jeffrey m. skerker, barrett s. perchuk, albert siryaporn, emma a. lubin, orr ashenberg, mark...

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 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

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 data

Spo0BLSpo0F model

Supplemental Data

Phosphorylation of target enzymes-only initial rate of phosphorylaton was

considered

Supplemental DataMore proof of specificity of chimeras