Dynamic map of protein interactions in the Escherichia coli chemotaxis pathway

Protein–protein interactions play key roles in virtually all cellular processes, often forming complex regulatory networks. A powerful tool to study interactions in vivo is fluorescence resonance energy transfer (FRET), which is based on the distance‐dependent energy transfer from an excited donor to an acceptor fluorophore. Here, we used FRET to systematically map all protein interactions in the chemotaxis signaling pathway in Escherichia coli, one of the most studied models of signal transduction, and to determine stimulation‐induced changes in the pathway. Our FRET analysis identified 19 positive FRET pairs out of the 28 possible protein combinations, with 9 pairs being responsive to chemotactic stimulation. Six stimulation‐dependent and five stimulation‐independent interactions were direct, whereas other interactions were apparently mediated by scaffolding proteins. Characterization of stimulation‐induced responses revealed an additional regulation through activity dependence of interactions involving the adaptation enzyme CheB, and showed complex rearrangement of chemosensory receptors. Our study illustrates how FRET can be efficiently employed to study dynamic protein networks in vivo. The chemotaxis system controlling the swimming behavior of the bacterium Escherichia coli is one of best‐studied model signaling pathways and has been used to illustrate many fundamental principles of biological signal processing (Sourjik, 2004; Wadhams and Armitage, 2004). The pathway regulates flagellar rotation dependent on chemotactic stimuli and consists of five ligand‐specific membrane‐associated receptors and six cytoplasmic proteins. Excitation signaling is mediated by histidine kinase CheA, coupling protein CheW and response regulator CheY, whereas CheY phosphatase CheZ plays the role in signal termination, and receptor methyltransferase CheR and methylesterase CheB constitute a simple adaptation system. Despite its simplicity, the pathway shows large signal amplification (Li and Weis, 2000; Gestwicki and Kiessling, 2002; Sourjik and Berg, 2002b, 2004; Lai et al, 2005) and impressive robustness against such perturbations as gene expression noise (Alon et al, 1999; Kollmann et al, 2005). Fluorescence resonance energy transfer (FRET), which is based on the distance‐dependent energy transfer from an excited donor to an acceptor fluorophore, allows the detection of intracellular interactions of fluorescently labeled proteins non‐invasively and with high selectivity (Wouters and Bastiaens, 2001). It was recently used to study stimulation dependence of several interactions in the chemotaxis pathway, and to investigate signal processing by receptor–kinase complexes (Sourjik and Berg, 2002a, 2002b; Vaknin and Berg, 2006, 2007; Sourjik et al, 2007). In the current study, we extend FRET‐based interaction mapping to the entire chemotaxis pathway, using the library of fusions to cyan and yellow fluorescent proteins (CFP and YFP, respectively) as donor–acceptor pairs. To reduce the chances of false negatives due to the large distance or unfavorable orientation of proteins in the complex and to find pairs with strongest FRET efficiency for subsequent investigation of stimulation dependence, the library contains both N‐ and C‐terminal fusions of CFP and YFP to all chemotaxis proteins, the aspartate receptor Tar and the serine receptor Tsr. Most fusions are functional and show the expected localization in the cell. Initial FRET mapping of protein interactions in our assay is made by acceptor photobleaching (Figure 2), whereby acceptor (YFP) is bleached by short high‐intensity laser illumination. After the identification of positive FRET pairs, the dependence of these interactions on chemotactic stimulation is examined using a flow assay (Figure 2B). This analysis enables us to picture the entire network of protein interaction in the chemotaxis pathway, with 19 positive FRET pairs being identified out of the 28 possible protein combinations (Figure 2C). Most positive pairs interact even in the absence of all other chemotaxis proteins (Figure 2C, solid lines), whereas interactions of other pairs depend on a common binding partner (Figure 2C, dotted and dashed lines). Nine FRET pairs are responsive to chemotactic stimulation, five of which clearly correspond to direct protein interactions (Figure 2C, black circles). Although stimulation dependence of most pairs could be expected based on existing biochemical data (Li et al, 1995; McEvoy et al, 1999) or FRET experiments (Sourjik and Berg, 2002a, 2002b; Vaknin and Berg, 2006), the observed attractant‐induced decrease in affinity between the adaptation enzyme CheB and both of its interaction partners, receptors and CheA, was novel. Such dependence implies an additional enhancement of a negative feedback from the level of kinase activity to that of receptor activity, which is provided by CheB phosphorylation. This feedback is essential to maintain robust output of the chemotaxis system under such perturbations as gene expression noise (Kollmann et al, 2005), and a recent computational analysis suggested that strong binding of phosphorylated CheB to receptors and weak binding of phosphorylated CheY to CheA are important for robust adaptation in chemotaxis (Matsuzaki et al, 2007). Equally important for the overall picture of the chemotaxis pathway is the observation that many pathway's interactions are not stimulation dependent. This allows us to rule out many potential regulation mechanisms, such as activity‐dependent CheZ oligomerization (Blat and Eisenbach, 1996) or large conformational changes in the sensory complexes. We further demonstrate differences in concentration dependence and kinetics of response to stimulation at different levels of signal processing. Upon a step‐like attractant stimulation, changes in kinase activity—reflected by the level of phosphorylation‐dependent FRET between CheY and CheZ—show fastest kinetics, followed by rearrangement of receptors in the sensory complexes, and then by changes in CheB binding to receptors and CheA. Receptor rearrangement shows different dependence on the stimulus strength than the downstream kinase regulation, confirming previous observations (Vaknin and Berg, 2007) and suggesting that amplification of chemotactic signals takes place among signaling domains of interacting receptors. Moreover, both dose–response and kinetics measurements reveal two distinct stimulation‐induced movements or rearrangements of receptors. Our study provides a holistic picture of chemotactic signaling in E. coli and demonstrates that FRET can be successfully used to systematically map and quantify all intracellular protein interactions in a signaling network, including transient interactions, with no false‐positives and nearly no false negatives. A strong advantage of FRET is that it enables systematic mapping of activity dependence of interactions and measuring response kinetics, and therefore yields a dynamic picture of the network. In addition to direct interactions, protein proximities—mediated by association with a common interaction partner—can be identified, facilitating the characterization of multiprotein complexes and their dynamics in vivo. FRET‐based interaction mapping thus stands as a simple and reliable means for the investigation of other protein networks in bacteria or eukaryotes.