The effects of KTKEGV repeat motif and intervening ATVA sequence on α-synuclein solubility and assembly

Alpha-synuclein (αS), the key protein in Parkinson's disease, is typically described as an intrinsically disordered protein. Consistent with this notion, several context-dependent folding states may coexist in neurons. Unfolded soluble monomers, helical monomers at membranes and helical multimers (soluble or at membranes) have all been reported and may be in an equilibrium with each other. We previously found that αS can be stabilized in its membrane-associated monomeric form by genetically increasing the hydrophobicity of the membrane-embedded half of the αS helix. αS amphipathic helix formation at membranes is governed by up to nine 11-amino acid repeats with the core motif KTKEGV. However, this repeat is only imperfectly conserved; for example, it consists of KAKEGV in repeat #1, KTKEQV in repeat #5, and AVVTGV in the poorly conserved repeat #6. Here we explored the effect of perfecting the αS core repeat to nine times KTKEGV ("9KV") and found by sequential protein extraction that this engineered mutant accumulates in the cytosolic phase of neural cells. Intact-cell cross-linking trapped a part of the cytosolic portion at multimeric positions (30, 60, 80, 100 kDa). Thus, compared to wild-type αS, αS 9KV seems less prone to populating the membrane-associated monomeric form. Removing the "ATVA" intervening amino-acid sequence between repeats 4 and 5 slightly increased cytosolic localization while adding "ATVA" in between all repeats 1-8 caused αS to be trapped as a monomer in membrane fractions. Our results contribute to an ongoing debate on the dynamic structure of αS, highlighting that wild-type αS is unlikely to be fully multimeric/monomeric or fully cytosolic/membrane-associated in cells, but protein engineering can create αS variants that preferentially adopt a certain state. Overall, the imperfect nature of the KTKEGV repeat motifs and the presence of ATVA in between repeats 4 and 5 seem to prevent a strong cytosolic localization of αS and thus play a major role in the protein's ability to dynamically populate cytosolic vs. membrane-associated and monomeric vs. multimeric states.