Deep mutational scanning reveals the structural basis for α-synuclein activity

Abstract

Defining the biologically active structures of proteins in their cellular environments remains challenging for proteins with multiple conformations and functions, where only a minor conformer might be associated with a given function. Here, we use deep mutational scanning to probe the structure and dynamics of α-synuclein, a protein known to adopt disordered, helical, and amyloid conformations. We examined the effects of 2,600 single-residue substitutions on the ability of intracellularly expressed α-synuclein to slow the growth of yeast. Computational analysis of the data showed that the conformation responsible for this phenotype is a long, uninterrupted, amphiphilic helix with increasing dynamics toward the C terminus. Deep mutational scanning can therefore determine biologically active conformations in cellular environments, even for a highly dynamic multi-conformational protein.

Robust Sequence Determinants of α-Synuclein Toxicity in Yeast Implicate Membrane Binding

“…To identify the conformational state(s) that drive toxicity in yeast, we compared the relative toxicity of 2,600 single missense mutants. 6 The results of that study inspired our work herein, so we will briefly summarize our previous findings, which led us to propose a membrane-bound helix as the toxic species in yeast (Figure 1). Proline substitutions in the first ~90 residues disrupted toxicity, as did incorporation of polar residues within the membrane-binding face.…”

“…In order to examine the relative toxicity of α-synuclein variants, we designed a Barcode Association (performed previously) 6 To link α-synuclein variants to specific barcode sequences, plasmids isolated from DH5α cells were amplified by PCR to isolate the α-synuclein-GFP-barcode DNA sequence using primers that append Illumina adapter sequences. This product was gel purified and sequenced on an Clusters with a greater than 5% chance of at least 1 error in the barcode sequence, judged by quality scores, were rejected from further analysis.…”

“…We recently used DMS to identify biologically active conformation(s) of α-synuclein, 6 which extensive genetic and pathological evidence implicates as a driver of Parkinson's disease (PD).…”

“…Previously mapped sequence-toxicity landscape of α-synuclein identifies a toxic molecular conformation. 6 (A) Fitness scores (defined as the slope of the line describing change in log-transformed variant frequencies over time, represented by a red-white-blue color scale) for expression of α-synuclein missense mutants in yeast. 6 (B) Average fitness scores of mutants with hydrophobic (W, Y, F, L, I, V, M, C, A; red), polar (S, T, N, Q, H, R, K, D, E; blue), or proline (green) residues.…”

“…6 (A) Fitness scores (defined as the slope of the line describing change in log-transformed variant frequencies over time, represented by a red-white-blue color scale) for expression of α-synuclein missense mutants in yeast. 6 (B) Average fitness scores of mutants with hydrophobic (W, Y, F, L, I, V, M, C, A; red), polar (S, T, N, Q, H, R, K, D, E; blue), or proline (green) residues. 6 (C) The structural model derived from the sequence-toxicity landscape in panel A: an extended, membrane-bound 11/3-helix with increasing dynamics toward the C terminus.…”