Quantitative Exchange NMR-Based Analysis of Huntingtin-SH3 Interactions Suggests an Allosteric Mechanism of Inhibition of Huntingtin Aggregation

Abstract

Huntingtin polypeptides (htt^ex1), encoded by exon 1 of the htt gene and containing an expanded polyglutamine tract, form fibrils that accumulate within neuronal inclusion bodies, resulting in the fatal neurodegenerative condition known as Huntington's disease. Htt^ex1 comprises three regions: a 16-residue N-terminal amphiphilic domain (NT), a polyglutamine tract of variable length (Q_n), and a polyproline-rich domain containing two polyproline tracts. The NT region of htt^ex1 undergoes prenucleation transient oligomerization on the sub-millisecond time scale, resulting in a productive tetramer that promotes self-association and nucleation of the polyglutamine tracts. Here we show that binding of Fyn SH3, a small intracellular proline-binding domain, to the first polyproline tract of htt^ex1Q₃₅ inhibits fibril formation by both NMR and a thioflavin T fluorescence assay. The interaction of Fyn SH3 with htt^ex1Q₇ was investigated using NMR experiments designed to probe kinetics and equilibria at atomic resolution, including relaxation dispersion, and concentration-dependent exchange-induced chemical shifts and transverse relaxation in the rotating frame. Sub-millisecond exchange between four species is demonstrated: two major states comprising free (P) and SH3-bound (PL) monomeric htt^ex1Q₇, and two sparsely populated dimers in which either both subunits (P₂L₂) or only a single subunit (P₂L) is bound to SH3. Binding of SH3 increases the helical propensity of the NT domain, resulting in a 25-fold stabilization of the P₂L₂ dimer relative to the unliganded P₂ dimer. The P₂L₂ dimer, in contrast to P₂, does not undergo any detectable oligomerization to a tetramer, thereby explaining the allosteric inhibition of htt^ex1 fibril formation by Fyn SH3.

Figure 1.

Inhibition of aggregation and fibril formation of htt^ex1Q₃₅ by Fyn SH3. (A) Time course of the integrated intensity of the amide proton envelope of 0.1 mM htt^ex1Q₃₅ in the absence (red) and presence (blue) of 3 mM Fyn SH3, obtained from the first increment of serial ¹H–¹⁵N correlation experiments recorded at 600 MHz and 5 °C. The inset is a picture of the NMR tubes after 70 h in the absence (left) and presence (right) of SH3. (B) Negative stain EM of the NMR sample after 70 h in the presence of SH3. (C) Concentration dependence of aggregation kinetics of htt^ex1Q₃₅ monitored by ThT fluorescence at 37 °C. (D) Effect of SH3 on aggregation of 50 μM htt^ex1Q₃₅ monitored by ThT fluorescence at 37 °C.

Figure 2.

Chemical shift perturbation mapping of interaction sites between htt^ex1Q₇ and Fyn SH3. (A) Weighted ¹H_N/¹⁵N perturbation profile (Δδ_H/N = {[ΔδN²/5² + ΔδH²]/2}^1/2) obtained for 0.16 mM ¹⁵N-labeled SH3 in the presence of 0.56 mM unlabeled htt^ex1Q₇. The inset shows the Δδ_H/N values for the side chain nitrogen atoms of W36 and N53. (B) Model of polyproline P₁₁ binding to Fyn SH3 derived from the NMR structure of a SH3-polyproline-containing peptide complex (PDB code 1AZG); side chains of residues with Δδ_H/N > 0.22 ppm are colored in red (see Experimental Section for details of modeling). (C) Δδ_H/N profile for 0.1 mM ¹⁵N/¹³C-labeled htt^ex1Q₇ in the presence of 0.4 mM SH3. The domain organization of htt^ex1Q₇ is depicted above the plot, and the inset shows progressive line broadening of ¹³C cross sections through the ¹H/¹³Cα cross-peaks of the prolines upon addition of SH3. All NMR data were recorded at 5 °C at either 600 (A) or 900 (C) MHz.

Figure 3.

