Emerging β-Sheet Rich Conformations in Supercompact Huntingtin Exon-1 Mutant Structures

Abstract

There exists strong correlation between the extended polyglutamines (polyQ) within exon-1 of Huntingtin protein (Htt) and age onset of Huntington's disease (HD); however, the underlying molecular mechanism is still poorly understood. Here we apply extensive molecular dynamics simulations to study the folding of Htt-exon-1 across five different polyQ-lengths. We find an increase in secondary structure motifs at longer Q-lengths, including β-sheet content that seems to contribute to the formation of increasingly compact structures. More strikingly, these longer Q-lengths adopt supercompact structures as evidenced by a surprisingly small power-law scaling exponent (0.22) between the radius-of-gyration and Q-length that is substantially below expected values for compact globule structures (∼0.33) and unstructured proteins (∼0.50). Hydrogen bond analyses further revealed that the supercompact behavior of polyQ is mainly due to the "glue-like" behavior of glutamine's side chains with significantly more side chain-side chain H-bonds than regular proteins in the Protein Data Bank (PDB). The orientation of the glutamine side chains also tend to be "buried" inside, explaining why polyQ domains are insoluble on their own.

Figure 1

Secondary structural analysis shows that the β-sheet content of Huntingtin (Htt) increases with Q-length faster than the α-helical content. (A) Absolute secondary structure content of the full Htt exon-1 for Q22, Q36, Q40, Q46, and Q56 (B) The average secondary structure scores of first 17 residues near N-terminus (N17) and polyglutamine (polyQ) regions of Htt for all Q-lengths. A sharp increase in β-sheet content emerges between Q22 and the pathogenic threshold Q-length, Q36. The estimated errors are less than 3%. (C) Absolute secondary structure content of the proline-rich segment near C-terminus (C38) for all Q-lengths. Because the content of the polyproline II (PPII) helices is so high, the inset shows other secondary structural motifs on a reduced scale.

Figure 2

Representative structures determined from clustering analysis of Q22, Q36, and Q46 datasets. The populations of each cluster, as a percentage from sampled conformations, is shown above each structure. In general, as the Q-length increases, the β-sheet content of the polyQ region increases. The N17 domain can also form more β-sheet structures as the Q-length increases. The N17 region is shown in tan, the polyQ in red, and the C38 in cyan. The proteins are rendered using the New Cartoon representation.

Figure 3

Hydrogen bond analysis shows that the contacts between N17 and C38 are reduced as the Q-length is increased. Panel (A) Pairwise residue hydrogen bonding probability map for all Q-lengths. Panel (B) (Left) Average number of hydrogen bonds in the polyQ region for all Q-lengths (orange circles). For comparison, the average numbers of hydrogen bonds in globular proteins selected from PDB structures are also plotted (brown crosses). The dashed line is a power-law fit with the scaling exponent of 1.28. As seen in the figure, the number of hydrogen bonds within the polyQ region increases at a faster rate than in other globular proteins. (Middle) Average number of sidechain-sidechain (blue square), backbone-sidechain (green triangle) and backbone-backbone (red circle) hydrogen bonds for polyQ and PDB proteins. The sidechains of polyQ show more hydrogen-bonding propensity than the PDB proteins with comparable sizes. (Right) Sidechain orientation with respect to protein surface. θ is an angle between a vector from center of mass of a protein to C_α atom and another vector from C_α to C_δ. Sidechain of GLN in polyQ tends to point inward as compared to general PDB proteins, suggesting that the GLN residues will not be available to form favorable interactions with the solvent.

Figure 4

Two-dimensional PMFs projected onto R_ee and R_g for the full exon-1 across all Q-lengths. The PMFs are plotted as differences from a global PMF maximum. Q22 has much wider distributions of R_g and R_ee values than the other Q-lengths, indicating a more flexible structure. The reduction in the sampled R_g and R_ee regions indicates that increases in Q-length lead to more compact structures. For Q22, the overlaid representative structures from the highlighted regions show that the maximum R_g values can be sampled from both the minimum and maximum R_ee values, indicating that the extended C38 region is very flexible. On the other hand, the representative structure highlighted in Q56 shows that the protein is relatively compact with the end-end distance less variant even for maximum R_g structures.

Figure 5

Increasing Q-length leads to super-compact conformations in the polyQ region. (A) Radii of gyration for the polyQ region (orange ⊙) and structured proteins from the PDB (brown ×), as a function of chain length. The dashed lines indicate power-law fitting. The scaling exponents are displayed in the figure, showing that the polyQ will adopt super-compact structures at longer Q-lengths. (B) Relative anisotropy, κ, for all Q-lengths. κ roughly decreases as Q-length increases except for Q40, which indicates that the chain becomes more spherical. (C) Ensemble of polyQ structures. 40 randomly selected polyQ structures are rendered together to illustrate the morphological change in polyQ with increasing Q-length. Q22 has a more aspherical shape than the other mtHtts, and the shape of the protein becomes more spherical as Q-length increases.