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. 2002 Nov 26;99(24):15404-9.
doi: 10.1073/pnas.202608799. Epub 2002 Nov 18.

Control of biochemical reactions through supramolecular RING domain self-assembly

Alex Kentsis  1 , Ronald E Gordon, Katherine L B Borden
Affiliations

Control of biochemical reactions through supramolecular RING domain self-assembly

Alex Kentsis et al. Proc Natl Acad Sci U S A. .

Abstract

RING domains act in a variety of unrelated biochemical reactions, with many of these domains forming key parts of supramolecular assemblies in cells. Here, we observe that purified RINGs from a variety of functionally unrelated proteins, including promyelocytic leukemia protein, KAP-1TIF1beta, Z, Mel18, breast cancer susceptibility gene product 1 (BRCA1), and BRCA1-associated RING domain (BARD1), self-assemble into supramolecular structures in vitro that resemble those they form in cells. RING bodies form polyvalent binding surfaces and scaffold multiple partner proteins. Separation of RING bodies from monomers reveals that self-assembly controls and amplifies their specific activities in two unrelated biochemistries: reduction of 5' mRNA cap affinity of eIF4E by promyelocytic leukemia protein and Z, and E3 ubiquitin conjugation activity of BARD1:BRCA1. Functional significance of self-assembly is underscored by partial restoration of assembly and E3 activity of cancer predisposing BRCA1 mutant by forced oligomerization. RING self-assembly creates bodies that act structurally as polyvalent scaffolds, thermodynamically by amplifying activities of partner proteins, and catalytically by spatiotemporal coupling of enzymatic reactions. These studies reveal a general paradigm of how supramolecular structures may function in cells.

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Figures

Fig 1.
Fig 1.
A variety of unrelated RING domains self-assemble into spherical and ring-shaped bodies, and scaffold multiple RING partner proteins on their surface. (A) Single-particle EM micrographs of RING bodies at a nominal magnification of ×80,000, using uranyl acetate counterstain. (Bottom Right) Micrograph is of RING of Mel18, whereas the rest are of PML, Z, and KAP-1/TIF1β and their site I and site II mutants. (B) Single-particle EM micrographs of BARD1 homomeric bodies, BRCA1 homomeric bodies, BARD1:BRCA1 heteromeric bodies, and BARD1:BRCA1 C64G at a nominal magnification of ×80,000, using methylamine tungstenate counterstain. (C and D) EM micrographs of Z and gold-eIF4E (C) and PML and gold-eIF4E (D), counterstained with uranyl acetate. Multiple gold-eIF4E molecules are scaffolded on the surface of both Z bodies and PML bodies, in agreement with gel filtration measurements (Fig. 7A). Note that heterogeneity in the number of molecules scaffolded by RING bodies is likely due to low (femtomolar) protein concentrations required for single-particle EM measurements that are several orders of magnitude below Kd for the respective associations, as well as stochastic heterogeneity on the microscopic level. The median diameter of gold particles is 6 nm.
Fig 2.
Fig 2.
Z bodies amplify specific activity of individual Z monomers as a result of thermodynamic linkage between self-assembly and RING partner binding and convert Z from a low-affinity low-cooperativity eIF4E inhibitor into a high-affinity switch-like antagonist. (A) Normalized corrected Trp fluorescence emission quenching curves of eIF4E and their fits, in free form (green ▪) and when bound to kinetically captured Z monomers (red ♦) and Z bodies (blue •) in the presence of increasing concentrations of m7GpppG. All proteins were used at 2 μM. (B) Thermodynamic linkage model describing the relationship among RING body assembly, RING partner binding, and RING partner activity for Z, eIF4E, and eIF4E ligand m7GpppG. Fraction of eIF4E (ƒ) bound to m7GpppG (y axis) and m7GpppG binding curves (x axis) are plotted as a function of increasing concentrations of Z (z axis) that lead to self-assembly of Z monomers into bodies, which scaffold multiple molecules of eIF4E.
Fig 3.
Fig 3.
BARD1:BRCA1 support Ubq polymerization by self-assembling into bodies that spatiotemporally couple multiple molecules of UbcH5C and form a catalytic polyvalent binding surface. (A) Size exclusion chromatography of BARD1:BRCA1 as a function of elution volume (Ve) immediately on initiation of assembly to kinetically capture dimers and tetramers (dashed), and, after incubation for 24 h, obtain bodies (solid). Bodies elute with globular molecular mass of 150 kDa, corresponding to 12 mers, as determined by using molecular weight standards represented by arrowheads (left to right: thyroglobulin, 667 kDa; catalase, 232 kDa; albumin, 67 kDa; chymotrypsinogen A, 25 kDa; RNase A, 14 kDa). (B) Relative activation of UbcH5C and poly-Ubq formation by BARD1:BRCA1 dimers, tetramers, and bodies as purified by gel filtration. Body′ represents freshly purified BARD1:BRCA1 12 mers, whereas Body represents 12 mers stored at −80°C, demonstrating that storage conditions do not affect activity. (C) Single-particle EM micrographs of products of ubiquitination assay of gel filtration purified BARD1:BRCA1:gold-UbcH5C complex using nanogold-Ubq, counterstained with methylamine tungstenate and methylamine vanadate, allowing visualization of both colloidal and nanogold particles. Multiple molecules of gold-UbcH5C (arrow, median diameter of 6 nm) are scaffolded by BARD1:BRCA1 ring-shaped bodies ≈13 nm in diameter, with multiple polynanogold-Ubq chains in the vicinity (arrowhead, 1.4-nm diameter). Heterogeneity in the number of molecules scaffolded by RING bodies is likely due to low (femtomolar) protein concentrations required for single-particle EM measurements that are several orders of magnitude below Kd for the respective associations, as well as stochastic heterogeneity on the microscopic level. Note that the difference in contrast between gold and nanogold is due to the difference in electron scattering power of the gold and nanogold particles.
Fig 4.
Fig 4.
Self-assembly of carcinogenic BRCA1 C64G mutant can be restored by forced oligomerization, leading to restoration of its ubiquitination activity. (A) Size exclusion chromatography profiles as a function of elution volume (Ve) of BARD1:BRCA1 C64G (dashed line) and BARD1:GST-BRCA1 C64G (solid line). Fusion of BRCA1 C64G to GST leads to formation of 170- to 500-kDa molecular mass species. (B) EM micrographs of BARD1:GST-BRCA1 C64G (GST-C64G) and BARD1:BRCA1 C64G (C64G), both at a nominal magnification of ×15,000, counterstained with uranyl acetate. Fusion of BRCA1 C64G to GST leads to formation of amorphous aggregates and round bodies ≈15 nm in diameter (arrowheads) that resemble those formed by wild-type BARD1:BRCA1 (Fig. 1B), whereas BARD1:BRCA1 C64G does not assemble. (C) Activation of UbcH5C and Ubq polymerization by BARD1/BRCA1 C64G (C64G), BARD1:GST-BRCA1 C64G (GST-C64G), as visualized by SDS/PAGE/Western blotting. Fusion of BRCA1 C64G to GST restores partial ubiquitination activity, as compared with wild-type soluble BARD1:BRCA1 (Body), and GST-BARD1:GST-BRCA1 immobilized on glutathione-Sepharose (Beads:WT). Interestingly, Sepharose-immobilized BARD1:GST-BRCA1 C64G (Beads:C64G) does not support Ubq polymerization, in agreement with other reports (7).
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