Transcript Rewiring the Specificity of Two-Component Signal

Rewiring the Specificity of TwoComponent 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
 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
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 × 10
5
-
-
Chim1
1408
5 × 10
5
-
-
Mut4
206
7 × 10
4
-
-
Mut5
94
3 × 10
4
-
-
CpxA
-
-
296
2 × 10
5
Chim2
-
-
690
5 × 10
5
histidine kinase
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