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

Significance Huntington’s disease is a fatal neurodegenerative condition arising from polyglutamine expansion within the Huntingtin protein leading to fibril accumulation in neurons. The initial multimerization events occur on the submillisecond timescale and involve sparsely populated species that can be probed at atomic resolution by NMR. Using a minimalistic construct comprising the N-terminal amphiphilic domain and seven glutamines, we uncover a branched oligomerization pathway, one leading to a tetramer comprising a dimer of coiled-coil helical dimers, and the other resulting in a nonproductive, partially helical, dimer. The results illuminate the contribution of the N-terminal amphiphilic domain in prenucleation events that precede fibril formation. The N-terminal region of the huntingtin protein, encoded by exon-1, comprises an amphiphilic domain (httNT), a polyglutamine (Qn) 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, httNTQ7, 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 (τex ∼ 30 μs, Kdiss ∼ 0.1 M) that goes on to form a tetramer (τex≲25 μs; Kdiss ∼ 22 μM), or exchanges with a “nonproductive” dimer that does not oligomerize further (τex ∼ 400 μs; Kdiss ∼ 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 D2 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 httNT domain in the earliest stages of prenucleation and oligomerization, before fibril formation.