rewiring the specificity of two-component signal transduction systems
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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. - PowerPoint PPT PresentationTRANSCRIPT
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
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 specificityChimeras made with CA domain of E.
coli kinase EnvZ and DHp domains of other kinasesShowed 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 specificityRstB 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 0.0031 1 0.0013 1RstB 2566 8 × 105 - -
Chim1 1408 5 × 105 - -
Mut4 206 7 × 104 - -
Mut5 94 3 × 104 - -
CpxA - - 296 2 × 105
Chim2 - - 690 5 × 105
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
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
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
Supplemental dataSpo0BLSpo0F model
Supplemental Data
Phosphorylation of target enzymes-only initial rate of phosphorylaton was
considered
Supplemental DataMore proof of specificity of chimeras