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Rewiring the Specificity of Two-Component Signal Transduction Systems

Rewiring the Specificity of Two-Component Signal Transduction Systems. 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. Background: Two-component signal transduction.

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Rewiring the Specificity of Two-Component Signal Transduction Systems

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  1. Rewiring the Specificity of Two-Component Signal Transduction Systems 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

  2. 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

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

  4. 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

  5. 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

  6. Summary of kinetic preferences

  7. 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

  8. 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

  9. 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

  10. Supplemental data Spo0BLSpo0F model

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

  12. Supplemental Data • More proof of specificity of chimeras

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