Disordered Nanostructure in Huntingtin Interacting Protein K Acts as a Stabilizing Switch To Prevent Protein Aggregation

Abstract

Protein misfolding due to mutation(s) and/or generation of unstable intermediate state(s) can be the cause of aberrant aggregations, leading to cellular degeneration. While molecular signatures like amyloidogenic regions cause aggregation, other features in proteins, like disorder and unique complexity regions, regulate and restrict such adhesive accumulation processes. Huntingtin interacting protein K (HYPK) is an aggregation-prone protein. Using various biophysical, microscopy, and computational techniques, we have deciphered how HYPK's N-terminal nanodisordered region plays a significant modulatory role in preventing its own aggregation and that of other proteins. HYPK's C-terminal hydrophobic regions lead to annular oligomerization and intermolecular charge interactions among the residues of low-complexity region (LCR) generate amorphous aggregates. The N-terminal disordered nanostructure loops toward the C-terminus, and a negative charge-rich patch in this region interacts with the LCR to shield LCR's positive charges. This interaction is required to prevent HYPK aggregation. Loss of this interaction causes partial unfolding of the structured C-terminus, resulting in HYPK's molten globule-like state and rapid annular oligomerization. The N-terminus also determines the specificity to mediate the differential bindings with aggregation-prone and wild type Huntingtin-exon1 proteins (Huntingtin97Q-exon1 and Huntingtin25Q-exon1). A sliding interaction of the specific N-terminal segment of HYPK along the extended polyglutamine region of Huntingtin-exon1 is responsible for HYPK's higher affinity for aggregation-prone Huntingtin than for its non-aggregating counterpart. Overall, our study provides evidence of the existence of disordered nanostructure in HYPK protein that mechanistically plays a decisive role in preventing both self and non-self protein aggregation.

HYPK scaffolds the Nedd8 and LC3 proteins to initiate formation of autophagosome around polyneddylated huntingtin exon1 aggregates

“…We understand that the partially reduced affinity of HYPK for Nedd8 was due to the interference of HYPK’s N-terminus in the interaction. We had previously shown that the N-terminus of HYPK was a disordered nanostructure ( 21 ). It could bend towards the C-terminus and interact with the C-terminal low complexity region (LCR) of HYPK ( 21 ).…”

“…We had previously shown that the N-terminus of HYPK was a disordered nanostructure ( 21 ). It could bend towards the C-terminus and interact with the C-terminal low complexity region (LCR) of HYPK ( 21 ). Such an interaction could lower the affinity of UBA of full-length HYPK for Nedd8.…”

“…The annular oligomers of HYPK, termed as H-granule, are involved in sequestering the protein aggregates ( 20 ). The balance of structural convolution of HYPK oligomers arises due to complex intra- and inter-molecular interactions of the hydrophobic regions, low complexity region and disordered nanostructure of HYPK ( 21 ). HYPK also shows chaperone-like activity ( 22 ) at ribosome where it interacts with the N-acetyl transferase and eIF1A1 proteins ( 23 ).…”

T54R mutation destabilizes the dimer of superoxide dismutase 1T54R by inducing steric clashes at the dimer interface

“…The simulations were performed in the same process that was done by us in our earlier studies. 21,35,36 There were three sequential steps in the MDS process -(a) protein structure preparation: in this step, optimization of protein structure was done to add missing hydrogen atoms, generate missing interatomic bonds and assign correct bond orders. Protein structure preparation was done in the protein preparation wizard of Maestro 9.2 (Schrödinger Incorporation) by using the following parameters -OPLS_2005 force eld, 0.3Å convergence heavy atom root mean square deviation.…”

“…The method of CD spectroscopy was similar to the process that was described in one of our earlier studies. 36 Cell culture IMR-32 cells were obtained from National Centre for Cell Science (India). Cells were cultured in Dulbecco's Modied Eagle Medium that was supplemented with 10% fetal bovine serum, 2 mM L-glutamine and penicillin/streptomycin solution.…”