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Systematic dissection and trajectory-scanning mutagenesis of the molecular interface that ensures specificity of two-component signaling pathways.

Capra EJ, Perchuk BS, Lubin EA, Ashenberg O, Skerker JM, Laub MT - PLoS Genet. (2010)

Bottom Line: The same approach was used for the response regulators OmpR and RstA.Collectively, the results begin to reveal the molecular mechanism by which a small set of amino acids enables an individual kinase to discriminate amongst a large set of highly-related response regulators and vice versa.Our results also suggest that the mutational trajectories taken by two-component signaling proteins following gene or pathway duplication may be constrained and subject to differential selective pressures.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA.

ABSTRACT
Two-component signal transduction systems enable bacteria to sense and respond to a wide range of environmental stimuli. Sensor histidine kinases transmit signals to their cognate response regulators via phosphorylation. The faithful transmission of information through two-component pathways and the avoidance of unwanted cross-talk require exquisite specificity of histidine kinase-response regulator interactions to ensure that cells mount the appropriate response to external signals. To identify putative specificity-determining residues, we have analyzed amino acid coevolution in two-component proteins and identified a set of residues that can be used to rationally rewire a model signaling pathway, EnvZ-OmpR. To explore how a relatively small set of residues can dictate partner selectivity, we combined alanine-scanning mutagenesis with an approach we call trajectory-scanning mutagenesis, in which all mutational intermediates between the specificity residues of EnvZ and another kinase, RstB, were systematically examined for phosphotransfer specificity. The same approach was used for the response regulators OmpR and RstA. Collectively, the results begin to reveal the molecular mechanism by which a small set of amino acids enables an individual kinase to discriminate amongst a large set of highly-related response regulators and vice versa. Our results also suggest that the mutational trajectories taken by two-component signaling proteins following gene or pathway duplication may be constrained and subject to differential selective pressures. Only some trajectories allow both the maintenance of phosphotransfer and the avoidance of unwanted cross-talk.

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Converting the phosphotransfer specificity of EnvZ to match RstB and vice versa.(A) Converting the phosphotransfer specificity of EnvZ to that of RstB. Wild-type EnvZ and each single, double, and triple mutant on the trajectory from EnvZ to RstB were autophosphorylated and then incubated alone or with one of three response regulators, as indicated, for 10 seconds. Wild-type RstB (far right) is shown for comparison to EnvZ(VYR). (B) Converting the phosphotransfer specificity of RstB to that of EnvZ. Wild-type RstB and each single, double, and triple mutant on the trajectory from RstB to EnvZ was autophosphorylated and then incubated alone or with one of three response regulators, as indicated, for 60 seconds. Wild-type EnvZ (far left) is shown for comparison to RstB(TLA). Arrows connect profiles of mutants differing by a single amino acid substitution.
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pgen-1001220-g004: Converting the phosphotransfer specificity of EnvZ to match RstB and vice versa.(A) Converting the phosphotransfer specificity of EnvZ to that of RstB. Wild-type EnvZ and each single, double, and triple mutant on the trajectory from EnvZ to RstB were autophosphorylated and then incubated alone or with one of three response regulators, as indicated, for 10 seconds. Wild-type RstB (far right) is shown for comparison to EnvZ(VYR). (B) Converting the phosphotransfer specificity of RstB to that of EnvZ. Wild-type RstB and each single, double, and triple mutant on the trajectory from RstB to EnvZ was autophosphorylated and then incubated alone or with one of three response regulators, as indicated, for 60 seconds. Wild-type EnvZ (far left) is shown for comparison to RstB(TLA). Arrows connect profiles of mutants differing by a single amino acid substitution.

Mentions: Although alanine-scanning provides some insight into specificity, an alanine substitution does not necessarily result in a simple loss of functionality, especially considering that EnvZ has a specificity residue that is already an alanine. In addition, as noted, there may be non-additive interdependencies between residues such that individual substitutions have minimal effect. We therefore sought to characterize the role of specificity-determining residues by examining the complete set of mutational intermediates between two histidine kinases with different specificities. For this analysis we focused on the paralogous systems EnvZ/OmpR and RstB/RstA, and term the approach trajectory-scanning. We constructed each possible specificity intermediate between EnvZ and RstB. This was feasible as the conversion of EnvZ phosphotransfer specificity to match that of RstB required only three substitutions, T250V, L254Y, and A255R [9]; the other major specificity residues identified by coevolution analysis are identical between EnvZ and RstB. In addition, we were able to rewire the specificity of RstB to match that of EnvZ by mutating the same three sites (Figure 4). The triple mutant RstB(V228T, Y232L, and R233A) no longer phosphorylated RstA and, instead, efficiently phosphorylated OmpR. These three residues thus play the dominant roles in dictating the specificity of both EnvZ and RstB. Other residues may make minor contributions.


