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

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 (tau(ex) <= 50 to 70 mu s), while leaving the population of the off-pathway (nonproductive) dimeric species largely unaffected (tau(ex) similar to 750 mu s). Next, we show that the affinity of a single molecule of profilin to the polyproline tracts is in the micromolar range (K-diss similar to 17 and similar to 31 mu M), but binding of a second molecule of profilin is negatively cooperative, with the affinity reduced similar to 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 (tau(ex) similar to 600 mu s and K-diss similar to 50 mu M). 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).