Mechanism of an ATP-independent protein disaggregase: II. distinct molecular interactions drive multiple steps during aggregate disassembly

Abstract

The ability of molecular chaperones to overcome the misfolding and aggregation of proteins is essential for the maintenance of proper protein homeostasis in all cells. Thus far, the best studied disaggregase systems are the Clp/Hsp100 family of "ATPases associated with various cellular activities" (AAA(+)) ATPases, which use mechanical forces powered by ATP hydrolysis to remodel protein aggregates. An alternative system to disassemble large protein aggregates is provided by the 38-kDa subunit of the chloroplast signal recognition particle (cpSRP43), which uses binding energy with its substrate proteins to drive disaggregation. The mechanism of this novel chaperone remains unclear. Here, molecular genetics and structure-activity analyses show that the action of cpSRP43 can be dissected into two steps with distinct molecular requirements: (i) initial recognition, during which cpSRP43 binds specifically to a recognition motif displayed on the surface of the aggregate; and (ii) aggregate remodeling, during which highly adaptable binding interactions of cpSRP43 with hydrophobic transmembrane domains of the substrate protein compete with the packing interactions within the aggregate. This establishes a useful framework to understand the molecular mechanism by which binding interactions from a molecular chaperone can be used to overcome protein aggregates in the absence of external energy input from ATP.

Keywords: ATP-independent Disaggregase; Enzyme Mechanisms; Kinetics; Membrane Proteins; Molecular Chaperone; Mutagenesis Mechanisms; Protein Aggregation; Protein Biogenesis; Signal Recognition Particle.

FIGURE 1.

cpSRP43 makes highly sequence-specific interactions with the FDPLGL motif in the L18 sequence. A–F, alanine-scanning mutagenesis of the entire LHCP (A-C), and alanine-, glycine-, and lysine-scanning mutagenesis within the L18 sequence of LHCP (D–F). The aggregation prevention activity of cpSRP43 was assayed at 1:1 (A and D) and 1:3 (B and E) molar ratios of LHCP to cpSRP43. aa, amino acids; mut, mutant. In C and F, the disaggregase activity was measured at 1:6 molar ratio of LHCP to cpSRP43. All assays were performed in 384-well plates using a Tecan Freedom EVO liquid-handling robot, as described under “Experimental Procedures.” G and H, single-cysteine substitutions at individual residues in L18 were tested for their ability to prevent the aggregation of LHCP (G) and to resolubilize existing LHCP aggregates (H). In G, a 1:1 ratio of cpSRP43 and LHCP was used. In H, a 5:1 ratio of cpSRP43 relative to LHCP (in aggregates) was used.

FIGURE 2.

cpSRP43 can interact with a variety of LHCP TM mutants. A, binding of cpSRP43 to TM mutants as measured by changes in anisotropy. Fits of data gave K_d values of 22 nm for ΔTM3 and 713 nm for 1-2-2. For comparison, the K_d^app values measured by light scattering were 26 and 489 nm, respectively (Table 4). B–E, binding of cpSRP43 to LHCP and its TM mutants as measured by the ability of cpSRP43 to prevent the aggregation of substrate proteins (see “Experimental Procedures”). The data were fit to Equation 1 (see “Experimental Procedures”) and gave K_d^app values that are summarized in Table 4. F, summary of the K_d^app values of all the LHCP TM mutants characterized in this study. Values of K_d^app were determined by a combination of light scattering and fluorescence anisotropy assays.

FIGURE 3.

Schematics depicting quantitative analysis of the cpSRP43-mediated disaggregation reaction. Concentration dependences of the kinetics (A) and equilibrium (B) of the cpSRP43-mediated reversal of wild-type LHCP aggregate yield important parameters that report on the energetics of different steps of the disaggregation pathway (C).

FIGURE 4.

