Probing initial transient oligomerization events facilitating Huntingtin fibril nucleation at atomic resolution by relaxation-based NMR

Abstract

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 (≲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 (τex ∼ 30 μs, Kdiss ∼ 0.1 M) that goes on to form a tetramer (τex≲25 μs; Kdiss ∼ 22 μM), or exchanges with a “nonproductive” dimer that does not oligomerize further (τex ∼ 400 μs; Kdiss ∼ 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 D2 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.

Site‐Specific Phosphorylation of Huntingtin Exon 1 Recombinant Proteins Enabled by the Discovery of Novel Kinases

“…These observations suggest that S13 phosphorylation primes phosphorylation at S16, similar to what has been shown for other proteins. [18,44] Additionally, these findings were in accordance with our previous cellular and in vivo observation where we showed that TBK1 phosphorylated mainly S13. [28] Figure 6.…”

Unraveling the structure and dynamics of the human DNAJB6b chaperone by NMR reveals insights into Hsp40-mediated proteostasis

“…4 A and B, cyan circles), the CTD domain was represented by an ensemble of varying size (N), ranging from 1 to 8 (46,47). Since the population of the closed state giving rise to the interdomain PREs is unknown a priori, we made use of a PRE target function that maximizes the correlation coefficient between observed and calculated PREs (48). The PRE data were supplemented by highly ambiguous distance restraints (49) from all atoms of residues 34 and 36 in the JD domain, identified from 15 N CPMG relaxation dispersion experiments (see following section) to be involved in interdomain contacts, to the atoms of all residues in the CTD domain.…”

Side chain to main chain hydrogen bonds stabilize a polyglutamine helix in a transcription factor

“…A number of in vitro experiments have established that the formation of fibrillar aggregates by proteins bearing polyQ tracts can proceed via oligomers 55 , potentially liquid-like 56 , stabilized by intermolecular interactions between flanking regions of polyQ tracts and equivalent to those stabilizing coiled coils 2,57,58 . In proteins bearing polyQ tracts such as huntingtin 59,60 and AR 20,21 extending the length of the tract increases its helicity and, as we have now shown, that of the sequence immediately flanking them at the N terminus.…”