UCSF

Robert Newberry’s α-Synuclein deep mutational scan paper out in Nature Chem Bio

α-Synuclein in yeast.

Deep mutational scan of α-Synuclein in yeast reveals the structural conformation contributing to its toxicity

DGL post-doc Robert Newberry published his paper in Nature Chemical Biology this week which reveals the molecular determinants of α-Synuclein’s toxicity to yeast by performing a deep mutational scan of the protein. The work was done in very close collaboration with Martin Kampmann (co-corresponding author) and his lab, as well as Eric Chow (Director of the UCSF Center for Advanced Technology (CAT)). Also, Newberry, Kampmann, and others used this work as the basis for the PUBS hands-on laboratory class taken by incoming PhD students in iPQB - leading to a companion paper to be published soon. Our neighbors at Twist Biosciences also generously donated DNA technology to enable the project!

diagram.

Potential toxicity-inducing structural states of α-Synuclein

Newberry and co-workers sought determine the structural state that contributes to the toxicity of α-Synuclein. This posed a difficult task since α-Synuclein has been hypothesized and observed to form many conformations in different cellular and in vitro contexts: intrinsically disordered, disordered aggregate, water-soluble helical bundle, amyloid fiber, membrane-bound helix. Each structural state offers distinct and often conflicting rational for α-Synuclein’s different biological roles in both healthy function and in disease, namely neurodegeneration (Parkinson's).

Through a pooled DNA library approach, Robert made every possible single-point mutant of α-Synuclein and measured each mutant’s effect on host yeast cell growth in efforts to discover the conformation responsible for inducing a toxicity, i.e. slow growing phenotype. The results were a striking pattern of mutational sensitivity in the first 90 amino acids with a clear amphipathic repeat pattern, which were best explained if the toxicity-inducing state is a continuous membrane-bound α-helix. Robert supported this hypothesis with direct membrane binding of select α-Synuclein mutants to vesicles in vitro.

diagram.

Deep mutational scanning data is most consistent with a membrane-bound helix being the toxicity-inducing conformation, with the most crucial residues at the α-Synuclein N-terminus

These results reveal key biophysical insights concerning α-Synuclein’s interactions with lipid membranes, particularly those with curvature. In all, the authors demonstrate an highly instructive experimental platform for understanding proteins’ like α-Synuclein role in neurodegeneration in the cellular context of choice.

Link to paper

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