Functional role of TRIM E3 ligase oligomerization and regulation of catalytic activity

Abstract

TRIM E3 ubiquitin ligases regulate a wide variety of cellular processes and are particularly important during innate immune signalling events. They are characterized by a conserved tripartite motif in their N-terminal portion which comprises a canonical RING domain, one or two B-box domains and a coiled-coil region that mediates ligase dimerization. Self-association via the coiled-coil has been suggested to be crucial for catalytic activity of TRIMs; however, the precise molecular mechanism underlying this observation remains elusive. Here, we provide a detailed characterization of the TRIM ligases TRIM25 and TRIM32 and show how their oligomeric state is linked to catalytic activity. The crystal structure of a complex between the TRIM25 RING domain and an ubiquitin-loaded E2 identifies the structural and mechanistic features that promote a closed E2~Ub conformation to activate the thioester for ubiquitin transfer allowing us to propose a model for the regulation of activity in the full-length protein. Our data reveal an unexpected diversity in the self-association mechanism of TRIMs that might be crucial for their biological function.

Keywords: TRIM25; TRIM32; enzyme mechanism; protein structure; ubiquitin ligase.

Figure 1. Domain structures of TRIM25 and TRIM32 and oligomerization

1. A

   Domain organization of TRIM25 and TRIM32 including fragments used in this study.

2. B, C

   (B) SEC‐MALLS traces of different TRIM25 and (C) TRIM32 constructs. The proteins are colour coded, and the domain architecture is reported next to the respective MALLS curve. The constructs were analysed over different concentration ranges, and the data are reported for: T25R, 4 mg/ml; T25RB1, 3.2 mg/ml; T25RB1B2, 3.6 mg/ml; T25RBCC, 0.5 mg/ml; T32R (core and extended), 3 mg/ml; T32RB, 4 mg/ml; T32RBCC, 4 mg/ml.

Figure 2. Relationship between oligomeric state and catalytic activity

1. A–D

   (A, C) UBE2D3˜Ub discharge assays with different TRIM25 and TRIM32 constructs, respectively. Assays were carried out with TRIM constructs as indicated and the reaction was monitored over 30 min. The asterisk indicates the band for the TRIM construct. (B, D) Quantification of the discharge assays using UBE2D1˜Ub^Atto and TRIM25 or TRIM32, respectively. The loss of UBE2D1˜Ub^Atto is plotted as the average of experimental triplicates (± s.d.).

2. E–H

   (E, G) K63 poly‐ubiquitination assays using UBE2N/UBE2V1 and TRIM25 and TRIM32, respectively. Reactions were incubated for 30 min and samples taken at the indicated times. The asterisk indicates the band for the TRIM construct. (F, H) Assays were quantified (see Materials and Methods) by supplementing the reaction with 1 μM Ub^Atto and integrating the loss of free Ub^Atto for TRIM25 and TRIM32, respectively. Assays were carried out in triplicate (± s.d.).

Source data are available online for this figure.

Figure EV1. E2~Ub discharge and poly‐ubiquitination assays using a fluorescently labelled ubiquitin Ub^Atto

1. Representative gels of the discharge assays with UBE2D1˜Ub^Atto and different TRIM25 constructs. The gels were scanned with a Storm 869 Scanner and the bands for free Ub^Atto integrated.

2. Representative gels of the discharge assays with UBE2D1˜Ub^Atto and different TRIM32 constructs. The gels were scanned with a Storm 869 Scanner and the bands for free Ub^Atto integrated.

3. Poly‐ubiquitination assays using UBE2N/UBE2V1 and different TRIM25 constructs supplemented with fluorescent Ub^Atto.

4. Poly‐ubiquitination assays using UBE2N/UBE2V1 and different TRIM32 constructs supplemented with fluorescent Ub^Atto.

Figure 3. Structure of the RING dimers and interaction with the E2~Ub intermediate

1. Structure of the TRIM32 RING dimer in ribbon representation with each RING monomer coloured in cyan and blue and the Zn²⁺ ions as grey spheres.

2. Overlap of the RING dimers of TRIM32 (cyan, 5FEY.pdb), TRIM25 (yellow, 5FER.pdb) and TRIM5α (orange, 4TKP.pdb). The structures were overlapped on the circled RING domain. This overlap shows that the structures of the RINGs are very similar but that there are differences in the relative orientations of the two RINGs.

3. Structure of the TRIM25 RING/E2˜Ub complex with the RING domains in the same colour scheme as in (A), UBE2D1 in grey and the ubiquitin molecules in salmon and red.

Figure 4. RING dimerization and the interaction with ubiquitin

1. Close‐up of the TRIM25 RING dimer interface highlighting the hydrophobic interactions made between the four α‐helices (left). Close‐up of the interface between each RING monomer and the proximal ubiquitin (right).

2. UBE2D1˜Ub^Atto discharge assays with TRIM25 wild‐type RING, the fused RING constructs and different mutants important for dimerization or the interaction with ubiquitin. Time point zero for the T25‐R Linker and T25‐R Linker V72R samples was taken before the addition of E3 as discharge is very fast. The loss of UBE2D1˜Ub^Atto is plotted as the average of experimental duplicates (± s.d.).

