Kinetic mechanism of p53 oncogenic mutant aggregation and its inhibition

Kinetics of aggregation from light scattering.

Aggregation was measured at 37 °C, by monitoring light scattering at 500 nm (Fig. 1A). There was an initial lag period, which is usually taken as indicating nucleation events taking place; a growth period; and then a leveling off as substrate was depleted. The lag time shortened with increasing concentrations of p53. This was also shown by monitoring the absorbance at 360 nm (Fig. 1C). Light scattering is greatly weighted to the contribution of large aggregates, as the magnitude of Rayleigh scattering varies as the size raised to the power of 6, which overemphasizes the lag.

Kinetics of aggregation from thioflavin T binding.

Thioflavin T (ThT) binds to amyloid fibrils, and can give a good signal that is relatively independent of size and so shows early aggregation events that precede the formation of large aggregates (14, 20, 21). Indeed, the lag was considerably reduced (Fig. 1B). Normalizing the ThT signal to the concentration of p53 (Fig. 1D) showed that the amplitude of the signal was constant, and not dependent on the size of particles, in contrast to light scattering. Remarkably, the kinetics fitted to the very simple Scheme 1 of a two-step reaction of sequential first-order reactions (where A is the monomeric protein, B is a monomeric intermediate, and C is a product that has the ThT-fluorescence signal), which has the solution Eq. 1 for the formation of C (22)

[1]

In practice, we fitted the data to:

[2]

where F_t is the intensity of ThT fluorescence at time t, and m, the amplitude, = [A]₀f, where f is the specific fluorescence of ThT bound to the aggregate. A small linear drift term of k₃t was added to Eq. 1 to allow for any drift in signal strength because of machine drift or slow settling of particles. k₃ was close to zero. Note that Eq. 1 is unchanged if k₁ and k₂ are interchanged, so we cannot assign from the curve fitting whether the numerically greater rate constant precedes the lower or vice versa. We solve the assignment problem later in this paper from inhibition studies.

The values of k₂ and k₁ in min^-1 derived from the fits to Eq. 2 were, respectively: 3 μM p53, 0.32 ± 0.03 and 0.064 ± 0.0016; 6 μM, 0.36 ± 0.02 and 0.067 ± 0.001; 9 μM, 0.37 ± 0.03 and 0.063 ± 0.0007; and 12 μM, 0.37 ± 0.015 and 0.071 ± 0.007 (means and standard errors for single runs). Both rate constants were virtually independent of protein concentration. The residuals between calculated curves and data are good (Fig. S1). There may be a low amplitude faster phase that we missed in the analysis, possibly that of the initial unfolding of the protein prior to slower aggregation events. But the above equation accounts for the major phases of aggregation. The mean and standard error for 9 measurements at 3 μM protein in the inhibition experiments below gave 0.314 ± 0.018 and 0.0545 ± 0.0017 min^-1.

Fluorescence spectra and kinetics.

Fluorescence spectra at various time points during aggregation (Fig. 2A) showed apparently only two major fluorescent species during the process: the native protein (λ_max = 305 nm) being lost and the aggregate state (λ_max = 340 nm) being formed with an isoemission wavelength of 314 nm and with the same rate constants (Fig. 2C). The process is not simple denaturation of Y220C, since the experiments were at 7.5 °C below its T_m and the fluorescence spectrum of the product is not that of the denatured state. [The denatured state has λ_max = 356 nm (3, 4) and Fig. 2B.] The kinetics of fluorescence 340 nm also fitted reasonably well to Eq. 1 to give similar values of k₂ and k₁ in min^-1: 1 μM p53, 0.20 ± 0.01 and 0.084 ± 0.0009; 2 μM, 0.47 ± 0.02 and 0.067 ± 0.0006; 3 μM, 0.45 ± 0.02 and 0.066 ± 0.0006; and 5 μM, 0.61 ± 0.04 and 0.074 ± 0.006. The formation of large aggregates causes the noise in the latter stages of all the kinetic runs and a slow decrease in signal (Fig. 2D). The mean rate constants are 0.43 ± 0.08 and 0.073 ± 0.004 min^-1, averaged from the singles runs at each concentration.

ThT has minimal perturbation of kinetics.

The near coincidence of ThT and 340-nm kinetic data strongly suggests that ThT does not affect the kinetics significantly. We showed directly that varying ThT between 5–20 μM has minimal effects on 340 nm-monitored kinetics (Fig. S2A) and on light scattering (Fig. S2B). The rate constants for ThT kinetics remained constant within experimental error between 5 and 25 μM (Fig. S2C), but the amplitudes increased, fitting a binding isotherm of 10 ± 0.6 μM (Fig. S2D), indicating a reversible binding of ThT. We discuss further in the accompanying paper why different techniques give different kinetics (23).

Lack of seeding by aggregated Y220C.

Nucleation mechanisms are characterized by being seeded by already aggregated protein. (24) The addition of 1 μM aggregated Y220C to 2 μM fresh p53 (Fig. S3A) had only a small effect on light scattering relative to that of 2 μM fresh protein alone, and the initial rate was similar to that of 3 μM fresh protein. The addition of 1 μM aggregated Y220C to 2 μM fresh p53 monitored by ThT fluorescence (Fig. S3B) gave no detectable change in the kinetics.

Electron microscopy studies.

During the lag period, we saw only tiny particles in electron micrographs. These covered much of the grid, but during the growth period they progressively coalesced to form amorphous aggregates that grew larger with increasing time (Fig. S4A, and see electron microscopy studies of inhibition below, Fig. S4B).

Light scattering.

