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

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(ex1)Q(35) inhibits fibril formation by both NMR and a thioflavin T fluorescence assay. The interaction of Fyn SH3 with htt(ex1)Q(7) 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(ex1)Q(7), 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 P-2 dimer. The P2L2 dimer, in contrast to P-2, does not undergo any detectable oligomerization to a tetramer, thereby explaining the allosteric inhibition of htt(ex1) fibril formation by Fyn SH3.

Automated summary

Huntingtin polypeptides (htt(ex1)), containing an expanded polyglutamine tract encoded by exon 1 of the htt gene, form fibrils that accumulate in neuronal inclusion bodies, leading to the fatal neurodegenerative condition Huntington's disease. The binding of Fyn SH3 to the first polyproline tract of htt(ex1)Q(35) inhibits fibril formation, showcasing potential therapeutic implications in Huntington's disease.