Rewiring the specificity of two-component signal transduction systems.
In a research published at Cell, Skerker et al. performs a computational co-variation analysis on a dataset of two-component system sequences. This results in a set of putative specificity determining residues. The authors then demonstrate how they can successfully use these to modulate Histidine Kinase-Response Regulator interactions both in-vitro and in-vivo. By Nir London
Skerker JM, Perchuk BS, Siryaporn A, Lubin EA, Ashenberg O, Goulian M, Laub MT.
Cell. 2008 Jun 13;133(6):1043-54
Two component signaling systems are found in nearly all bacteria. These signaling pathways typically consist of a sensor histidine kinase (HK) that autophosphorylates and then transfers the phosphoryl group to a cognate response regulator (RR) that can effect changes in cellular physiology or behavior, often by changing gene expression. Most histidine kinases have one or two cognate response regulators, and there appears to be minimal crosstalk between different pathways.
Histidine kinases and response regulators encoded in the same operon typically interact with one another in an exclusive, one-to-one fashion. Using nearly 200 sequenced bacterial genomes, the authors identified nearly 1300 HK-RR pairs. These cognate pairs were treated as a single sequence and then aligned. To identify positions in this multiple sequence alignment that covary, mutual information was calculated between each pair of sites in the alignment. This procedure identified approximately 40 pairs of residues with intermolecular mutual information scores greater than 0.35, 16 within the histidine kinase and 12 within the response regulator. Most of the pairs involved residues of the DHp domain of the kinase and the response regulator. These analyses suggest that the DHp domain dictates the interaction between a histidine kinase and its cognate substrate.
No high-resolution crystal structure of a histidine kinase in complex with a response regulator has been solved. However, the structure of a histidine phosphotransferase, Spo0B, in complex with a response regulator, Spo0F, is considered a reasonable proxy. Mapping the putative specificity-determining residues onto the Spo0B:Spo0F crystal structure, showed that nearly all of these residues are solvent exposed in the individual molecules, but they are buried in the interface of the protein complex. At a score threshold of 0.35, there is clear enrichment for residues in close proximity, with an average distance of 10 Å. In summary, the mutual information analysis identifies a set of covarying residues that are located at, or very near, the molecular interface of an HK-RR complex. There are two apparent clusters of covarying residues in each molecule. In the histidine kinase, one cluster lies below the phosphotransfer active site, and the second lies just above the active site histidine.
To test whether the highest-scoring residues confer specificity to the HK-RR interaction, the authors constructed a series of chimeric and mutant histidine kinases. A first set of chimeras was composed of the DHp domains of heterologous kinases fused to the CA domain of the Escherichia coli kinase EnvZ. With respect to phosphotransfer specificity, each chimera behaved according to the identity of its DHp domain; These results indicate that the phosphotransfer specificity of a histidine kinase is dictated almost exclusively by its DHp domain, consistent with the mutual information analysis.
The covariation analysis identified a cluster of 7 residues below the histidine active site of the DHp domain. To test whether this cluster is important for specificity, the auothors produced subdomain chimeras in which only a short segment of the EnvZ DHp domain was replaced with the corresponding sequence of five E. coli histidine kinases, which share varying levels of homology with EnvZ. The transplanted segment included 7 of the putative specificity-determining residues. The substrate specificity of each chimeric kinase was assessed by comparing its phosphotransfer activity toward OmpR (cognate substrate of EnvZ) and the new, desired target. In all of the chimeras strong phosphorylation of the new response regulator was observed and no residual phosphorylation of OmpR. This data indicates that the amino acids at the base of the DHp domain, which includes 7 of the highest-scoring residues from the computational analysis, are sufficient to determine the phosphotransfer specificity of a histidine kinase.
The authors therefore sought to test whether mutating only these 7 residues would be sufficient to change the substrate selectivity of EnvZ. For these experiments, mutations were made in EnvZ in which putative specificity residues were replaced with the corresponding residues from RstB. Two of the single mutants generated, showed significant phosphorylation of both RstA and OmpR. The double mutant preferentially phosphorylated RstA, but it still retained some residual activity toward OmpR. Subsequent inclusion of another 3rd mutation, however, eliminated the phosphorylation of OmpR but maintained robust phosphotransfer to RstA. Meaning, changing as few as three residues is sufficient to change the substrate preference of EnvZ to that of RstB.
The authors conclude with a striking example of an in-vivo experiment, in which by the mutation of EnvZ they were able to phosphorylate a non-cognate target and as a result to modulate the expression of GFP in the cell.
By Nir London
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