Quantitative NMR analysis of the kinetics of prenucleation oligomerization and aggregation of pathogenic huntingtin exon-1 protein

Abstract

The N-terminal region of the huntingtin protein, encoded by exon-1 (htt^ex1) and containing an expanded polyglutamine tract, forms fibrils that accumulate in neuronal inclusion bodies, resulting in Huntington's disease. We previously showed that reversible formation of a sparsely populated tetramer of the N-terminal amphiphilic domain, comprising a dimer of dimers in a four-helix bundle configuration, occurs on the microsecond timescale and is an essential prerequisite for subsequent nucleation and fibril formation that takes place orders of magnitude slower on a timescale of hours. For pathogenic htt^ex1, such as htt^ex1Q₃₅ with 35 glutamines, NMR signals decay too rapidly to permit measurement of time-intensive exchange-based experiments. Here, we show that quantitative analysis of both the kinetics and mechanism of prenucleation tetramerization and aggregation can be obtained simultaneously from a series of ¹H-¹⁵N band-selective optimized flip-angle short-transient heteronuclear multiple quantum coherence (SOFAST-HMQC) correlation spectra. The equilibria and kinetics of tetramerization are derived from the time dependence of the ¹⁵N chemical shifts and ¹H-¹⁵N cross-peak volume/intensity ratios, while the kinetics of irreversible fibril formation are afforded by the decay curves of ¹H-¹⁵N cross-peak intensities and volumes. Analysis of data on htt^ex1Q₃₅ over a series of concentrations ranging from 200 to 750 μM and containing variable (7 to 20%) amounts of the Met⁷O sulfoxide species, which does not tetramerize, shows that aggregation of native htt^ex1Q₃₅ proceeds via fourth-order primary nucleation, consistent with the critical role of prenucleation tetramerization, coupled with first-order secondary nucleation. The Met⁷O sulfoxide species does not nucleate but is still incorporated into fibrils by elongation.

Keywords: NMR spectroscopy; aggregation kinetics; huntingtin exon-1; nucleation; short-lived excited states.

Fig. 1.

Simultaneous determination of the kinetics and mechanism of tetramerization and fibril formation of htt^ex1Q₃₅ from serially recorded ¹H-¹⁵N SOFAST-HMQC spectra. The kinetics and equilibria of tetramerization are derived from the time dependence of the ¹⁵N chemical shifts and ¹H_N/¹⁵N cross-peak V/I ratios, which provide the dependence of these spectral parameters on the concentration of free monomer, while the kinetics and mechanism of fibril formation are determined from analysis of the decay of ¹H_N/¹⁵N cross-peak intensities and volumes. Inhibition of tetramer formation, either directly by modification of residues within the NT region (24, 25) or sequestration of the monomer by binding of the NT region to chaperones (26) or indirectly via an allosteric mechanism by binding of cellular proteins such as profilins and SH3 domains to the C-terminal polyproline rich domain (22, 27), blocks fibril formation.

Fig. 2.

Overall aggregation profiles of 750 μM ¹⁵N-labeled htt^ex1Q₃₅ monitored by acquisition of serial ¹H-¹⁵N SOFAST-HMQC spectra at 5 °C and 800 MHz. (A) Time courses of normalized cross-peak intensities (red circles) and volumes (blue circles) for residues in the NT, polyQ, and PRD domains. The data are normalized to the first (reference) experiment recorded immediately upon dissolution and pH adjustment (t ∼0 h). (B) Domain organization of htt^ex1Q₃₅ (Top) and residual ¹H-¹⁵N cross-peak intensities after 15 h (Bottom). No cross-peaks are detectable above the noise level for residues in either the NT or polyQ regions, indicating that these regions are fully immobilized within the htt^ex1Q₃₅ fibrils. ¹H-¹⁵N cross-peaks attributable to residues in the PRD, however, are present with the intensity increasing toward the C terminus, indicative of significant mobility/disorder of the PRD within the htt^ex1Q₃₅ fibrils. The error bars represent the SDs for the cross-peak intensities determined from three ¹H-¹⁵N SOFAST-HMQC spectra recorded at 14.8, 15.0, and 15.2 h. Note that no cross-peaks can be detected for the two proline repeats (shaded in green) in a ¹H-¹⁵N correlation spectrum since the proline nitrogen atom is not bonded to a proton. (C) Correlation between the normalized average ¹H-¹⁵N cross-peak intensities for residues within the PRD (x axis) and the average normalized ¹H-¹⁵N cross-peak volumes (y axis) for residues in the NT (Top) and PRD (Bottom) domains. corr. coeff., correlation coefficient.

Fig. 3.

Impact of the oxidation of Met⁷ to a sulfoxide (Met⁷O) on prenucleation tetramerization and fibril formation. (A) Ser¹²/Ser¹⁵ region of the ¹H-¹⁵N SOFAST-HMQC spectrum of 750 μM ¹⁵N-labeled htt^ex1Q₃₅. Cross-peaks arising from native (reduced) and Met⁷O oxidized forms are labeled in black and red, respectively. In this sample, the fraction of the Met⁷O sulfoxide form is 20 ± 2%. (B) 100% Met⁷O oxidized htt^ex1Q₃₅ (150 μM) does not aggregate as monitored from the time course of the integrated intensity (from 7.9 to 8.5 ppm) of the amide proton envelope (obtained from the first increment of a ¹H-¹⁵N SOFAST-HMQC spectrum. The inset shows a one-dimensional spectrum of the amide proton envelope recorded at 0 (red) and 10 (blue) h. (C) Concentration dependence of ¹⁵N exchange-induced shifts (¹⁵N-δ_ex) for Ser12 in the native reduced (black) and Met⁷O oxidized (red) forms of htt^ex1Q₃₅ referenced to the shifts of a 10-μM sample. The total concentration of htt^ex1Q₃₅ at t = 0 h was 750 μM, corresponding to 600-μM native and 150-μM Met⁷O oxidized forms, and the shifts are followed by serial ¹H-¹⁵N SOFAST-HMQC spectra as the concentration of monomeric htt^ex1Q₃₅ decreases. The concentration is determined from the time dependence of the average cross-peak intensities for residues within the PRD domain. (D) Time dependence of the ¹H-¹⁵N cross-peak intensities for Ser¹² in the native reduced (black) and Met⁷O oxidized (red) forms observed for a 750-μM sample of ¹⁵N-labeled htt^ex1Q₃₅ with 20% in the Met⁷O oxidized form. All NMR data were recorded at 5 °C and 800 MHz.

