Structural and functional asymmetry of RING trimerization controls priming and extension events in TRIM5α autoubiquitylation

Fig. 1. Recombinant RING constructs mimic distinct relative RING arrangements in TRIM5α. a Conserved tripartite motif (TRIM) domain arrangement in TRIM5α. b NMR structure of the human TRIM5α RING domain monomer (PDB: 2ECV). c Cartoon representation of the TRIM5α dimer, in which the two RING domains are presumed to exist as monomers. d The monomeric R1-WT RING construct used in this study. e Crystal structure of the rhesus TRIM5α RING (PDB: 4TKP) reveals the four-helix-bundle dimer interface. The isoleucine residue in the core of the four-helix bundle is highlighted in cyan here (I77) and in panel a (I76). f RING dimerization is thought to occur when multiple TRIM dimers come into proximity upon association with their binding partners. g RING dimerization is stabilized in the R2-WT tandem RING construct. h Crystal structure of the B-box trimer (PDB: 5IEA) reveals the structural arrangement of three B-box domains at the vertices of the honeycomb-like TRIM5α assemblies (i)19, 26. j Proximity of three RING domains at the vertices of the TRIM5α honeycomb was mimicked in the R3-WT construct by fusing RING domain to the T4 fibritin foldon, a compact trimerization domain.

Fig. 2. Conformational repertoire of distinct RING constructs as revealed by NMR spectroscopy and circular dichroism. a 15N-TROSY HSQC spectra of different RING constructs reveal spectral signatures of RING dimerization. b Three representative spectral regions from (a) showing NMR signals that display distinctive chemical shift changes upon transition between the monomeric and the dimeric states of the RING domain. c Dependence of molar ellipticity on protein concentration reveals that almost three-quarters of total alpha-helical content is lost in the R1-WT construct as protein concentration is lowered from 100 μM to 6 μM (n = 2 independent experiments). d Cartoon representation of the distinct conformational and oligomeric states observed in different RING constructs.

Fig. 3. The contribution of RING dimerization to the catalytic enhancement of ubiquitin discharge from the UBE2N~Ub and UBE2W~Ub conjugates can be quantified using a FRET ubiquitin discharge assay. a A cartoon schematic of the FRET ubiquitin discharge assay used in this study. b Kinetics of RING-catalyzed ubiquitin discharge from the heterodimeric UBE2N~Ub/V2 (0.1 µM) is well approximated by an exponential decay function in agreement with the Michaelis-Menten equation (Supplementary Methods). c In contrast, ubiquitin discharge from UBE2W~Ub (0.1 µM) is not well described by the Michaelis-Menten model. d UBE2N/V2 ubiquitin discharge rates are plotted against RING concentration and the initial slopes (kcat/Km) can be used for quantification of the catalytic activity (n = 2 independent experiments). e Quantification of UBE2W discharge (see “Methods” and Supplementary Methods for more details) establishes that UBE2W is also activated by RING dimerization, albeit less potently than UBE2N. [RING] concentration is shown here as total concentration of RING domains in each sample irrespective of the oligomerization state (e.g., 15 μM sample of the tandem RING construct R2-WT has [RING] = 30 μM)…

Fig. 4. Proximity of three RING domains promotes each of the two distinct steps in the autoubiquitylation of TRIM5α: the N-terminal monoubiquitylation (priming) and the extension of the TRIM5α-anchored K63-linked polyubiquitin chain. a, b RING autoubiquitylation products visualized by WB of FLAG-tagged RING constructs. [RING] = 5 µM, [Ub] = 10 µM, [E2] = 0.1; 0.2; 0.5; 1 µM. 30 min incubation. a Monoubiquitylation of RING in the presence of the increasing amounts of UBE2W. b RING polyubiquitylation in the presence of both UBE2W and the heterodimeric UBE2N/V2. c Ubiquitylation products of different E2/E3 combinations analyzed side-by-side on the same SDS-PAGE gel. [RING] = 5 µM, [Ub] = 10 µM, [E2] = 1 µM. 30 min incubation. WB was performed with either anti-FLAG antibodies (top panel) or anti-K63-linked-Ub antibodies (bottom panel). d N-terminal anchoring of ubiquitin in the R3 WT construct is confirmed by mass spectrometry. e N-terminal monoubiquitylation is preferred over other modification sites by more than two orders of magnitude. f Kinetics and products of ubiquitin discharge from the AF594-UBE2W~AF488-Ub conjugate (0.1 μM) catalyzed by R2-WT ([RING] = 1 μM; [dimer] = 0.5 μM) or…

Fig. 5. Transient proximity of three RING domains promoted by the association of TRIM5α with the retroviral capsid explains how TRIM5α autoubiquitylation is activated by capsid binding. The honeycomb-like TRIM5α assembly on the surface of the retroviral capsid is required for the antiviral activity of the protein (see a recent comprehensive review for detailed bibliography10). The architecture of the TRIM5α assembly suggests that the cooperativity between SPRY:capsid interactions and B-box:B-box interactions forms the basis of the pattern recognition functionality and promotes capsid binding22. B-box trimerization at the vertices of the TRIM5α honeycomb brings three N-terminal RING domains into proximity. Here we show that proximity of three RINGs creates an asymmetric structural arrangement, which explains how RING can act as both the catalytic activator and the substrate in the ubiquitin transfer reactions. Fluorescent ubiquitin discharge assays reveal that proximity of three RINGs strongly enhances the rates of the two distinct ubiquitin transfer processes involved in the autoubiquitylation of TRIM5α: the N-terminal priming of TRIM5α with a single ubiquitin by UBE2W and the subsequent…