Effects of the Flanking polyQ Regions and Membrane Physical Properties on Huntingtin Binding to Lipid Vesicles

Abstract

The pathological expansion of the polyglutamine (polyQ) repeat within the first exon of huntingtin (HTTex1) protein is a hallmark of Huntington’s disease (HD). Multiple evidences support that the membrane interaction of huntingtin is critical for its misfolding and aggregation in HD. Here, we focus on obtaining a detailed understanding of the initial steps of HTTex1-lipid interaction. This characterization has been exceptionally challenging using traditional ensemble methods due to its heterogeneous nature and the high propensity of HTTex1 to aggregate. To overcome these limitations, we used single-molecule approaches based mainly on fluorescence correlation spectroscopy (FCS) to monitor the binding of HTTex1 to synthetic lipid vesicles. The dependence on membrane curvature and lipid composition was studied, considering as models anionic lipids, raft-mimicking mixtures, and total brain lipid extract. The FCS results show a preferential binding of HTTex1 towards POPS (anionic lipid)-containing vesicles of small curvature. Moreover, time-resolved anisotropy measurements reveal distinct conformational dynamics of the adjacent polyQ regions in the HTTex1 membrane-bound state. Notably, the proline-rich domain remains highly flexible and solvent exposed even upon membrane binding. instead, the N17 segment converts into a less dynamic state. Our findings provide unique insight into how membrane curvature/composition and flanking polyQ sequences modulate the early stages of HTTex1 aggregation at the membrane surface. Work supported by FCT-Portugal (PTDC/BIA-BFS/30959/2017 grant and CEECIND/00884/2017 contract to AMM, and UIDB/04565/2020 funding to iBB-IST).