The effects of potential drugs on aggregation were monitored at 500 nm to avoid any light absorbance by them. The addition of 5174 at up to 120 μM significantly inhibited aggregation (Fig. 3A). However, the shape of the curve changed with increasing concentration, having a sloping phase before leveling off. Such curves can be fitted to empirical equations. Following the procedure of Wetzel and coworkers (25, 26), we used a simple model-free method and plotted the initial rates of scattering against t², where t is time [a generally useful plot (27)]. The rate of addition of monomer to growing oligomers varies as t² and the slope of the plot is V₀. If a ligand binds to the protein with dissociation constant K_I and the protein-ligand complex is fully inhibited from aggregation, V₀ depends on the concentration of unbound protein and is given by:

[3]

More generally, if the ligand-bound protein also aggregates with an initial rate of V_0BL, then:

[4]

The slopes of the inhibition plots plotted against concentrations of ligand fitted saturation curves, with V_0BL being undetectably small. Variation of 5174 (Fig. 3A) gave a K_I of 19 ± 4 μM (Fig. 3C). Compound 5201 was particularly effective: the calculated K_I = 6 ± 1 μM, and the lag period for aggregation at 120 μM drug was increased to nearly an hour (Fig. 3B and D). Similar plots were performed for other ligands (Fig. S6).

Thioflavin T binding.

The rate of increase of fluorescence of ThT was inhibited by the ligands and gave curves that could individually be well fitted to Eq. 2 (Fig. S7). The inhibition of k₁ (Fig. 4) and k₂ (Fig. 5) fitted to simple binding isotherms that did not tend to zero as the concentration of ligand increased. We first fitted the data to Eq. 4 (substituting k for V₀) without any constraints. The curves generated large standard errors (Table 1), so we then refitted assuming a common value for k₁ in the absence of ligand, the average of all the curves (0.0557 ± 0.002 min^-1), and for the value for ligand-bound protein (0.0187 ± 0.0027 min^-1), so that the only variable was the constant K_I (Fig. 4). The values thus derived were similar to the free-fitted, but with much better standard errors (Table 1). These values were very similar to those measured directly by isothermal titration calorimetry (ITC). (19) The same was done for k₂ using values of (0.319 ± 0.001 min^-1) and for the value for ligand-bound protein (0.055 ± 0.015 min^-1, Fig. 5), to generate values of K_I that were, generally, significantly higher, apart from 5176, where the data were poorer.

The product of the two rate constants k₁k₂ also fitted a simple isotherm. The initial rate of aggregation, according to Eq. 1 as t tends to 0, is given by: V₀ = 0.5k₁k₂t²[A]₀. That equation is analogous to that for the initial rate of the light scatter plots. The initial rate of increase of ThT fluorescence versus t² may be derived from the product of k₁ and k₂ measured from the lag kinetics. A plot of k₁k₂ versus [ligand] is analogous to Eqs. 3 and 4 for calculating a K_D for inhibition (Fig. 6). The derived values were close to those obtained from ITC (Table 1). We could not analyze the data for the binding of 83 because it binds to ThT.

340 nm fluorescence.

The kinetics of appearance of aggregate was followed by fluorescence at 340 nm. The absorbance of the ligands at the excitation wavelength as well as the emission caused significant loss of signal at higher concentrations, so the data were less accurate. But the same trends as for the ThT-monitored kinetics were observed, and the limited analysis of k₁k₂ gave dissociation constants consistent with the other methods (Table 1).

Electron microscopy.

140 μM 5174 and 400 μM 83 inhibited the formation and growth of aggregates in good qualitative agreement with the light scattering studies (Fig. S4B).

Light scattering.

To measure the effect of compounds on the kinetics of aggregation of Y220C, we monitored protein aggregation by measuring light scattering at 37 °C at 500 nm as excitation and emission wavelengths (excitation slit width 0.8 nm, emission slit width 2 nm), using a Horiba FluoroMax-3 spectrophotometer. Experiments were generally performed with a protein concentration of 3 μM in 25 mM potassium or sodium phosphate, pH 7.2, 150 mM NaCl, 5 mM DTT or 1 mM TCEP, and 5% DMSO. The kinetics at the single wavelength of 340 nm was measured using the same setup as light scattering above, but excitation and emission wavelengths were set to 285 nm and 340 nm, respectively, with slit widths set to 3 nm (excitation) and 4 nm (emission). To obtain homogenous mixing, Scienceware (Bel-Art Products) spectrometer cell spinbars were used. Effects of aggregation seeds were minimized using disposable Fisher Scientific UV grade PMMA cuvettes and storing the spinbars in nitric acid when not in use. Data analysis was performed using KaleidaGraph (Synergy Software).

Thioflavin T assays.

As for detecting other amyloid aggregates, we measured the ThT fluorescence at 482 nm upon excitation at 450 nm (33) (excitation/emission slits are 3 nm/4 nm) using a Horiba FluoroMax-3 Spectrofluorometer. Time-resolved fluorescence was recorded immediately after adding 3 μM Y220C to pre-equilibrated buffer (25 mM sodium phosphate, pH 7.2, 150 mM NaCl, 1 mM TCEP, 5% DMSO, 20 μM ThT).

Time-resolved fluorescence spectra.

The intrinsic fluorescence spectra of Y220C (excited at 280 nm, emission between 300 nm and 500 nm) were recorded with a Horiba FluoroMax-4 Spectrofluorometer immediately after the addition of protein to pre-equilibrated buffer (25 mM sodium phosphate, pH 7.2, 150 mM NaCl, 1 mM TCEP, 5% DMSO). Both the slits for excitation and emission were 5 nm.