Cells have evolved extraordinarily complex and sophisticated signalling mechanisms to monitor and adapt to their environments. The capability of processing information from diverse internal and external cues is crucial to the fitness and survival of cells. As for numerous different signalling pathways, it is a common theme that certain domains with similar core structures and conserved functions, such as phosphorylation, methylation and protein recognition, have been repeatedly exploited as the building blocks for signalling proteins. The modular design of these signalling protein families allows the coupling of arrays of different input and output domains with the central conserved core function to form distinct and versatile pathways responding to a wide variety of signals. Conversely, as the sequence and structural similarity of the conserved core domains may be advantageous for interactions and communications between different signalling pathways, it also creates significant challenges to maintain the signalling specificity and fidelity among large numbers of similar signalling proteins within the same family. Thus, it becomes increasingly important to understand the interactions and specificity within a single family, which could lead to insights regarding the evolution of structure–function relationships of these signalling proteins as well as the discovery of novel cross-regulation between different pathways.
Two-component signal transduction is one of the most prevalent signalling schemes in bacteria, participating in various cell regulatory tasks such as chemotaxis, nutrition utilization, virulence, quorum sensing and cell cycle regulation. Typical two-component systems consist of two major families of signalling proteins, sensor histidine protein kinases (HKs) and response regulators (RRs) (see reviews by Stock et al., 2000
; Gao et al., 2007
). HKs share a kinase domain that catalyses autophosphorylation at a conserved histidine residue, while the conserved receiver domain of RRs catalyses transfer of a phosphoryl group from the phosphoHis of the HK to one of its own aspartate residues (). Signal perception by the sensing domain of the HK regulates the kinase activity and, in some bifuncitional HKs, the phosphatase activity as well, to mediate the phosphorylation level of its cognate RR. The phosphorylated RR functions as the ultimate control element that modulates the activity of its effector domain and elicits the particular response. The sensing domain of HKs and the effector domain of RRs show great diversity, which allows the utilization of the same His–Asp phosphotransfer scheme in distinct HK–RR pairs to couple diverse input stimuli, such as nutrients, redox state, osmolarity and antibiotics, to an equally diverse range of output responses, most frequently through transcriptional regulation, but also by mediation of protein–protein interactions and enzyme activities.
Two-component signal transduction and the OmpR/PhoB subfamily of response regulators.
Most sequenced bacterial genomes encode multiple two-component proteins, with the number positively correlating with the genome size (Galperin, 2005
; Ulrich and Zhulin, 2007
). There are 30 HKs and 32 RRs in Escherichia coli
, and the total number of two-component proteins exceeds 200 in Myxococcus xanthus
and some cyanobacteria species. Both lineage-specific expansion and horizontal gene transfer are thought to contribute to the evolution of diverse two-component pathways that allow bacteria to adapt to complex environments (Alm et al., 2006
). The presence of many paralogous HK/RR proteins in the same cell requires individual pathways to be insulated from one another to ensure signal transmission fidelity and avoid detrimental cross-talk despite their highly similar sequences and structures. This is especially demanding for the OmpR/PhoB subfamily of RRs; they share not only similar structures for individual domains but also a common active dimer state that is believed to be conserved within the subfamily (Bachhawat et al., 2005
; Toro-Roman et al., 2005a
Response regulators are classified into different subfamilies according to their effector domains. The OmpR/PhoB subfamily of RRs is the largest subfamily, characterized by a winged helix–turn–helix effector domain for DNA binding. They account for ~30% of all RRs and half of the RRs possessing a DNA-binding domain (Galperin, 2006
). Fourteen out of 32 RRs in E. coli
belong to this subfamily. OmpR and PhoB, the eponymous members of this subfamily, are well-studied RRs responsible for osmoregulation and phosphate assimilation in E. coli
respectively (Pratt and Silhavy, 1995
; Wanner, 1996
). The RR receiver domain is an α/β domain with a conserved activation mechanism in which phosphorylation of the aspartate residue allosterically affects a distant surface, primarily the α4-β5-α5 face, to mediate inter- or intra-protein interactions. The sequence of the α4-β5-α5 region is highly conserved within the OmpR/PhoB subfamily with a ~60% sequence identity compared with the 20–30% identity typically observed over the entire length of RRs. This represents a significant difference that distinguishes the OmpR/PhoB subfamily from other subfamilies, such as the NtrC/DctD and the NarL/FixJ subfamilies. Structural characterization of a few RRs from the OmpR/PhoB subfamily reveals that the α4-β5-α5 face is the dimerization interface () with a common set of hydrophobic and charged residues involved in van der Waals contact and salt bridges () (Toro-Roman et al., 2005a
The sequence conservation of the contacting residues suggests a similar dimerization interface for most OmpR/PhoB subfamily members. Given the prevalence of this subfamily in bacteria, it is important to question the specificity of the dimerizing interactions among subfamily members and whether RR heterodimers can form. While unproductive RR heterodimers could be problematic for the fidelity of signal transmission, particular heterodimer pairs might be valuable to the integration of different pathways via transcription of a distinct set of heterodimer-regulated genes for a co-ordinated response to complex environmental conditions. In contrast to eukaryotic signalling pathways in which the regulatory roles of both homodimers and heterodimers are well documented, e.g. the receptor tyrosine kinase family and the nuclear hormone receptor superfamily (Schlessinger, 2000
; Aranda and Pascual, 2001
; de Lera et al., 2007
), RR heterodimers are rarely reported (Knutsen et al., 2004
) and the dimerization specificity of RRs is uncharacterized. Here we report development of a strategy to investigate RR dimerization via measuring the Förster resonance energy transfer (FRET) between cyan fluorescent protein (CFP)- and yellow fluorescent protein (YFP)-fused RRs in vitro
. The FRET method allowed us to systematically analyse all possible protein–protein interactions among 14 OmpR/PhoB subfamily members from E. coli
. We demonstrate that these RRs show significant dimerization specificity in spite of the conserved residues at the dimerization interface. Further, interactions between several different RRs do occur, suggesting potential cross-regulation between some two-component pathways.