Probing transient pre-nucleation oligomerization of huntingtin at atomic resolution by NMR G. Marius Clore NIDDK, National Institutes of Health, USA Huntington’s disease is a fatal, autosomal, neurodegenerative condition that arises from CAG expansion within exon-1 of the huntingtin gene that encodes a polyglutamine (polyQ) repeat. Although the huntingtin protein is very large (~350 kDa), proteolysis and/or incomplete mRNA splicing generates mutated N-terminal fragments encoded by exon-1 that aggregate to form neuronal inclusion bodies in pathological states. The N-terminal region of huntingtin encoded by exon 1, htt ex1 , comprises three distinct regions or domains: a 16-residue N-terminal amphiphilic sequence (htt NT ), a polyQ tract of variable length, and a proline rich domain (PRD) with two polyproline repeats of 11 (P 11 ) and 10 (P 10 ) residues. In this talk we will summarize out work on the earliest pre-nucleation transient oligomerization events involving htt ex1 using NMR experiments designed to probe rapidly exchanging systems (sub-millisecond time range) involving sparsely-populated excited states, and how reversible submillisecond tetramerization triggers much slower (minutes to hours time scale) nucleation and fibril formation. The importance of pre-nucleation tetramerization is evidenced by the fact that inhibition of tetramer formation blocks fibrillization. Tetramerization constitutes the molecular switch that increases the probability of occurrence of intermolecular polyQ contacts (by effectively increasing the local concentration of the polyQ tracts) and hence polyQ fibril formation. Blocking tetramer formation may provide a fruitful avenue for preventing or delaying the onset of Huntington’s disease. Inhibition of pre-nucleation oligomerization can be achieved in a number of ways: (a) perturbing the productive dimer and/or tetramer interface; (b) sequestration of htt ex1 through binding of the htt NT sequence to chaperones; and (c) allosteric, long-range inhibition by interaction of intracellular proline-binding proteins with the proline rich domain (PRD). References 1. Kotler, S.A., Tugarinov, V., Schmidt, T., Ceccon, A., Libich, D.S., Ghirlando, R., Schwieters, C.D. and Clore, G.M. (2019) Probing initial transient oligomerization events facilitating Huntingtin fibril nucleation at atomic resolution by relaxation-based NMR. Proc. Natl. Acad. Sci. U. S. A. 116 , 3562-3571. 2. Ceccon, A., Tugarinov, V., Ghirlando, R. & Clore, G.M. (2020) Abrogation of pre-nucleation, transient oligomerization of the Huntingtin exon-1 protein by human profilin-I. Proc. Natl. Acad. Sci. U.S.A. 117, 5844-5852. 3. Wälti, M.A., Kotler, S.A. & Clore, G.M. (2021) Probing the interaction of huntingtin exon-1 polypeptides with the chaperonin nanomachine GroEL. ChemBioChem 22, 1985-1991. 4. Ceccon, A., Tugarinov, V. & Clore, G.M. (2021) Quantitative exchange NMR-based analysis of huntingtin-SH3 interactions suggests an allosteric mechanism of inhibition of huntingtin aggregation . J. Am. Chem. Soc. 143, 9672-9681. 5. Ceccon, A., Tugarinov, V., Toricella, F. & Clore, G.M. (2022) Quantitative NMR analysis of the kinetics of pre-nucleation oligomerization and aggregation of pathogenic huntingtin exon-1 protein. Proc. Natl. Acad. Sci. U.S.A. 119 , e2207690119.
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