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

Identification of coevolving amino acids in cognate pairs of histidine kinases and response regulators.(A) Residues in histidine kinases and response regulators that strongly coevolve (adjusted MI score >3.5) are listed with lines connecting covarying pairs. Residues are numbered according to their position in E. coli EnvZ and OmpR. (B–C) Residues in histidine kinases that coevolve with residues in response regulators are shown on a primary sequence alignment of HK853 from T. maritima and EnvZ, RstB, and CpxA from E. coli. Residues in response regulators that strongly coevolve with residues in histidine kinases are shown on a primary sequence alignment of RR468 from T. maritima and OmpR, RstA, and CpxR from E. coli. Residues highly conserved across all two-component signaling proteins are shaded in grey. Coevolving residues are shown in orange and red for the kinase and regulator, respectively. Secondary structure elements, based on the co-crystal structure of HK853 and RR468 from T. maritima [13], are shown beneath the sequences. (D) Coevolving residues mapped onto the HK853-RR468 structure. Coevolving residues are shown by space-filling and colored as in panels A–C. The side chains of the conserved phosphorylatable histidines and aspartate are shown as magenta sticks. The HK853-RR468 complex is shown in the center with each individual molecule rotated 90° and shown separately.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2991266&req=5

pgen-1001220-g001: Identification of coevolving amino acids in cognate pairs of histidine kinases and response regulators.(A) Residues in histidine kinases and response regulators that strongly coevolve (adjusted MI score >3.5) are listed with lines connecting covarying pairs. Residues are numbered according to their position in E. coli EnvZ and OmpR. (B–C) Residues in histidine kinases that coevolve with residues in response regulators are shown on a primary sequence alignment of HK853 from T. maritima and EnvZ, RstB, and CpxA from E. coli. Residues in response regulators that strongly coevolve with residues in histidine kinases are shown on a primary sequence alignment of RR468 from T. maritima and OmpR, RstA, and CpxR from E. coli. Residues highly conserved across all two-component signaling proteins are shaded in grey. Coevolving residues are shown in orange and red for the kinase and regulator, respectively. Secondary structure elements, based on the co-crystal structure of HK853 and RR468 from T. maritima [13], are shown beneath the sequences. (D) Coevolving residues mapped onto the HK853-RR468 structure. Coevolving residues are shown by space-filling and colored as in panels A–C. The side chains of the conserved phosphorylatable histidines and aspartate are shown as magenta sticks. The HK853-RR468 complex is shown in the center with each individual molecule rotated 90° and shown separately.

Mentions: At an adjusted score threshold of 3.5, we found 12 coevolving pairs, comprising 9 residues in the histidine kinases and 7 in the response regulators (Figure 1A–1C). These residues form a single, densely-interconnected cluster of coevolving residues. The residues are all solvent-exposed in the individual molecules, but buried within the molecular interface formed in a co-crystal structure of T. maritima HK853 and RR468 (Figure 1D) [13]. The residues identified here overlap substantially with, but are not identical to, those we identified previously [9]. Of the coevolving residues in the kinase, all are in the DHp domain, consistent with this domain being the primary site of interaction with the response regulator. Within the DHp domain, the coevolving residues are found on both alpha helices and are located below the histidine phosphorylation site (Figure 1D). The covarying residues in the response regulator are spatially near the conserved aspartic acid phosphorylation site (Figure 1D), predominantly on a single face of alpha helix-1 in the receiver domain with one additional residue within the β5-α5 loop. At lower score thresholds, an additional cluster of coevolving residues are found (Figure S2), but we focus here on the set of 16 residues identified at a threshold of 3.5.


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)

Identification of coevolving amino acids in cognate pairs of histidine kinases and response regulators.(A) Residues in histidine kinases and response regulators that strongly coevolve (adjusted MI score >3.5) are listed with lines connecting covarying pairs. Residues are numbered according to their position in E. coli EnvZ and OmpR. (B–C) Residues in histidine kinases that coevolve with residues in response regulators are shown on a primary sequence alignment of HK853 from T. maritima and EnvZ, RstB, and CpxA from E. coli. Residues in response regulators that strongly coevolve with residues in histidine kinases are shown on a primary sequence alignment of RR468 from T. maritima and OmpR, RstA, and CpxR from E. coli. Residues highly conserved across all two-component signaling proteins are shaded in grey. Coevolving residues are shown in orange and red for the kinase and regulator, respectively. Secondary structure elements, based on the co-crystal structure of HK853 and RR468 from T. maritima [13], are shown beneath the sequences. (D) Coevolving residues mapped onto the HK853-RR468 structure. Coevolving residues are shown by space-filling and colored as in panels A–C. The side chains of the conserved phosphorylatable histidines and aspartate are shown as magenta sticks. The HK853-RR468 complex is shown in the center with each individual molecule rotated 90° and shown separately.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1001220-g001: Identification of coevolving amino acids in cognate pairs of histidine kinases and response regulators.(A) Residues in histidine kinases and response regulators that strongly coevolve (adjusted MI score >3.5) are listed with lines connecting covarying pairs. Residues are numbered according to their position in E. coli EnvZ and OmpR. (B–C) Residues in histidine kinases that coevolve with residues in response regulators are shown on a primary sequence alignment of HK853 from T. maritima and EnvZ, RstB, and CpxA from E. coli. Residues in response regulators that strongly coevolve with residues in histidine kinases are shown on a primary sequence alignment of RR468 from T. maritima and OmpR, RstA, and CpxR from E. coli. Residues highly conserved across all two-component signaling proteins are shaded in grey. Coevolving residues are shown in orange and red for the kinase and regulator, respectively. Secondary structure elements, based on the co-crystal structure of HK853 and RR468 from T. maritima [13], are shown beneath the sequences. (D) Coevolving residues mapped onto the HK853-RR468 structure. Coevolving residues are shown by space-filling and colored as in panels A–C. The side chains of the conserved phosphorylatable histidines and aspartate are shown as magenta sticks. The HK853-RR468 complex is shown in the center with each individual molecule rotated 90° and shown separately.
Mentions: At an adjusted score threshold of 3.5, we found 12 coevolving pairs, comprising 9 residues in the histidine kinases and 7 in the response regulators (Figure 1A–1C). These residues form a single, densely-interconnected cluster of coevolving residues. The residues are all solvent-exposed in the individual molecules, but buried within the molecular interface formed in a co-crystal structure of T. maritima HK853 and RR468 (Figure 1D) [13]. The residues identified here overlap substantially with, but are not identical to, those we identified previously [9]. Of the coevolving residues in the kinase, all are in the DHp domain, consistent with this domain being the primary site of interaction with the response regulator. Within the DHp domain, the coevolving residues are found on both alpha helices and are located below the histidine phosphorylation site (Figure 1D). The covarying residues in the response regulator are spatially near the conserved aspartic acid phosphorylation site (Figure 1D), predominantly on a single face of alpha helix-1 in the receiver domain with one additional residue within the β5-α5 loop. At lower score thresholds, an additional cluster of coevolving residues are found (Figure S2), but we focus here on the set of 16 residues identified at a threshold of 3.5.

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