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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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Related in: MedlinePlus

Complete trajectory-scanning mutagenesis of EnvZ and OmpR.Each histidine kinase, indicated on the far right, was autophosphorylated and tested for phosphotransfer to each of the response regulators listed across the top. Mutants of EnvZ are named according to the identity of the three specificity residues being examined; for instance, wild-type EnvZ is ‘TLA’ whereas the mutant T250V is ‘VLA’. Mutants of OmpR are named similarly. All phosphotransfer reactions were incubated for 10 seconds with the exception of RstB and CpxA, which were examined at both 10 seconds and 1 minute. Each kinase profile was composed of two separate gels that were run, exposed to phosphor screens, and scanned in parallel. The resulting two gel images were treated identically and then stitched together between OmpR(EVAPFN) and OmpR(EVATTP).
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pgen-1001220-g005: Complete trajectory-scanning mutagenesis of EnvZ and OmpR.Each histidine kinase, indicated on the far right, was autophosphorylated and tested for phosphotransfer to each of the response regulators listed across the top. Mutants of EnvZ are named according to the identity of the three specificity residues being examined; for instance, wild-type EnvZ is ‘TLA’ whereas the mutant T250V is ‘VLA’. Mutants of OmpR are named similarly. All phosphotransfer reactions were incubated for 10 seconds with the exception of RstB and CpxA, which were examined at both 10 seconds and 1 minute. Each kinase profile was composed of two separate gels that were run, exposed to phosphor screens, and scanned in parallel. The resulting two gel images were treated identically and then stitched together between OmpR(EVAPFN) and OmpR(EVATTP).

Mentions: The mutational trajectory scanning done for both EnvZ and RstB was extended to the response regulator OmpR. Converting OmpR to have the phosphotransfer specificity of RstA required 3 mutations in alpha helix-1 and 3 mutations in the β5-α5 loop (Figure 2A). We treated the loop as a single entity and made the 15 possible OmpR-RstA intermediates: 4 single, 6 double, 4 triple, and 1 quadruple mutant. We then examined phosphotransfer from each of the 7 EnvZ-RstB mutants (Figure 4A), as well as wild type EnvZ, RstB, and CpxA, to each of the 15 OmpR mutants and to wild-type OmpR, RstA, and CpxR, for a total of 180 pairwise combinations. The complete data are shown in Figure 5 and Figure 6. All phosphotransfer reactions were run for 10 seconds, except for RstB and CpxA, which were run for 10 seconds and for 1 minute. To evaluate phosphotransfer, we quantified the relative intensity of each response regulator band for a given histidine kinase, yielding a profile of phosphotransfer activity for each kinase. From the comprehensive profiles, several observations and trends emerged (Figure 5 and Figure 6).


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)

Complete trajectory-scanning mutagenesis of EnvZ and OmpR.Each histidine kinase, indicated on the far right, was autophosphorylated and tested for phosphotransfer to each of the response regulators listed across the top. Mutants of EnvZ are named according to the identity of the three specificity residues being examined; for instance, wild-type EnvZ is ‘TLA’ whereas the mutant T250V is ‘VLA’. Mutants of OmpR are named similarly. All phosphotransfer reactions were incubated for 10 seconds with the exception of RstB and CpxA, which were examined at both 10 seconds and 1 minute. Each kinase profile was composed of two separate gels that were run, exposed to phosphor screens, and scanned in parallel. The resulting two gel images were treated identically and then stitched together between OmpR(EVAPFN) and OmpR(EVATTP).
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1001220-g005: Complete trajectory-scanning mutagenesis of EnvZ and OmpR.Each histidine kinase, indicated on the far right, was autophosphorylated and tested for phosphotransfer to each of the response regulators listed across the top. Mutants of EnvZ are named according to the identity of the three specificity residues being examined; for instance, wild-type EnvZ is ‘TLA’ whereas the mutant T250V is ‘VLA’. Mutants of OmpR are named similarly. All phosphotransfer reactions were incubated for 10 seconds with the exception of RstB and CpxA, which were examined at both 10 seconds and 1 minute. Each kinase profile was composed of two separate gels that were run, exposed to phosphor screens, and scanned in parallel. The resulting two gel images were treated identically and then stitched together between OmpR(EVAPFN) and OmpR(EVATTP).
Mentions: The mutational trajectory scanning done for both EnvZ and RstB was extended to the response regulator OmpR. Converting OmpR to have the phosphotransfer specificity of RstA required 3 mutations in alpha helix-1 and 3 mutations in the β5-α5 loop (Figure 2A). We treated the loop as a single entity and made the 15 possible OmpR-RstA intermediates: 4 single, 6 double, 4 triple, and 1 quadruple mutant. We then examined phosphotransfer from each of the 7 EnvZ-RstB mutants (Figure 4A), as well as wild type EnvZ, RstB, and CpxA, to each of the 15 OmpR mutants and to wild-type OmpR, RstA, and CpxR, for a total of 180 pairwise combinations. The complete data are shown in Figure 5 and Figure 6. All phosphotransfer reactions were run for 10 seconds, except for RstB and CpxA, which were run for 10 seconds and for 1 minute. To evaluate phosphotransfer, we quantified the relative intensity of each response regulator band for a given histidine kinase, yielding a profile of phosphotransfer activity for each kinase. From the comprehensive profiles, several observations and trends emerged (Figure 5 and Figure 6).

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