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.