Marius Clore

NIH, NIDDK, Laboratory of Chemical Physics

Monday 09.08.

8:00 PDT -  11:00 EDT - 15:00 UTC

16:00 BST - 17:00 CEST - 18:00 IDT

Probing transient pre-nucleation oligomerization of huntingtin at atomic resolution by NMR

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, httex1, comprises three distinct regions or domains: a 16-residue N-terminal amphiphilic sequence (httNT), a polyQ tract of variable length, and a proline rich domain (PRD) with two polyproline repeats of 11 (P11) and 10 (P10) residues. In this talk we will summarize out work on the earliest pre-nucleation transient oligomerization events involving httex1 using NMR experiments designed to probe rapidly exchanging systems (sub-millisecond time range) involving sparsely-populated excited states. We show that a branched assembly mechanism is involved comprising on- and off-pathways. In the on-pathway branch, the major monomeric species self-associates to form a productive helical coiled-coil dimer of the NT region that goes on to form a four-helix bundle tetramer comprising a dimer-of-dimers. In the off-pathway branch, a “non-productive” dimer ensemble with partially helical character is formed that does not undergo further oligomerization. The importance of pre-nucleation tetramerization is evidenced by the fact that inhibition of tetramer formation blocks fibrillization. Thus on-pathway tetramerization constitutes the prenucleation trigger or molecular switch that hugely increases the probability of occurrence of intermolecular polyQ contacts (by effectively increasing the local concentration of the polyQ tracts) and hence polyQ fibril formation.  It is therefore clear that blocking productive dimer and/or 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 httex1 through binding of the httNT sequence to chaperones; and (c) allosteric, long-range inhibition by interaction of intracellular proline-binding proteins with the proline rich domain (PRD).