Jeffrey M. Skerker, Barrett S. Perchuk, Albert Siryaporn, Emma A. Lubin, Orr Ashenberg, Mark Goulian, and Michael T. Laub Cell 2008 Presented by: Amber Lin Rewiring the Specificity of Two- Component Signal Transduction Systems

Background: Two-component signal transduction Histidine kinase – response regulator pairings HK exhibit large kinetic preference towards in vivo cognate RR Pairings often encoded in same operon

Background: Specificity-Determining residues Found 43 pairs with high mutual information scores (>0.35) 33 pairs from DHp domain Avg distance btwn residues: 10Å

Residue determination of HK-RR specificity Chimeras made with CA domain of E. coli kinase EnvZ and DHp domains of other kinases Showed similar specificity, implying kinase domain is main determinant

Residue determination of HK-RR specificity (cont.) Tested point mutations of amino acids predicted by analysis to determine specificity RstB showed increasing specificity with increasing combination of mutations

Summary of kinetic preferences histidine kinase kinetic preference (RstA v. OmpR) * * kinetic preference relative to EnvZ kinetic preference (CpxR v. OmpR) # # kinetic preference relative to EnvZ EnvZ RstB25668 × Chim × Mut42067 × Mut5943 × CpxA × 10 5 Chim × 10 5

Residue determination of HK-RR specificity (cont.) Mutant kinases for CpxA, PhoR, AtoS, and PhoQ showed only partial specificity Chimeras showed almost complete switch loop located near C-terminal end of helix 1 also specificity- determining

In Vivo testing of rewiring Tested EnvZ MI+loop mutants for phosphorylation ability in E. coli GFP/YFP promoters stimulated by phosphorylated RR Used Western Blot to confirm levels were <= WT EnvZ control

Discussion Results Determined way to rationally rewire two-component signal system HK-RR Identified 2 primary clusters of HK-RR aa covarying pairs Successfully changed specificity of EnvZ to match other kinases Potential Uses Enhance use of HK-RR in synthetic signaling circuits Identification could lead to further understanding of system properties and their evolution

Supplemental data Spo0BLSpo0F model

Supplemental Data Phosphorylation of target enzymes -only initial rate of phosphorylaton was considered

Supplemental Data More proof of specificity of chimeras