Quantitative analysis of the kinetics and equilibria of Fyn SH3 binding to htt^ex1Q₇ and subsequent dimerization. (A) Examples of the dependence of δ_ex and R_2,eff (at two spin lock fields, 750 and 3000 Hz) measured for 0.1 mM ¹⁵N/¹³C-labeled htt^ex1Q₇ on the concentration of added unlabeled SH3. The data were acquired at 900 MHz. (B) Examples of ¹⁵N (800 MHz) and ¹³Cα (600 and 800 MHz) CPMG relaxation dispersion profiles for 0.3 mM ¹⁵N/¹³Cα-labeled htt^ex1Q₇ in the presence of 0.3 mM (¹⁵N and ¹³Cα) and 0.6 (¹⁵N) mM unlabeled SH3. (C) ¹⁵N-δ_ex observed at 800 MHz for 0.3 mM ¹⁵N/¹³Cα-labeled htt^ex1Q₇ in the presence of 0.3 mM unlabeled SH3. (D) Examples of the dependence of ¹⁵N- and ¹H_N-δ_ex measured at 600 MHz for 0.16 mM ¹⁵N-labeled SH3 on the concentration of added unlabeled htt^ex1Q₇. The experimental data are shown as circles in panels A, B, and D and as filled-in red circles in panel C; the global best-fit curves to the scheme shown in Figure 4 are displayed as continuous lines in panels A, B and D, and as filled-in blue circles in panel C. All data were recorded at 5 °C. The complete experimental data sets and best-fit curves are provided in Figures S3–S6.

Figure 4.

Scheme for the binding of Fyn SH3 to htt^ex1Q₇ and subsequent dimerization viewed from the perspective of (A) htt^ex1Q₇ and (B) Fyn SH3. The bold font is used to denote magnetization of the isotopically labeled species. Δω_i are the differences in chemical shift to the major NMR observable, either monomeric htt^ex1Q₇ (P) or free SH3 (L). ki and k_–i are second-order association and first-order dissociation rate constants, respectively, and k_–4 = (k₁k₄k_–3k_–2)/(k_–1k₂k₃). The prefactors in front of the rate constants relate to the differential equations (eq 1) describing the time-dependence of magnetizations. The species populations, shown in parentheses in panel A, relate to 0.1 mM htt^ex1Q₇ in the presence of 0.3 mM SH3.

Figure 5.

¹⁵N and ¹³Cα Δω profiles within the NT region of htt^ex1Q₇ attributable to binding of Fyn SH3 (Δω_B) to the first polyproline tract of the PRD and to dimerization of the NT (Δω_D) obtained from the global fit to the experimental NMR data. The Δω_B and Δω_D values are displayed as gray and black circles, respectively. Note that Gln19 exhibits no observable ¹³Cα exchange-induced shifts or ¹³Cα-CPMG relaxation dispersion.

Figure 6.

Simulation of the populations of free monomer htt^ex1Q₇ (P) and monomeric (PL) and dimeric (P₂L and P₂L₂) states of SH3-bound htt^ex1Q₇ as a function of total Fyn SH3 concentration. (A) [P]_total = 0.1 or 0.3 mM with [SH3]_total up to 0.6 mM. (B) [P]_total = 0.1 mM with [SH3]_total up to 3 mM. Note the population scale on the left panels ranges from 0 to 100%, while that on the right panels is from 0% to 6%. The equilibrium dissociation constants for the four-state binding scheme are given in Table 1.

Figure 7.

Probing putative tetramerization of htt^ex1Q₇ in the presence of Fyn SH3. (A) Scheme for the oligomerization of htt^ex1Q₇ upon binding of SH3 that considers the existence of a putative tetramer P₄L₄ formed by self-association of the P₂L₂ dimer and characterized by the equilibrium dissociation constant K_D5. The expression for the dependence of the population of P₄L₄ on the population of the bound monomer PL is provided. For simplicity the singly bound dimer, P₂L, shown in the scheme of Figure 4A has been omitted, as its equilibrium population is ~20 times smaller than that of P₂L₂. (B) Examples of the dependence of ¹⁵N-δ_ex on the concentration of ¹⁵N/¹³C-labeled htt^ex1Q₇ in the presence of 5 mM SH3. The experimental data, recorded at 800 MHz and 5 °C, are displayed as red circles, and simulations of ¹⁵N-δ_ex for a range of values of K_D5 are shown as dashed lines. The calculated curve for K_D5 ≥ 1 mM (red continuous line) is indistinguishable from that without tetramerization. Simulations were performed using the values of the rate constants provided in Table 1 and the values of Δω_B and Δω_D reported in Table S1. The ¹⁵N chemical shifts within the NT region of the P₂L₂ dimer and P₄L₄ tetramer were assumed to be the same (i.e., $\Delta {\omega }_{{\text{P}}_4{\text{L}}_4}=\Delta {\omega }_{{\text{P}}_2{\text{L}}_2}=\Delta {\omega }_{\text{B}}+\Delta {\omega }_{\text{D}}$). The populations indicated above the species in panel A correspond to those at the highest concentration of htt^ex1Q₇ (0.4 mM) employed in the experiments with a SH3 concentration of 5 mM. Under these conditions the population of free htt^ex1Q₇ (P) is only ~5%.