L18-binding mutants uncouple initial recognition of the aggregate from its subsequent solubilization. A, binding of cpSRP43 to wild-type Lhcb₅ and L18 mutants H160C and H170C. The data were fit to Equation 1 (see “Experimental Procedures”) and gave K_d^app values of 10 nm for wild-type Lhcb₅ (black), 30 nm for Lhcb₅-H160C (blue), and 1.1 μm for Lhcb₅-L160C (red). B, concentration dependences for the equilibrium of disaggregation of LHCP by wild-type cpSRP43 (black) or mutant cpSRP43(R161A) (magenta). C and D, chaperone concentration dependences for the equilibrium (C) and kinetics (D) of disaggregation of Lhcb₅ (black), Lhcb₅-H160C (blue), and Lhcb₅-L170C (red) by wild-type cpSRP43.

FIGURE 5.

LHCP TM mutants exhibit a wide range of disaggregation efficiencies. A–H, representative concentration dependences of the equilibrium (A, C, E, and G) and kinetics (B, D, F, and H) for disassembly of the aggregates formed by the LHCP TM mutants. The data for wild-type LHCP (black) were shown as a reference of comparison in all four sets. The data in A, C, E, and G were fit to Equation 4 (black, blue, and green) or Equation 6 (red) to obtain K_max values and extract K_app values at 4 μm cpSRP43. The data in B, D, F, and H were fit to Equation 5 (black, blue, and green) or Equation 7 (red) to obtain k_max, 〈K_m〉, and h values and to extract k_app values at 4 μm cpSRP43. All the thermodynamic and kinetic parameters were reported in Table 4.

FIGURE 6.

Time courses for the alkylation reactions of cysteine residues in the L18 shows accessibility of WT (A) and mutant (B and C) LHC proteins. A–C, Lhcb5 L170C (A), ΔTM2 G158C (B), and ΔTM3 G158C (C) were labeled with 30-fold excess N-ethyl-maleimide in denaturant guanidinium hydrochloride (GdmHCl), pH 7.5 (black traces) and in aqueous buffer, pH 7.5 (red traces), and the reactions were quenched at different time points with DTT and subjected to intact protein mass spectrometry as in the accompanying manuscript (34).

FIGURE 7.

The irreversible TM mutants form ultrastable aggregates. A, sedimentation analysis of the ability of guanidinium chloride (GdmHCl) and urea to resolubilize LHCP aggregates. M denotes the protein marker lane. B–E, urea solubilization curves of LHCP and its TM mutants. The data were fit to Equation 8 (see “Experimental Procedures”) and gave U₅₀ values (Table 4).

FIGURE 8.

Linear free energy analysis of the cpSRP43-mediated disaggregation reaction. A, the maximal disaggregation rate constant strongly correlates with a weighted combination of the U₅₀ and K_d^app values. Values for the analysis are from Table 4. The black line represents a linear fit to the data (R² = 0.96). ΔTM1 and ΔTM3 (blue) were marked as outliers and were not included in the correlation. B, ϕ value analysis of LHCP disaggregation. The values of K_app and k_app were calculated from fits of disaggregation equilibrium and kinetic data to Equations 4 and 5 (“Experimental Procedures”), respectively, and the concentration of cpSRP43 was chosen at 4 mm. Linear fit of the data (black line, R² = 0.98) gave a slope (ϕ value) of 0.73.

FIGURE 9.

A, Working model for cpSRP43-mediated disaggregation reaction. Step 1 depicts initial binding of cpSRP43 (magenta) to the LHCP aggregate (green), which occurs via recognition of the solvent-exposed L18 motif (red). Step 2 depicts the cooperative action of cpSRP43 molecules to compete with and disrupt the packing interactions between the LHCP TM segments within the aggregate, leading to its resolubilization. B and C, qualitative free energy diagrams summarizing the effects of the L18-binding mutants that disrupt the initial binding step (B) and the irreversible TM mutants that disrupt the remodeling step (C), as described under “Results.” The figures are not drawn to scale.