Systematic dissection and trajectory-scanning mutagenesis of the molecular interface that ensures specificity of two-component signaling pathways.

Capra EJ, Perchuk BS, Lubin EA, Ashenberg O, Skerker JM, Laub MT - PLoS Genet. (2010)

Converting the phosphotransfer specificity of EnvZ to match RstB and vice versa.(A) Converting the phosphotransfer specificity of EnvZ to that of RstB. Wild-type EnvZ and each single, double, and triple mutant on the trajectory from EnvZ to RstB were autophosphorylated and then incubated alone or with one of three response regulators, as indicated, for 10 seconds. Wild-type RstB (far right) is shown for comparison to EnvZ(VYR). (B) Converting the phosphotransfer specificity of RstB to that of EnvZ. Wild-type RstB and each single, double, and triple mutant on the trajectory from RstB to EnvZ was autophosphorylated and then incubated alone or with one of three response regulators, as indicated, for 60 seconds. Wild-type EnvZ (far left) is shown for comparison to RstB(TLA). Arrows connect profiles of mutants differing by a single amino acid substitution.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2991266&req=5

pgen-1001220-g004: Converting the phosphotransfer specificity of EnvZ to match RstB and vice versa.(A) Converting the phosphotransfer specificity of EnvZ to that of RstB. Wild-type EnvZ and each single, double, and triple mutant on the trajectory from EnvZ to RstB were autophosphorylated and then incubated alone or with one of three response regulators, as indicated, for 10 seconds. Wild-type RstB (far right) is shown for comparison to EnvZ(VYR). (B) Converting the phosphotransfer specificity of RstB to that of EnvZ. Wild-type RstB and each single, double, and triple mutant on the trajectory from RstB to EnvZ was autophosphorylated and then incubated alone or with one of three response regulators, as indicated, for 60 seconds. Wild-type EnvZ (far left) is shown for comparison to RstB(TLA). Arrows connect profiles of mutants differing by a single amino acid substitution.
Mentions: Although alanine-scanning provides some insight into specificity, an alanine substitution does not necessarily result in a simple loss of functionality, especially considering that EnvZ has a specificity residue that is already an alanine. In addition, as noted, there may be non-additive interdependencies between residues such that individual substitutions have minimal effect. We therefore sought to characterize the role of specificity-determining residues by examining the complete set of mutational intermediates between two histidine kinases with different specificities. For this analysis we focused on the paralogous systems EnvZ/OmpR and RstB/RstA, and term the approach trajectory-scanning. We constructed each possible specificity intermediate between EnvZ and RstB. This was feasible as the conversion of EnvZ phosphotransfer specificity to match that of RstB required only three substitutions, T250V, L254Y, and A255R [9]; the other major specificity residues identified by coevolution analysis are identical between EnvZ and RstB. In addition, we were able to rewire the specificity of RstB to match that of EnvZ by mutating the same three sites (Figure 4). The triple mutant RstB(V228T, Y232L, and R233A) no longer phosphorylated RstA and, instead, efficiently phosphorylated OmpR. These three residues thus play the dominant roles in dictating the specificity of both EnvZ and RstB. Other residues may make minor contributions.

Bottom Line: The same approach was used for the response regulators OmpR and RstA.Collectively, the results begin to reveal the molecular mechanism by which a small set of amino acids enables an individual kinase to discriminate amongst a large set of highly-related response regulators and vice versa.Our results also suggest that the mutational trajectories taken by two-component signaling proteins following gene or pathway duplication may be constrained and subject to differential selective pressures.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA.

ABSTRACT
Two-component signal transduction systems enable bacteria to sense and respond to a wide range of environmental stimuli. Sensor histidine kinases transmit signals to their cognate response regulators via phosphorylation. The faithful transmission of information through two-component pathways and the avoidance of unwanted cross-talk require exquisite specificity of histidine kinase-response regulator interactions to ensure that cells mount the appropriate response to external signals. To identify putative specificity-determining residues, we have analyzed amino acid coevolution in two-component proteins and identified a set of residues that can be used to rationally rewire a model signaling pathway, EnvZ-OmpR. To explore how a relatively small set of residues can dictate partner selectivity, we combined alanine-scanning mutagenesis with an approach we call trajectory-scanning mutagenesis, in which all mutational intermediates between the specificity residues of EnvZ and another kinase, RstB, were systematically examined for phosphotransfer specificity. The same approach was used for the response regulators OmpR and RstA. Collectively, the results begin to reveal the molecular mechanism by which a small set of amino acids enables an individual kinase to discriminate amongst a large set of highly-related response regulators and vice versa. Our results also suggest that the mutational trajectories taken by two-component signaling proteins following gene or pathway duplication may be constrained and subject to differential selective pressures. Only some trajectories allow both the maintenance of phosphotransfer and the avoidance of unwanted cross-talk.

Show MeSH
Related in: MedlinePlus