Self-assembly properties of a model RING domain

Abstract

RING domains act in a variety of essential cellular processes but have no general function ascribed to them. Here, we observe that purified arenaviral protein Z, constituted almost entirely by its RING domain, self-assembles in vitro into spherical structures that resemble functional bodies formed by Z in infected cells. By using a variety of biophysical methods we provide a thermodynamic and kinetic framework for the RING-dependent self-assembly of Z. Assembly appears coupled to substantial conformational reorganization and changes in zinc coordination of site II of the RING. Thus, the rate-limiting nature of conformational reorganization observed in the folding of monomeric proteins can also apply to the assembly of macromolecular scaffolds. These studies describe a unique mechanism of nonfibrillar homogeneous self-assembly and suggest a general function of RINGs in the formation of macromolecular scaffolds that are positioned to integrate biochemical processes in cells.

Figure 1

Purified LCMV and LFV Z self-assemble. (a) Alignment of lymphocytic choriomeningitis virus (LCMV) and Lassa fever virus (LFV) Z amino acid sequences and schematic of zinc binding by their RING domains using the cross-brace topology (6). Site I is in blue and site II is in red. Arrows delineate N- and C-terminal regions deleted in the RING construct. (b) Z is present in punctate nuclear and cytoplasmic bodies (indicated by arrow heads) and in diffuse pattern throughout the cytoplasm of NIH 3T3 cells 90 h postinfection with LCMV, stained with an antibody for Z, and visualized by using confocal laser scanning microscopy. Micrograph represents a single confocal section using an ×100 objective lens. (c) Electron micrograph of purified and negatively stained Z showing a morphologically homogeneous population of 300–500-Å spherical particles. Nominal magnification is ×100,000.

Figure 2

Assembly behavior of Z and its mutants and their secondary structure content. (a) Far-UV CD spectra of wild-type Z (solid black), first zinc-binding site mutant (Z1, red), second zinc-binding site mutant (Z2, blue), Z deletion mutant containing only the minimal RING domain (RING, orange), Z at 40°C (green), wild-type Z in the presence of 10-fold excess of EDTA (dashed black), and Z in the presence of 4 M guanidine hydrochloride (dotted black). (b Upper) Fractional sedimentation boundary plateau values as derived from sedimentation velocity (SV) data for assembly species for wild-type Z (Z), RING, fusion of Z with glutathione S-transferase (Z-GST), Z with 10-fold excess of EDTA, Z1, and Z2. Note that destabilization of site I in the Z1 leads to precipitation of most of the protein. SV profiles of the fusion of Z with GST and Z2 were fit to two sedimenting species, with error bars representing the uncertainty in estimating the plateau concentration caused by broad boundary widths. Asterisks represent inconsistencies between calculated sedimentation and diffusion coefficients (see Methods and Analysis and Table 2). (b Lower) Single-particle electron micrographs of negatively stained preparations showing fully assembled bodies of Z and Z2, lack of assembly of the minimal RING domain, Z1, and EDTA-treated Z as well as amorphous aggregation of the fusion of Z with GST. (Bar, 0.1 μm.) (c) Gel filtration profile as a function of elution volume (V_e) for Z (solid) and Z2 (dashed) at 6°C. Z elutes with three well separated peaks corresponding to molecular masses for monomer (M), tetramer (T), and body (B), whereas the Z2 exhibits broad overlapping tetramer and body peaks, suggesting fast assembly and disassembly kinetics. Note that peak heights, normalized to total eluted absorbance, correspond to SV assembly species' fractions and are in good agreement with relative species' concentrations as measured by SV at 4°C (Figs. 3b and 4c). Elution of globular molecular mass standards are represented by solid triangles (from left to right: thyroglobulin, 667 kDa; catalase, 232 kDa; albumin, 67 kDa; RNase A, 14 kDa). (d) SV profile of Z consisting of radial scans of absorbance as a function of sedimentation time and showing three sedimentation boundaries that move outward to the edge of the cell at 7.2 cm and become broader because of diffusion in the course of the experiment. Absorbance values of the plateaus of sedimentation boundaries are related directly to the concentration of the sedimenting species. The meniscus is located at 6.2 cm.

Figure 3

Thermodynamics and kinetics of Z assembly. (a) Guanidine denaturation profile of Z bodies (○), Z tetramers (◊), and Z monomers (□) as ascertained from SV boundary plateau concentrations. Reversibility is indicated by the formation of bodies (●) from monomers (■). Denaturation (★) and renaturation (⋆) profile of Z as monitored by CD at 222 nm. (b) van't Hoff plot of the apparent equilibrium constants (K) of assembly as derived from the plateau concentration analysis for the monomer-tetramer (dashed line, blue ◊) and tetramer-body (solid line, green ○) assembly steps. (c) Kinetics of Z assembly as monitored by CD at 222 nm (lines) and SV boundary plateau analysis (blue dots). Assembly was initiated by manual dilution of fully unfolded and reduced protein into native conditions in the presence of stoichiometric Zn²⁺ and reductant, upon which the protein folded and tetramerized within the experimental 30-s dead time (supporting Fig. 4e). Assembly kinetics of T → B for wild-type Z in 0.1 M guanidine (black) and 0.1 M guanidine in the presence of 1% fully assembled bodies (red, with the arrow indicating time of addition of seed) and Z2 in 0.1 M guanidine (green). Nucleation kinetics were tested by using both bodies assembled at pH 5 and 0.5 M guanidine hydrochloride and produced identical results (data not shown). Horizontal and vertical error bars reflect the uncertainty in estimating the time point of observation by SV and the error in estimating the fraction body from sedimentation boundary plateau concentrations, respectively. (d) Guanidine dependence of observed assembly (left limb) and disassembly rates (right limb) for Z (solid line, ●) and Z2 (dashed line, ○). Assembly rates scale linearly with guanidine concentration even in <0.5 M guanidinium, where rollover of rates would be expected because of the presence of the tetrameric intermediate. The lack of observation of such a rollover is likely caused by the experimental dead time of 30 s and subsecond interconversion of monomers and tetramers (Methods and Analysis). The apparent two-state nature of the kinetic pathway thus is due to preequilibration of monomer and tetramer as a result of current experimental limitations.