Huntingtin N-Terminal Monomeric and Multimeric Structures Destabilized by Covalent Modification of Heteroatomic Residues

Abstract

Early stage oligomer formation of the huntingtin protein may be driven by self-association of the 17-residue amphipathic α-helix at the protein's N-terminus (Nt17). Oligomeric structures have been implicated in neuronal toxicity and may represent important neurotoxic species in Huntington's disease. Therefore, a residue-specific structural characterization of Nt17 is crucial to understanding and potentially inhibiting oligomer formation. Native electrospray ion mobility spectrometry-mass spectrometry (IMS-MS) techniques and molecular dynamics simulations (MDS) have been applied to study coexisting monomer and multimer conformations of Nt17, independent of the remainder of huntingtin exon 1. MDS suggests gas-phase monomer ion structures comprise a helix-turn-coil configuration and a helix-extended-coil region. Elongated dimer species comprise partially helical monomers arranged in an antiparallel geometry. This stacked helical bundle may represent the earliest stages of Nt17-driven oligomer formation. Nt17 monomers and multimers have been further probed using diethylpyrocarbonate (DEPC). An N-terminal site (N-terminus of Threonine-3) and Lysine-6 are modified at higher DEPC concentrations, which led to the formation of an intermediate monomer structure. These modifications resulted in decreased extended monomer ion conformers, as well as a reduction in multimer formation. From the MDS experiments for the dimer ions, Lys6 residues in both monomer constituents interact with Ser16 and Glu12 residues on adjacent peptides; therefore, the decrease in multimer formation could result from disruption of these or similar interactions. This work provides a structurally selective model from which to study Nt17 self-association and provides critical insight toward Nt17 multimerization and, possibly, the early stages of huntingtin exon 1 aggregation.

Figure 1

False color IMS-MS distribution of Nt17 ions. Colored intensity is presented on a log scale.

Figure 2. Expanded regions of the

IMS-MS distribution (Figure 1) showing monomer (a.), dimer (b.), trimer (c.), and tetramer (d.) ions. Discrete features corresponding to separate conformations are labeled sequentially in monomer and dimer distributions.

Figure 3

Nt17 ribbon structures calculated from MDS. Pink regions are α-helical. Blue regions correspond to a 3–10 helix. a.) Extracted t_D distribution for [M+2H]²⁺ ions. b.) Compact conformer (1) at 11.0 ms. c.) Extended conformer (2) at 12.4 ms. See Table 1 for assigned collision cross-sections

Figure 4

Nt17 dimer structures calculated from MD simulation. Color schemes are the same as in Figure 3 a.) Extracted arrival time distribution. ‘3’, ‘4’, and ‘5’ correspond to relevant dimer structures, depicted in panels b., c., and d., respectively.

Figure 5

t_D distributions of covalently modified Nt17 [M+2H]²⁺ monomer at 5x (a.), 50x (b.), and 250x (c.) DEPC. t_D distributions are normalized to total peptide ion counts. Blue trace: unlabeled Nt17; green trace: singly modified Nt17; red trace: doubly modified. d. – f., mass spectra at each DEPC concentration showing monomer m/z range (light blue box) and dimer m/z range (light red box). The number of covalent modifications is shown next to each peak of interest in the mass spectra.

Figure 6

Mobility selected MS/MS analysis of the intermediate, doubly modified conformer. a.) Sequence coverage by ion. b.) b-ion series. c.) y-ion series. d.) Enhanced region showing the y₈ ion for clarity.