Fig. 4.

Quantitative analysis of prenucleation tetramerization of htt^ex1Q₃₅ from a global fit to concentration-dependent ¹⁵N exchange–induced shifts (¹⁵N-δ_ex) and ¹H-¹⁵N cross-peak V/I ratios. (A–C) ¹⁵N-δ_ex (A) and ¹H-¹⁵N cross-peak (B) V/I ratios as a function of the concentration of native monomeric htt^ex1Q₃₅ at 5 °C and 800 MHz. The concentrations are derived from the time dependence of the average cross-peak intensity for residues within the PRD. The total sample concentration is 750 μM, but only the native reduced form, corresponding to 600 μM, undergoes prenucleation oligomerization. The experimental data are shown as circles, and the best fits to the tetramerization scheme in (C) are represented by the continuous solid lines. The ¹⁵N-δ_ex data report only on the monomer ↔ dimer and dimer ↔ tetramer equilibria, while the cross-peak V/I ratios also report on the dissociation rate constant from dimer to monomer (k₋₁) and provide a lower limit on the dissociation rate constant from tetramer to dimer (k₋₂). (C) Kinetic scheme with values of equilibria and rate constants (E, monomer; E₂, dimer; E₄, tetramer). The populations listed above the species correspond to those at the highest concentration (600 μM at t = 0 h) of native htt^ex1Q₃₅. For errors of 0.5 Hz and 1 a.u. for ¹⁵N-δ_ex and ¹H-¹⁵N V/I, respectively, the value of the reduced χ² for the global fit is 0.86. a.u., arbitrary units.

Fig. 5.

Quantitative analysis of the mechanism and kinetics of htt^ex1Q₃₅ aggregation probed by serially acquired ¹H-¹⁵N SOFAST-HMQC spectra. (A) Time dependence of the average ¹H-¹⁵N cross-peak intensities for residues in the PRD domain (Top) and of the ¹H-¹⁵N cross-peak volume and intensity of Ser¹² in the native reduced and Met⁷O oxidized forms, respectively, (Bottom) of htt^ex1Q₃₅. The total concentrations of monomeric htt^ex1Q35 at t = 0 are indicated. For the 750-μM sample, the percentage of the Met⁷O oxidized form is 20 (±2)%; for the other samples, the percentage of the Met⁷O oxidized form is 7 to 8%. The experimental data (shown as circles, with the bars equal to 1 S.D.) were recorded at 5 °C and 800 MHz and normalized to the first time point (at t ∼0 h). The best-fit curves are shown as black continuous lines and were obtained from a global fit to the kinetic scheme described by the set of differential equations in Eq. 4, which incorporates fourth-order primary nucleation (nc = 4), elongation, and first-order secondary nucleation (n2 = 1) for the native reduced form and elongation for the Met⁷O oxidized form. (B) Schematic depiction of primary nucleation, elongation, and monomer-dependent surface-catalyzed secondary nucleation. [m_red] and [m_oxi] are the concentrations of free monomer in the native reduced and Met⁷O oxidized forms, respectively; [M] is the total fibril mass in monomer units; [P] is the number concentration of extendable ends of the fibril chain; k_n, k₊, and k₂ are the rate constants for primary nucleation, elongation, and secondary nucleation, respectively; and nc and n2 are the order of primary and secondary nucleation, respectively. (C) Grid search showing the dependence of the reduced χ² on the order of primary nucleation (nc). The minimum χ² is obtained for nc = 4, consistent with prenucleation tetramerization. Although the value of χ² for nc = 3 is only slightly higher than that for nc = 4, nc = 3 can be excluded as unrealistically high values of the initial fibril number concentration, P(0), about 20-fold higher than for nc = 4, are required to fit the data (SI Appendix, Fig. S4). (D) Log-log plot of aggregation t_1/2 versus concentration of htt^ex1Q₃₅ monomer (red) and population (p_tetramer) of tetramer (black, calculated using the equilibrium dissociation constants listed in Fig. 4C). corr. coeff., correlation coefficient.

Fig. 6.

Simulated time dependence of htt^ex1Q₃₅ fibrillization at 5 °C calculated using the optimized values of the rate constants given in Table 1. The fibril concentration in monomer units (M_red and M_oxi), the concentration of extendable ends (P), and the ratio of total fibril concentration (M_red + M_oxi) to extendable ends (P) are displayed. The latter ratio is proportional to fibril length. Assuming a separation of ∼4.8 Å (the distance between strands in a β-sheet) between monomer units along the length of the fibril, the fibril length reaches a maximum of approximately ∼1 μm, consistent with negative stain electron microscopy images of htt^ex1Q₃₅ fibrils (after 70-h incubation of 40 μM htt^ex1Q₃₅ at 5 °C), shown in the Inset.