title: 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 fluorescent-protein 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 buffer-insoluble 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.
authors.affiliation: Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115.
authors.forename: Ulf
authors.initials: U
authors.lastname: Dettmer
authors.affiliation: Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Newman
authors.affiliation: Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115.
authors.forename: Victoria E
authors.initials: VE
authors.lastname: von Saucken
authors.affiliation: Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115.
authors.forename: Tim
authors.initials: T
authors.lastname: Bartels
authors.affiliation: Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115 dselkoe@partners.org.
authors.forename: Dennis
authors.initials: D
authors.lastname: Selkoe
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D016923:Cell Death / Q000187:drug effects; D017124:Conserved Sequence; D003432:Cross-Linking Reagents / Q000494:pharmacology; D006801:Humans; D002479:Inclusion Bodies / Q000187:drug effects / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D055503:Protein Multimerization; D017207:Rats, Sprague-Dawley; D020449:Repetitive Sequences, Amino Acid; D017384:Sequence Deletion; D013329:Structure-Activity Relationship; D051844:alpha-Synuclein / Q000737:chemistry* / Q000235:genetics / Q000633:toxicity*
chemical_list: D003432:Cross-Linking Reagents; D050505:Mutant Proteins; D051844:alpha-Synuclein
keywords: Parkinson's disease; alpha-synuclein; multimer; neurotoxicity; tetramer