Fig. 1: Construction of an orthogonal PYR1/HAB1 dimerization module using an allele-specific suppression strategy.
From: An orthogonalized PYR1-based CID module with reprogrammable ligand-binding specificity
a, Design pipeline for identification of ‘orthogonalizing’ mutations. The WT ABA sensor contains an ABA receptor, PYR1, that binds to the PP2C HAB1 in response to ABA to form a stable complex. A two-step approach was taken to identify mutations that program an orthogonal PYR1/HAB1 interaction. Site saturation mutagenesis of both proteins was first used to systematically identify mutations at the PYR1/HAB1 binding interface that disrupt ABA-mediated dimerization using established Y2H assays; this identified 237 and 163 non-binding mutants in PYR1 and HAB1, respectively (Supplementary Tables 1 and 2). The collection of non-functional mutant alleles was co-transformed into a Y2H reverse two-hybrid strain, and mutually suppressive allele combinations were identified using a growth-based selection strategy. The best suppressor pair identified was PYR1T162D/HAB1V393R (Supplementary Table 3 shows all combinations identified). b, Orthogonal interactions between the WT and mutant modules. PYR1T162D does not bind to HAB1, nor does HAB1V393R bind to PYR1, as measured using Y2H assays of the ABA-stimulated interaction (ABA 5,000 nM; mock, DMSO carrier solvent control). Colony overlay assays with an X-gal substrate indicate Y2H circuit activation by β-galactosidase induction. c, HAB1 phosphatase inhibition assay for combinations of WT and mutant alleles using a 4-methylumbeliferyl phosphate (4-MUP) substrate. Data points indicate the mean of three technical replicates; error bars indicate the s.d. and are normalized to the HAB1 activity in the presence of the receptor tested but without ligand. The data demonstrate that PYR1T162D does not cross-activate WT HAB1, as expected for an orthogonalizing mutation.
