds the HIV-1 capsid, and this network promotes dimerization of the associated RING domains^7,11,12. The activated RING domains synthesize both self-attached and free polyUb chains to inhibit viral replication^13–15. Another example is TRIM25, whose catalytic activity is induced by multivalent binding to its ubiquitination substrate, RIG-I⁹. These examples illustrate how a general biochemical property can be translated into different pathway-specific mechanisms to control a common enzymatic activity.

In this study, we analyzed human TRIM23 in order to determine if this protein follows the model of catalytic activation described above. In contrast to the TRIM5α and TRIM25 RING domains that were observed to dimerize only in complex with E2 or E2-Ub^7–9, the isolated TRIM23 RING domain had a marked, although weak propensity to dimerize independently. We report crystal structures of the TRIM23 RING domain, both alone and in complex with a stable E2-Ub conjugate. In both structures, the RING domain adopts the same dimeric quaternary fold, indicating that TRIM23 also requires RING dimerization to catalyze polyUb synthesis. Our studies therefore support a general model wherein higher-order oligomerization or assembly regulates catalytic activation of TRIM proteins.

METHODS

RESULTS AND DISCUSSION

The human TRIM23 RING domain (residues 1-123) was expressed as a polyhistidine-tagged SUMO-fusion protein and purified to homogeneity after cleavage of the leader sequence (Fig. 1B). The purified RING domain was functional, and upon screening a panel of E2 conjugating enzymes was found to catalyze polyubiquitination reactions with the following E2s: UbcH5b, UbcH5c, UbcH5d, UbcH6, UbcH13/Mms2, and Ube2W (Fig. 1C). Previous studies have shown that the equivalent RING domains of TRIM5α and TRIM25 were monomeric in isolation^7–9, whereas the TRIM32 RING was dimeric⁸. We therefore analyzed the TRIM23 RING domain by using SEC-MALS (size exclusion chromatography coupled with multi-angle light scattering) to determine its oligomeric state (Fig. 1D). When injected at 20 μM concentration, the protein eluted as a single peak (green curve), with a calculated molecular weight (MW) close to that of a monomer (14.3 kDa, expected MW from sequence = 13.1 kDa). When injected at higher concentrations (100 μM, blue and 500 μM, maroon), the peak eluted at progressively earlier times with correspondingly higher apparent MW closer to that of a dimer. These results indicated that the purified TRIM23 RING domain was under dynamic monomer-dimer equilibrium in solution. On the basis of the above data, we estimate that the dimer dissociation constant is around 100–200 μM. Thus, like TRIM32 but unlike TRIM5α and TRIM25, the TRIM23 RING domain can independently dimerize in solution.

We obtained well-diffracting crystals of the isolated RING domain from primary crystallization screens. The refined structure consisted of 3 molecules in the asymmetric unit, with each monomer having well-defined electron density spanning residues 28–103. Of the three chains in the asymmetric unit, two formed a non-crystallographic dimer, whereas the third dimerized with its symmetry-related mate. The crystallographically distinct dimers were very similar with a root mean square deviation (RMSD) of 0.39 Å over equivalent Cα atoms and 0.43 Å over backbone (N, Cα, C, and O) atoms.

The TRIM23 RING dimer is organized around a 2-helix bundle made up of residues that are C-terminal to the canonical zinc-coordinating folds in primary sequence (Fig. 1E). Additional contacts are contributed by N-terminal residues, which insert hydrophobic sidechains into the grooves between the two α-helices. Thus, the N-terminal and C-terminal residues that flank the zinc lobes make up a “pedestal” upon which the zinc lobes sit. This arrangement is very similar to the structures of E2-bound RING dimers from other TRIM proteins, except that in those cases the “pedestal” is a 4-helix bundle because the N-terminal residues also fold into an α-helix^7–9. More importantly, these results suggested that the dimer made by the TRIM23 RING domain in solution is already in the same configuration as the E2-bound dimer.

To confirm that the E2-bound dimer is similar to the uncomplexed dimer, we also crystallized the TRIM23 RING domain in complex with UbcH5b conjugated to Ub. To prevent loss of the Ub moiety, we used an established strategy of stably conjugating Ub to the E2 enzyme through an isopeptide linkage, instead of the labile, native thioester²⁰. Despite local structural differences, isopeptide-linked E2-Ub conjugates have been shown to recapitulate their catalytically primed structures when crystallized with RING E3 ligases^8,9,20. Our final refined structure contained one RING dimer flanked by two UbcH5b-Ub conjugates in the asymmetric unit (Fig. 2A). This quaternary arrangement is very similar to equivalent RING/E2 and RING/E2-Ub structures of TRIM5α⁷, TRIM25^8,9, and the more distantly related RNF4²⁰ and BIRC7²¹ (Fig. 2B shows a superposition of the TRIM23 RING/E2-Ub structure with the TRIM25 and RNF4 complexes).

The unbound and E2-bound TRIM23 RINGs were highly similar, with average RMSD of 0.36 Å over equivalent Cα atoms and 0.37 Å over backbone atoms. Indeed, there were remarkably few differences between the bound and unbound states, apart from side chain rotamers buried within the contact sites. These results imply that the TRIM23 RING dimer has a relatively rigid quaternary fold, and that the E2 and Ub-binding surfaces are “pre-made” prior to E2-Ub binding.

The two RING domains have distinct interactions with the E2-Ub conjugate and cooperate in facilitating the ubiquitination reaction. One TRIM23 RING binds both the E2 and Ub moieties and provides an “arginine finger” (Arg76) to coordinate a hydrogen bond network that packs the Ub C-terminus against a shallow groove leading to the UbcH5b active site (Fig. 2C). This set of interactions is highly conserved, as seen in crystal structures of divergent dimeric RINGs in complex with various E2-Ub conjugates^8,9,20,21 (e.g., TRIM25 in Fig. 2D and RNF4 in Fig. 2E). On the other hand, the second RING domain interacts only with the Ub moiety, and this set of interactions is less conserved. In TRIM25, the second interface consists of a hydrogen bond network mediated by hydrophilic residues^8,9 (Fig. 2G). In contrast, the TRIM23 interface consists of a pi-stacking interaction between the sidechain of Phe93 and the Ub peptide bond between Glu34 and Gly35 (Fig. 2F). Interestingly, similar pi-stacking interactions involving a RING hydrophobic sidechain and the Ub backbone are found in structures of more distantly related RING domains, BIRC7 (phenylalanine)²¹ and RNF4 (tyrosine)²⁰ (Fig. 2H). Mutation of these residues abrogate ubiquitination by these RING domains^20,21, and we expect the same effect for TRIM23.

In summary, our structural analyses indicate that the TRIM23 RING domain is catalytically active as a dimer. Since full-length TRIM23 is expected to also form an elongated coiled-coil dimer with two RINGs on opposite ends, enzymatic activation will require self-association of at least two dimers in a higher-order oligomer or assembly state. In other TRIM proteins, RING activation is triggered by multivalent interactions with ubiquitination substrates or higher-order assembly platforms^7,9,12,15. TRIM23 itself is not yet well-characterized, but has been reported to function in multiple signaling pathways and to ubiquitinate different substrates to regulate antiviral responses and cell differentiation^22–24. It is likely that the mode of activation described here has an important role in defining the pathway specificity and timely occurrence of TRIM23-mediated polyubiquitination.