Transient Interdomain Interactions Modulate the Monomeric Structural Ensemble and Self-Assembly of Huntingtin Exon 1

Abstract

Polyglutamine (polyQ) tract length expansion (≥ 36 residues) within the N-terminal exon-1 of Huntingtin (Httex1) leads to Huntington's disease, a neurodegenerative condition marked by the presence of intranuclear Htt inclusions. Notably, the polyQ tract in Httex1 is flanked by an N-terminal coiled-coil domain -N17 (17 amino acids), which promotes the formation of soluble oligomers and brings the aggregation-prone polyQ tracts in close proximity. However, the molecular mechanisms underlying the conversion of soluble oligomers into insoluble β-rich aggregates with increasing polyQ length, remain unclear. In this study, extensive atomistic molecular dynamics (MD) simulations (aggregate time ≈0.7 milliseconds) are performed to uncover the interplay between structural transformation and domain "cross-talk" on the conformational ensemble and oligomerization of Httex1 due to polyQ expansion. Notably, MD-derived ensembles of N17-Q_n-P₅ monomers validated against NMR indicated that in addition to elevated α-helicity, polyQ expansion also favored transient, interdomain (N17/polyQ) interactions which resulted in the emergence of β-sheet conformations. Further, interdomain interactions modulated the stability of N17-mediated polyQ dimers and promoted a heterogeneous dimerization landscape. Finally, it is observed that the intact C-terminal proline-rich domain (PRD) promoted condensation of Httex1 through self-interactions involving its P₁₀/P₁₁ tracts while also interacting with N17 to suppress its α-helicity.

Keywords: huntingtin; molecular dynamics simulation; polyglutamine.

Figure 1

Validation of N17‐Q₁₆‐P₅ ensemble against solution NMR data and assessing the effect of N17 (¹⁴LKSF^{[ 17 ]}) mutants on polyQ helicity. A) Schematic showing the organization of Httex1 into three regions – N17, polyQ, and PRD (top), and the corresponding sequences of N17/PRD domains (bottom). The length of polyQ region in the wild‐type Huntingtin protein is ≈23 residues. B) Comparison of per‐residue α‐helix fractions for N17‐Q₁₆‐P₅ wild‐type and its deletion constructs with respect to NMR. Helical fractions were computed using the DSSP algorithm and compared to SSP scores for the helical propensity (grey) computed from experimental ¹³C chemical shifts. Error bars denote std. An error of the mean was calculated over three independent trajectories. C) Same as in panel B for wild‐type and ¹⁴LKSF^{[ 17 ]} mutants.

Figure 2

Structural cooperativity between N17 and polyQ domains. A) Secondary structure variation as a function of time in a representative N17‐Q₁₆‐P₅ (top) and Q₁₆ (bottom) trajectory. B) SS‐map of N17‐Q₁₆‐P₅ wild‐type computed from an aggregate trajectory (≈63 µs) indicating the probability of various helical lengths across N17 and polyQ regions. Representative α‐helical conformations corresponding to numbered regions of the SS‐map are shown in cartoon representation. C) Same as in E for ¹⁴LLLF^{[ 17 ]} mutant. D) Helix initiation times for each residue along the peptide sequence. Initiation time represents the earliest simulation frame (in ns) at which each residue adopts a helical conformation. Error bars indicate standard errors of the mean computed across three independent simulation trajectories. Residues in the central region (approximately residues 11–17) exhibit notably lower initiation times, indicating earlier helix formation compared to residues in the polyQ tract region (higher residue numbers). E) Histogram depicting the Bayesian‐inspired Monte Carlo sampling of initiation time differences between the polyQ region and the central region (Δt = t_avg(polyQ) – t_avg(central)). The red dashed line at zero indicates no difference between regions. Positive values demonstrate that the central region consistently forms helices earlier than the polyQ region.

