Limits...
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.

Show MeSH

Related in: MedlinePlus

Mutational trajectories from EnvZ/OmpR to RstB/RstA.EnvZ and OmpR can be converted by a series of single mutations to harbor the specificity residues found in RstB and RstA, respectively, without disrupting phosphotransfer in intermediate stages. (A) A series of single mutations can convert the specificity of EnvZ to match that of RstB and OmpR to match RstA. Starting with the wild type specificity residues in red text at the top, each subsequent line introduces a single mutation (shown in black text) until both sets of specificity residues have been completely changed. As noted in the text, we treated the loop as a single mutation. As shown in panel B, each kinase-regulator pair listed is capable of phosphotransfer and does not include a regulator that is phosphorylated by CpxA. (B) The complete set of intermediates between wild type OmpR (RLR/PFN) and the quadruple mutant (EVA/TTP) are listed. For wild type EnvZ (TLA), the single mutant EnvZ(TYA), the double mutant EnvZ(TYR), and the triple mutant EnvZ(VYR), the set of OmpR mutants recognized by each kinase are shaded, with a merge of all four at the bottom. Mutants that are phosphorylated by CpxA are listed in grey text, all others in black text. Bold lines connect the mutant series shown in panel A.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2991266&req=5

pgen-1001220-g007: Mutational trajectories from EnvZ/OmpR to RstB/RstA.EnvZ and OmpR can be converted by a series of single mutations to harbor the specificity residues found in RstB and RstA, respectively, without disrupting phosphotransfer in intermediate stages. (A) A series of single mutations can convert the specificity of EnvZ to match that of RstB and OmpR to match RstA. Starting with the wild type specificity residues in red text at the top, each subsequent line introduces a single mutation (shown in black text) until both sets of specificity residues have been completely changed. As noted in the text, we treated the loop as a single mutation. As shown in panel B, each kinase-regulator pair listed is capable of phosphotransfer and does not include a regulator that is phosphorylated by CpxA. (B) The complete set of intermediates between wild type OmpR (RLR/PFN) and the quadruple mutant (EVA/TTP) are listed. For wild type EnvZ (TLA), the single mutant EnvZ(TYA), the double mutant EnvZ(TYR), and the triple mutant EnvZ(VYR), the set of OmpR mutants recognized by each kinase are shaded, with a merge of all four at the bottom. Mutants that are phosphorylated by CpxA are listed in grey text, all others in black text. Bold lines connect the mutant series shown in panel A.

Mentions: Third, these profiles reveal mutational paths from the specificity of the EnvZ/OmpR pair to that of RstB/RstA in which phosphotransfer is maintained. In other words, there is an ordered series of single mutations that can be made in EnvZ and OmpR that convert them to the specificity of RstB and RstA, respectively, without disrupting their ability to phosphotransfer to one another along the way. For example, wild-type EnvZ phosphorylates OmpR and the single mutant OmpR(RLAPFN) to similar levels, and conversely the single mutant EnvZ(TLA) phosphorylates both OmpR and OmpR(RLAPFN). In Figure 7 we extend this example to show how EnvZ and OmpR could, in principle, change its specificity to that of the RstB/RstA system by a series of alternating mutations in the two molecules without ever severely disrupting their interaction. There are several such paths, although each path is not necessarily equivalent because CpxA phosphorylates some mutational intermediates of OmpR and some EnvZ mutants phosphorylate CpxR. For instance, EnvZ(TLR) phosphorylated CpxR, and OmpR(ELRPFN) was phosphorylated by CpxA (Figure 5, also see Figure 4). The avoidance of cross-talk may limit the possible evolutionary pathways between EnvZ/OmpR and RstA/RstB, or at least favor some relative to others (Figure 7).


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)

Mutational trajectories from EnvZ/OmpR to RstB/RstA.EnvZ and OmpR can be converted by a series of single mutations to harbor the specificity residues found in RstB and RstA, respectively, without disrupting phosphotransfer in intermediate stages. (A) A series of single mutations can convert the specificity of EnvZ to match that of RstB and OmpR to match RstA. Starting with the wild type specificity residues in red text at the top, each subsequent line introduces a single mutation (shown in black text) until both sets of specificity residues have been completely changed. As noted in the text, we treated the loop as a single mutation. As shown in panel B, each kinase-regulator pair listed is capable of phosphotransfer and does not include a regulator that is phosphorylated by CpxA. (B) The complete set of intermediates between wild type OmpR (RLR/PFN) and the quadruple mutant (EVA/TTP) are listed. For wild type EnvZ (TLA), the single mutant EnvZ(TYA), the double mutant EnvZ(TYR), and the triple mutant EnvZ(VYR), the set of OmpR mutants recognized by each kinase are shaded, with a merge of all four at the bottom. Mutants that are phosphorylated by CpxA are listed in grey text, all others in black text. Bold lines connect the mutant series shown in panel A.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1001220-g007: Mutational trajectories from EnvZ/OmpR to RstB/RstA.EnvZ and OmpR can be converted by a series of single mutations to harbor the specificity residues found in RstB and RstA, respectively, without disrupting phosphotransfer in intermediate stages. (A) A series of single mutations can convert the specificity of EnvZ to match that of RstB and OmpR to match RstA. Starting with the wild type specificity residues in red text at the top, each subsequent line introduces a single mutation (shown in black text) until both sets of specificity residues have been completely changed. As noted in the text, we treated the loop as a single mutation. As shown in panel B, each kinase-regulator pair listed is capable of phosphotransfer and does not include a regulator that is phosphorylated by CpxA. (B) The complete set of intermediates between wild type OmpR (RLR/PFN) and the quadruple mutant (EVA/TTP) are listed. For wild type EnvZ (TLA), the single mutant EnvZ(TYA), the double mutant EnvZ(TYR), and the triple mutant EnvZ(VYR), the set of OmpR mutants recognized by each kinase are shaded, with a merge of all four at the bottom. Mutants that are phosphorylated by CpxA are listed in grey text, all others in black text. Bold lines connect the mutant series shown in panel A.
Mentions: Third, these profiles reveal mutational paths from the specificity of the EnvZ/OmpR pair to that of RstB/RstA in which phosphotransfer is maintained. In other words, there is an ordered series of single mutations that can be made in EnvZ and OmpR that convert them to the specificity of RstB and RstA, respectively, without disrupting their ability to phosphotransfer to one another along the way. For example, wild-type EnvZ phosphorylates OmpR and the single mutant OmpR(RLAPFN) to similar levels, and conversely the single mutant EnvZ(TLA) phosphorylates both OmpR and OmpR(RLAPFN). In Figure 7 we extend this example to show how EnvZ and OmpR could, in principle, change its specificity to that of the RstB/RstA system by a series of alternating mutations in the two molecules without ever severely disrupting their interaction. There are several such paths, although each path is not necessarily equivalent because CpxA phosphorylates some mutational intermediates of OmpR and some EnvZ mutants phosphorylate CpxR. For instance, EnvZ(TLR) phosphorylated CpxR, and OmpR(ELRPFN) was phosphorylated by CpxA (Figure 5, also see Figure 4). The avoidance of cross-talk may limit the possible evolutionary pathways between EnvZ/OmpR and RstA/RstB, or at least favor some relative to others (Figure 7).

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