KTKEGV repeat motifs are key mediators of normal α-synuclein tetramerization: Their mutation causes excess monomers and neurotoxicity

Abstract

α-Synuclein (αS) is a highly abundant neuronal protein that aggregates into β-sheet-rich inclusions in Parkinson's disease (PD). αS was long thought to occur as a natively unfolded monomer, but recent work suggests it also occurs normally in α-helix-rich tetramers and related multimers. To elucidate the fundamental relationship between αS multimers and monomers in living neurons, we performed systematic mutagenesis to abolish self-interactions and learn which structural determinants underlie native multimerization. Unexpectedly, tetramers/multimers still formed in cells expressing each of 14 sequential 10-residue deletions across the 140-residue polypeptide. We postulated compensatory effects among the six highly conserved and one to three additional αS repeat motifs (consensus: KTKEGV), consistent with αS and its homologs β-and γ-synuclein all forming tetramers while sharing only the repeats. Upon inserting in-register missense mutations into six or more αS repeats, certain mutations abolished tetramer formation, shown by intact-cell cross-linking and independently by fluorescentprotein complementation. For example, altered repeat motifs KLKEGV, KTKKGV, KTKEIV, or KTKEGW did not support tetramerization, indicating the importance of charged or small residues. When we expressed numerous different in-register repeat mutants in human neural cells, all multimer-abolishing but no multimer-neutral mutants caused frank neurotoxicity akin to the proapoptotic protein Bax. The multimer-abolishing variants became enriched in bufferinsoluble cell fractions and formed round cytoplasmic inclusions in primary cortical neurons. We conclude that the αS repeat motifs mediate physiological tetramerization, and perturbing them causes PD-like neurotoxicity. Moreover, the mutants we describe are valuable tools for studying normal and pathological properties of αS and screening for tetramer-stabilizing therapeutics.alpha-synuclein | multimer | tetramer | Parkinson's disease | neurotoxicity

New insights into cellular α-synuclein homeostasis in health and disease

“…In addition, lysine-to-glutamate mutations in those repeats alter aSyn binding to membrane phospholipids [45]. A similar mutant, seven-times KLKEGV, has recently been described by us to abolish native aSyn tetramers/multimers in neurons [46 ]. Other repeat motif mutants, such as KTKEIV and KTKKGV, abolished native multimerization as well, and this was invariably accompanied by an enrichment of the protein in PBSinsoluble fractions that required Triton-X 100 for solubilization, presumably representing more membrane association.…”

Computational Analysis of Cholesterol Binding and Pore-Lining Regions in Alpha-Synuclein: Role in Mitochondrial Function

“…Trexler and Rhoades [31] have demonstrated that N-terminal acetylation is critical for forming α-helical oligomers of α-syn. Further evidence for the tetrameric conformation has been obtained by Bartels et al [32], Pochapsky [33] and Dettmer et al [34]. As noted by Trexler and Rhoades [31], acetylation removes the N-terminal charge, making this region more hydrophobic.…”

“…All α-syn isoforms also contain multiple highly conserved phospholipid-binding motifs (KTKE(Q) G(Q)V), thought to mediate binding to phospholipid membranes (e.g. [34]), which in the tetramer would involve up to 16 phospholipid binding (KTKE(Q)G(Q)V) motifs. Cholesterol-containing pore-lining regions are known to form Ca 2+ ion channels [18,26] which together with the EF-hand in the C-terminal region of the molecule would regulate Ca 2+ movement across the mitochondrial membrane.…”

Caspase-1 clipping causes complications for α-synuclein

“…These occur in the N-terminal third of the protein and may induce structural changes that decrease the intramolecular interaction between the N and C termini (3) and/or decrease the intermolecular propensity to form α-helical tetramers and related multimers (4). Interfering with the latter process can lead to an excess of unfolded monomers prone to abnormal oligomerization (5). In rare families that inherit αSyn gene multiplication, a causal link between the lifelong increase in monomer levels, neurotoxicity, and PD is likely (6).…”