3. UBE2D3˜Ub discharge assays with the same mutants as in (B), stained with InstantBlue.

4. K63 poly‐ubiquitination assays using UBE2N/UBE2V1. The asterisk indicates the band for the TRIM construct.

Source data are available online for this figure.

Figure EV2. UBE2D1~Ub^Atto discharge assays with TRIM25 and TRIM32 RING mutants

1. Representative gels of the discharge assays with UBE2D1˜Ub^Atto and different TRIM25 RING mutants. The gels were scanned with a Storm 869 Scanner and the bands for free Ub^Atto integrated.

2. Representative gels for the equivalent assays with TRIM32 RING mutants.

Figure 5. Stabilization of the closed E2~Ub conformation

1. A

   Role of E10 in the RING of TRIM25 (blue) in stabilizing the closed E2˜Ub conformation by contacting K11 from the proximal ubiquitin (salmon) and N71 of the opposite RING (cyan) which in turn contacts K33 of the same ubiquitin.

2. B

   E2˜Ub discharge and K63 poly‐ubiquitination assays to test the role of E9 and E10 in TRIM25 activity. Substitution of Glu9 with Arg has no significant effect on activity, whereas the E10R mutation almost completely abolishes catalytic activity.

3. C

   Discharge and K63 poly‐ubiquitination assays to test the role of the equivalent residue E16 in TRIM32 and the role of I85R. Mutation of E16R abolishes catalytic activity indicating that the role of the glutamate is conserved.

4. D, E

   Quantification of UBE2D1˜Ub^Atto discharge assays. The loss of E2˜Ub is plotted as the average of experimental duplicates (± s.d.).

Source data are available online for this figure.

Figure 6. Solution structure of TRIM25 and TRIM32

1. A–D

   2D ¹H‐¹⁵N‐HSQC spectra of the TRIM25 RING domain at a concentration of 125 μM (A) and 570 μM (B), the monomeric TRIM32 RING _core (C) and TRIM32 RING dimer (D).

2. E

   Isotropic rotational correlation times of the different TRIM25 and TRIM32 RING constructs, obtained from the relaxation analysis of resonances in the corresponding 2D ¹H‐¹⁵N HSQC spectra (dark grey) or calculated from available structures by HYDRONMR (light grey). Error bars are derived from relaxation rate analysis implemented in TENSOR2 (Dosset et al, 2000).

3. F

   Scattering profiles of RING constructs (left) and their normalized pair‐distribution functions P(r) (middle). The right‐hand panel shows low‐resolution ab initio models derived from the SAXS data analysis.

4. G

   Scattering profiles, their normalized pair‐distribution functions P(r) and low‐resolution ab initio models for RING and B‐box‐containing constructs.

5. H

   Scattering profiles, their normalized pair‐distribution functions P(r) and low‐resolution ab initio models for the RBCC domains of TRIM25 and TRIM32. The curves and envelopes are reported using the same colour scheme in all respective panels and the envelopes for the constructs are drawn to scale.

Figure EV3. SAXS data recording and modelling of TRIM25 and TRIM32

1. A, B

   Size‐exclusion chromatography in line with SAXS data recording for TRIM25 RBCC (A) and TRIM32 RBCC (B) at the SWING beamline at SOLEIL. The intensity (blue and green) and Rg (red and purple) profiles are reported as a function of the frames recorded at equal time intervals.

2. C

   Ab initio low‐resolution envelopes calculated with the program DAMAVER for TRIM25 RING (green), TRIM32 RING core (blue) and TRIM32 RING dimer superimposed to their structures by the program SUPCOMB. The values of the chi‐square are calculated by the program CRYSOL.

3. D

   Low‐resolution envelopes calculated by DAMAVER for TRIM25 RBCC (red) and TRIM32 RBCC (blue) in different orientations presented in scale with the structures of TRIM5α BCC (PDB 4TN3), TRIM25 CC (PDB 4LTB) and TRIM20 CC (PDB 4CG4).

Figure 7. Model of RING dimerization in different TRIM ligases

TRIM25 and TRIM32 use different mechanisms to allow their respective RING domains to dimerize: in TRIM25, the propensity to dimerize is low and happens in an intramolecular fashion possibly aided by E2~Ub and substrate binding. It is possible that the B‐box domains may contact the CC region to support dimerization. The RINGs of TRIM32 are constitutive dimers even in the absence of the rest of the remainder of the protein or additional binding partners. Tetramerization of TRIM32 may be important for the recognition of low‐affinity substrates or may allow co‐localization of different binding partners.

Figure EV4. Models for the domain architecture of TRIM25 and TRIM32 RBCC

1. A, B

   The internal volume of the low‐resolution average envelopes of TRIM25 (A) and TRIM32 (B) RBCC, obtained from the SAXS analysis, can accommodate the two monomeric TRIM25 RB1B2 envelopes (represented in blue, see Fig 6) and the two dimeric TRIM32 RB2 envelopes (in green, see Fig 6) at the N‐terminus on each side of the central coiled‐coil spanning the length of the molecule. The structure coordinates of TRIM25 CC and TRIM20 CC were used to model the central coiled‐coil region of TRIM25 RBCC and TRIM32 RBCC, respectively. For TRIM32RBCC, 2 copies of the same coiled‐coil coordinates (rotated of 180° relative to the log helical axis) were used.