期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 116, 期 9, 页码 3562-3571出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1821216116
关键词
prenucleation; kinetics; relaxation-based NMR; EPR; structure
资金
- Postdoctoral Research Associate Training Program Fellowship of the National Institute of General Medical Sciences [Fi2GM117609-01]
- Intramural Program of the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health
- NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES [ZIADK033007, ZIADK029023] Funding Source: NIH RePORTER
The N-terminal region of the huntingtin protein, encoded by exon-1, comprises an amphiphilic domain (httNT), a polyglutamine (Qn) tract, and a proline-rich sequence. Polyglutamine expansion results in an aggregation-prone protein responsible for Huntington's disease. Here, we study the earliest events involved in oligomerization of a minimalistic construct, httNTQ7, which remains largely monomeric over a sufficiently long period of time to permit detailed quantitative NMR analysis of the kinetics and structure of sparsely populated (less than or similar to 2%) oligomeric states, yet still eventually forms fibrils. Global fitting of concentration-dependent relaxation dispersion, transverse relaxation in the rotating frame, and exchange-induced chemical shift data reveals a bifurcated assembly mechanism in which the NMR observable monomeric species either self-associates to form a productive dimer (tau(ex) similar to 30 mu s, K-diss similar to 0.1 M) that goes on to form a tetramer (tau(ex) less than or similar to 25 mu s; K(dis)s similar to 22 mu M), or exchanges with a nonproductive dimer that does not oligomerize further (tau(ex) similar to 400 mu s; K-diss similar to 0.3 M). The excited state backbone chemical shifts are indicative of a contiguous helix (residues 3-17) in the productive dimer/tetramer, with only partial helical character in the nonproductive dimer. A structural model of the productive dimer/tetramer was obtained by simulated annealing driven by intermolecular paramagnetic relaxation enhancement data. The tetramer comprises a D-2 symmetric dimer of dimers with largely hydrophobic packing between the helical subunits. The structural model, validated by EPR distance measurements, illuminates the role of the httNT domain in the earliest stages of prenucleation and oligomerization, before fibril formation.
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