Identification and biochemical and structural characterization of A1aY1 binder. (A) Schematic overview of the strategy employed to identify binder from SSO7D yeast display library. The biotinylated TUBA1A with Y (green star) and mono-Glu and ΔY (yellow star) used for positive and negative selection, respectively. For a detailed description, see Materials and methods. (B) kd of A1aY1 binder against biotinylated α tubulin CTT peptides of human (Hs_TUBA1A; 1.6 ± 0.3 µM, Hs_TUBA1A-ΔY; >60 µM*, Hs_TUBA1A-mG; 13.6 ± 4.5 µM), Drosophila (Dm_Tub84B; 4.1 ± 0.9 µM) and C. elegans (Ce_TBA1; 1.0 ± 0.2 µM) measured using an SPR-based steady-state binding assay. Experiments were performed in triplicate with at least two different batches of the protein A1aY1 (asterisk represents that the binding affinities cannot be uniquely determined with the current fit). The inset shows the titration response up to 4 µM A1aY1 binder concentration. (C) The NMR structure of A1aY1 binder (magenta surface representation) bound to Hs_TUBA1A peptide (blue cartoon with key residues as stick representation). The key interacting residues from A1aY1 binder and α tubulin CTT are labeled in magenta and blue, respectively. (D) Sequence alignment of α tubulin CTTs from human (Hs), Drosophila (Dm), C. elegans (Ce), Arabidopsisthaliana (At), and Schizosaccharomyces pombe (Sp) with gene names as indicated. Asterisks indicate the residues involved in A1aY1 binder interaction, and the terminal tyrosine and alternating glutamate residues are indicated as n series.
