Spatial tethering of kinases to their substrates relaxes evolutionary constraints on specificity.

Signal transduction proteins are often multi-domain proteins that arose through the fusion of previously independent proteins. How such a change in the spatial arrangement of proteins impacts their evolution and the selective pressures acting on individual residues is largely unknown. We explored this problem in the context of bacterial two-component signalling pathways, which typically involve a sensor histidine kinase that specifically phosphorylates a single cognate response regulator. Although usually found as separate proteins, these proteins are sometimes fused into a so-called hybrid histidine kinase. Here, we demonstrate that the isolated kinase domains of hybrid kinases exhibit a dramatic reduction in phosphotransfer specificity in vitro relative to canonical histidine kinases. However, hybrid kinases phosphotransfer almost exclusively to their covalently attached response regulator domain, whose effective concentration exceeds that of all soluble response regulators. These findings indicate that the fused response regulator in a hybrid kinase normally prevents detrimental cross-talk between pathways. More generally, our results shed light on how the spatial properties of signalling pathways can significantly affect their evolution, with additional implications for the design of synthetic signalling systems.

Evolution of two-component signal transduction systems.

To exist in a wide range of environmental niches, bacteria must sense and respond to a variety of external signals. A primary means by which this occurs is through two-component signal transduction pathways, typically composed of a sensor histidine kinase that receives the input stimuli and then phosphorylates a response regulator that effects an appropriate change in cellular physiology. Histidine kinases and response regulators have an intrinsic modularity that separates signal input, phosphotransfer, and output response; this modularity has allowed bacteria to dramatically expand and diversify their signaling capabilities. Recent work has begun to reveal the molecular basis by which two-component proteins evolve. How and why do orthologous signaling proteins diverge? How do cells gain new pathways and recognize new signals? What changes are needed to insulate a new pathway from existing pathways? What constraints are there on gene duplication and lateral gene transfer? Here, we review progress made in answering these questions, highlighting how the integration of genome sequence data with experimental studies is providing major new insights.

Adaptive mutations that prevent crosstalk enable the expansion of paralogous signaling protein families.

Orthologous proteins often harbor numerous substitutions, but whether these differences result from neutral or adaptive processes is usually unclear. To tackle this challenge, we examined the divergent evolution of a model bacterial signaling pathway comprising the kinase PhoR and its cognate substrate PhoB. We show that the specificity-determining residues of these proteins are typically under purifying selection but have, in α-proteobacteria, undergone a burst of diversification followed by extended stasis. By reversing mutations that accumulated in an α-proteobacterial PhoR, we demonstrate that these substitutions were adaptive, enabling PhoR to avoid crosstalk with a paralogous pathway that arose specifically in α-proteobacteria. Our findings demonstrate that duplication and the subsequent need to avoid crosstalk strongly influence signaling protein evolution. These results provide a concrete example of how system-wide insulation can be achieved postduplication through a surprisingly limited number of mutations. Our work may help explain the apparent ease with which paralogous protein families expanded in all organisms.

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

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.

Comparative developmental expression profiling of two C. elegans isolates.

Gene expression is known to change during development and to vary among genetically diverse strains. Previous studies of temporal patterns of gene expression during C. elegans development were incomplete, and little is known about how these patterns change as a function of genetic background. We used microarrays that comprehensively cover known and predicted worm genes to compare the landscape of genetic variation over developmental time between two isolates of C. elegans. We show that most genes vary in expression during development from egg to young adult, many genes vary in expression between the two isolates, and a subset of these genes exhibit isolate-specific changes during some developmental stages. This subset is strongly enriched for genes with roles in innate immunity. We identify several novel motifs that appear to play a role in regulating gene expression during development, and we propose functional annotations for many previously unannotated genes. These results improve our understanding of gene expression and function during worm development and lay the foundation for linkage studies of the genetic basis of developmental variation in gene expression in this important model organism.