Figure 3

Effect of polyQ length expansion on N17‐Q_n‐P₅ structural ensemble. A) Comparison of per‐residue α‐helix fractions for N17‐polyQ_n‐P₅ constructs with increasing polyQ length (Q_16‐46) against NMR‐derived helical propensities (SSP scores). Error bars denote std. The error of the mean was calculated over three independent trajectories for N17‐Q_16‐32 constructs and over four intervals (150 ns each) of the PT‐WTE replica trajectory (293 K) for N17‐Q₄₆‐P₅. B) SS‐map of N17‐Q₄₆‐P₅ wild‐type (left) from the PT‐WTE aggregate trajectory (600 ns) indicating the probability of α‐helices formed of varying lengths across N17 and polyQ regions. Representative helical conformations corresponding to numbered regions of the SS‐map are shown in cartoon representation. C) Secondary structure variation as a function of time for two representative N17‐Q₄₆‐P₅ trajectories showing the formation of two‐stranded β‐sheet structures (black boxes). D) 2D intramolecular contact maps calculated over six N17‐Q₄₆‐P₅ independent trajectories indicating the low population of β‐conformations (marked as *) relative to α‐helices. E) Representative β‐sheet structures (5 in total) from the trajectory periods highlighted in C (black boxes) involving either direct (i) or indirect (ii) contacts between the N17 and Q₄₆ domains. The coloring scheme for the structures is as follows: N17 – purple, Q₄₆ – white P₅ – Gold, β‐strand – red. F) Pairswise 2D contact maps calculated for the two β‐sheet structural ensembles shown in (E).

Figure 4

Competition between structural transformation and interdomain interactions shape the dimerization landscape of N17‐polyQ constructs. A) NMR structure of N17‐Q₇ (H7) tetramer showing interactions (sidechains shown as sticks) in the hydrophobic core. The anti‐parallel dimer was extracted from the structure for MD simulations. B) Comparison of per‐residue α‐helix fractions for N17‐Q₇/N17‐Q₁₆‐P₅ chains from dimer simulations against monomer simulations and NMR. Error bars denote std. The error of mean was calculated over six independent trajectories. The comparison indicates significantly elevated helicity of the N17 region in N17‐Q_7/16 dimers compared to their respective monomers. C) 2D PMF (potential of mean force) plots as a function of two order parameters – (i) total number of intermolecular N17 contacts and (ii) the inter‐helical angle (°) between N17 domains, which characterize the association free energy landscape for four N17 dimer variants. The plots show the combined data derived from 6 independent dimer trajectories (≈2.3 µs) for each variant. The position of the NMR – N17 dimer (initial structure) is marked on the PMF plots as (*). Representative dimer MD structures from the basins marked (1, 2) in PMF plots are shown on the left with interfacial hydrophobic residues shown as grey sticks.

Figure 5

PRD‐mediated interactions stabilize the N17‐Q₁₆‐PRD condensate and suppress N17 ɑ‐helicity. A) All‐atom MD simulation snapshots of N17‐Q₁₆‐P₅ (left) and N17‐Q₁₆‐PRD (right) condensate systems over 2 µs of their respective trajectories. Protein chains are shown as ribbons and ions (Na⁺/Cl⁻) are shown as spheres. The snapshots indicate destabilization of the N17‐Q₁₆‐P₅ dense phase (0.2–1.0 µs) and its complete dissolution (2.0 µs), resulting in a homogeneous system. B) Pairwise 2D‐intermolecular contact map averaged over all chain pairs reveals significant contributions of the PRD toward stabilization of the N17‐Q₁₆‐PRD. C) Mean fractional ɑ‐helicity per‐residue (computed over all dense phase chains) shows reduced ɑ‐helical formation within N17 for the N17‐Q₁₆‐PRD condensate compared to a homogenous N17‐Q₁₆‐P₅ condensate system (i.e., lacking protein‐solvent interface). Error bars denote std. The error of mean overall chains.

Figure 6

polyQ expansion promotes “domain crosstalk” which modulates the structural ensemble and oligomerization landscape of Httex1 to favor fibrillar aggregation. Schematic illustrating the effect of polyQ length expansion on the conformation and oligomerization of Httex1. polyQ expansion promotes inter‐domain N17/polyQ interactions. These interactions can (i) induce the emergence of transient β‐sheet conformers in monomers which may fold to form the critical nucleus and initiate protofibril formation (oligomer‐independent pathway), and (ii) counteract stabilization of N17‐mediated oligomers to favor polyQ/polyQ interactions which lead to nucleation and fibrillation. The nucleus size is shown as a monomer (n = 1) and is representative of Httex1‐Q_>23.