Probing initial transient oligomerization events facilitating Huntingtin fibril nucleation at atomic resolution by relaxation-based NMR

The N-terminal region of the huntingtin protein, encoded by exon-1, comprises an amphiphilic domain (htt ^NT ), a polyglutamine (Q _n ) tract, and a proline-rich sequence. Polyglutamine expansion results in an aggregation-prone protein responsible for Huntington’s disease. Here, we study the earliest events involved in oligomerization of a minimalistic construct, htt ^NT Q ₇ , which remains largely monomeric over a sufficiently long period of time to permit detailed quantitative NMR analysis of the kinetics and structure of sparsely populated (≲2%) oligomeric states, yet still eventually forms fibrils. Global fitting of concentration-dependent relaxation dispersion, transverse relaxation in the rotating frame, and exchange-induced chemical shift data reveals a bifurcated assembly mechanism in which the NMR observable monomeric species either self-associates to form a productive dimer (τ _ex ∼ 30 μs, K _diss ∼ 0.1 M) that goes on to form a tetramer (τ _ex ≲ 25 μs; K _diss ∼ 22 μM), or exchanges with a “nonproductive” dimer that does not oligomerize further (τ _ex ∼ 400 μs; K _diss ∼ 0.3 M). The excited state backbone chemical shifts are indicative of a contiguous helix (residues 3–17) in the productive dimer/tetramer, with only partial helical character in the nonproductive dimer. A structural model of the productive dimer/tetramer was obtained by simulated annealing driven by intermolecular paramagnetic relaxation enhancement data. The tetramer comprises a D ₂ symmetric dimer of dimers with largely hydrophobic packing between the helical subunits. The structural model, validated by EPR distance measurements, illuminates the role of the htt ^NT domain in the earliest stages of prenucleation and oligomerization, before fibril formation.