Abrogation of prenucleation, transient oligomerization of the Huntingtin exon 1 protein by human profilin I.

Abstract

Human profilin I reduces aggregation and concomitant toxicity of the polyglutamine-containing N-terminal region of the huntingtin protein encoded by exon 1 (htt^ex1) and responsible for Huntington's disease. Here, we investigate the interaction of profilin with htt^ex1 using NMR techniques designed to quantitatively analyze the kinetics and equilibria of chemical exchange at atomic resolution, including relaxation dispersion, exchange-induced shifts, and lifetime line broadening. We first show that the presence of two polyproline tracts in htt^ex1, absent from a shorter huntingtin variant studied previously, modulates the kinetics of the transient branched oligomerization pathway that precedes nucleation, resulting in an increase in the populations of the on-pathway helical coiled-coil dimeric and tetrameric species (τ_ex ≤ 50 to 70 μs), while leaving the population of the off-pathway (nonproductive) dimeric species largely unaffected (τ_ex ∼750 μs). Next, we show that the affinity of a single molecule of profilin to the polyproline tracts is in the micromolar range (<i>K</i>_diss ∼ 17 and ∼ 31 μM), but binding of a second molecule of profilin is negatively cooperative, with the affinity reduced ∼11-fold. The lifetime of a 1:1 complex of htt^ex1 with profilin, determined using a shorter huntingtin variant containing only a single polyproline tract, is shown to be on the submillisecond timescale (<i>τ</i>_ex ∼ 600 μs and <i>K</i>_diss ∼ 50 μM). Finally, we demonstrate that, in stable profilin-htt^ex1 complexes, the productive oligomerization pathway, leading to the formation of helical coiled-coil htt^ex1 tetramers, is completely abolished, and only the pathway resulting in "nonproductive" dimers remains active, thereby providing a mechanistic basis for how profilin reduces aggregation and toxicity of htt^ex1.