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Jeffrey M. Skerker, Barrett S. Perchuk, Albert Siryaporn, Emma A. Lubin, Orr Ashenberg, Mark
Goulian, and Michael T. LaubCell 2008
Presented by: Amber Lin
Rewiring the Specificity of Two-Component Signal Transduction
Systems
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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
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Background: Specificity-Determining residues
Found 43 pairs with high mutual information scores (>0.35)
33 pairs from DHp domain
Avg distance btwn residues: 10Å
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Residue determination of HK-RR specificityChimeras made with CA domain of E.
coli kinase EnvZ and DHp domains of other kinasesShowed similar specificity, implying kinase
domain is main determinant
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Residue determination of HK-RR specificity (cont.)Tested point mutations of amino acids
predicted by analysis to determine specificityRstB showed increasing specificity with increasing
combination of mutations
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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 0.0031 1 0.0013 1
RstB 2566 8 × 105 - -
Chim1 1408 5 × 105 - -
Mut4 206 7 × 104 - -
Mut5 94 3 × 104 - -
CpxA - - 296 2 × 105
Chim2 - - 690 5 × 105
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Residue determination of HK-RR specificity (cont.)Mutant kinases for CpxA, PhoR, AtoS, and
PhoQ showed only partial specificityChimeras showed almost complete switch
loop located near C-terminal end of helix 1 also specificity-determining
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In Vivo testing of rewiringTested EnvZ MI+loop mutants for
phosphorylation ability in E. coliGFP/YFP promoters stimulated by
phosphorylated RRUsed Western Blot to confirm levels were <=
WT EnvZ control
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DiscussionResults
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 UsesEnhance use of HK-RR in synthetic signaling circuitsIdentification could lead to further understanding of
system properties and their evolution
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Supplemental data
Spo0BLSpo0F model
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Supplemental Data
Phosphorylation of target enzymes-only initial rate of phosphorylaton was
considered
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Supplemental DataMore proof of specificity of chimeras