Variable active site positions ‘59’ and ‘89’ strongly influence autodephosphorylation rate
During signal transduction, response regulators generally switch from the active to inactive state via dephosphorylation. It is critical that the kinetics of this conversion match the timescale of the biological process involved. In spite of extensive structural and functional similarities, response regulators exhibit a range of more than 104-fold in autodephosphorylation rates (Table 1). We found that two variable residues, located in close spatial proximity to the phosphoryl group (Fig. 1), contribute substantially to this reaction rate. Twenty different pairs of amino acids at positions ‘59’ and ‘89’ tested in the CheY and/or Spo0F response regulators resulted in sets of proteins spanning ∼100-fold ranges of autodephosphorylation rates for each response regulator (Tables 2 and 3). Although the 20 different pairs examined here comprise only 5% of the 400 mathematically possible combinations, amino acids do not occur at equal frequencies at positions ‘59’ and ‘89’ in response regulators. The tested combinations actually occur in 24% of the entries in a database of 6310 non-redundant response regulators (K. Wuichet and I. Zhulin, pers. comm.), and therefore reflect a significant fraction of the ‘59’/‘89’ pairs found in nature. Substitutions tested in CheY, which has a relatively fast autodephosphorylation rate, resulted in modest rate increases or larger rate decreases. Many tested substitutions in Spo0F, which has a relatively slow autodephosphorylation rate, resulted in considerable rate enhancements. No substitutions that retard Spo0F autodephosphorylation were observed among the limited test set, although response regulators with slower autodephosphorylation rates do exist (Table 1).
Location of β4/α4 loop explains the non-interactive tendencies of positions ‘59’ and ‘89’ observed in Spo0F mutants
Comparison of autodephosphorylation rates in proteins bearing single or double amino acid substitutions suggest that CheY positions 59 and 89 influence one another whereas Spo0F positions ‘59’ and ‘89’ do not (Table 4). The interactive effects (or lack thereof) between positions ‘59’ and ‘89’ on autodephosphorylation rates may arise from differences in the distances between the ‘59’ and ‘89’ side-chains in CheY and Spo0F. Structures of CheY and several other response regulators in the phosphorylated (or BeF3- bound) versus unphosphorylated state show a substantial (> 4 Å) movement of the β4/α4 loop, which contains position ‘89’, toward the active site. The shift is accompanied by a change in the ‘89’ side-chain from an outward to an inward orientation, thereby considerably decreasing the distance between residues ‘59’ and ‘89’ (Birck et al., 1999; Lee et al., 2001; Hastings et al., 2003; Bachhawat et al., 2005). Although the side-chains at positions 59 and 89 are ∼5 Å apart in wild-type CheY·BeF3- (Lee et al., 2001), in CheY 59NR·BeF3- a salt bridge exists between Arg59 and Glu89 (Silversmith et al., 2003), which suggests that, at least for CheY derivatives, the amino acids present at positions 59 and 89 determines whether an interaction occurs. Similarly, in the PhoB·BeF3- (Bachhawat et al., 2005), ArcA·BeF3- (Toro-Roman et al., 2005), Spo0A·PO32− (Lewis et al., 1999), DctD·BeF3- (Park et al., 2002) and FixJ·PO32− (Birck et al., 1999) structures, the side-chains of positions ‘59’ and ‘89’ are in close proximity (< 4 Å) (Fig. 3), suggesting that interactions between these two positions commonly occur in phosphorylated receiver domains. In contrast, the β4/α4 loop is in a different position in Spo0F·BeF3- structures (Gardino et al., 2003; Varughese et al., 2006), thereby maintaining the outward orientation of residue ‘89’, which in turn results in a separation of > 8 Å between positions ‘59’ and ‘89’ (Fig. 3). The ability of position ‘89’ to influence Spo0F autodephosphorylation rate in spite of its distance from the phosphoryl group suggests an indirect interaction. One possibility is that position ‘89’ may alter the mobility of the β4/α4 loop in Spo0F (Feher and Cavanagh, 1999) and thereby affect the location of other side-chains such as Glu‘91’, which projects into the same space as position ‘89’ of CheY. The potential role of position ‘91’ in Spo0F autodephosphorylation has not yet been investigated.
Figure 3. Orientations of residues at positions ‘59’ and ‘89’ in various activated receiver domain structures. Sections of superimposed backbones are displayed for (A) CheY·BeF3- (blue), Spo0F·BeF3- (green), PhoB·BeF3- (magenta), ArcA·BeF3- (grey) and Spo0A·PO32− (orange) and (B) CheY·BeF3- (blue), Spo0F·BeF3- (green), DctD·BeF3- (yellow), FixJ·PO32− (purple). For clarity, only the β1-α1, β3-α3 and β4-α4 loops surrounding the BeF3-/PO32− group (red) are shown. Side-chains are shown for position ‘59’, located at the C-terminal end of β3, and position ‘89’, on the β4-α4 loop. Note that the distance between positions ‘59’ and ‘89’ in Spo0F is greater than the ∼4 Å distance observed in other receiver domains (distances not shown). The specific software, method of superimposition, literature references and pdb files used to create this figure are described in Experimental procedures.
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Position ‘14’ does not appear to influence autodephosphorylation rate
The apparent correlation between the amino acids at position ‘14’ and autodephosphorylation rate in Table 1 made position ‘14’ a plausible candidate as an additional factor that influences response regulator phosphoryl group stability. However, the amino acid substitutions tested at position ‘14’ had little actual effect on rate (Tables 2 and 3, Table S1). This apparent discrepancy may arise from the high specificity that response regulators show toward their paired sensor kinases, with little cross-talk from other kinases (Skerker et al., 2005; Yamamoto et al., 2005). There is structural evidence suggesting that position ‘14’ is important for response regulator interaction with paired kinases. Although high-resolution structural details of the interaction between response regulators and histidine phosphorylation sites are not available for sensor kinases, presumably similar interactions with histidine phosphotransferase proteins have been described. For example, Gln‘14’ of Spo0F (Varughese et al., 2006) forms a hydrogen bond with Spo0B, a dimeric phosphotransferase whose structure resembles that of histidine kinases. Furthermore, Asn‘14’ of SLN1 (Xu et al., 2003) forms a hydrogen bond with the monomeric phosphotransferase YPD1. In all three types of proteins (dimeric sensor kinases, dimeric histidine phosphotransferases and monomeric histidine phosphotransferases), the histidine phosphorylation site is located on a four-helix bundle. If the hydrogen bond interactions observed between response regulators and phosphotransferase proteins also occur between response regulators and sensor kinases, then it may account for the prevalence of Asn, Asp, Gln, Glu and His residues at position ‘14’ in transcriptional response regulators (Table 1). On the other hand, chemotaxis sensor kinases display a distinct organization from transcriptional sensor kinases (Dutta et al., 1999; Grebe and Stock, 1999). Therefore, the different sets of amino acids observed at position ‘14’ in chemotaxis response regulators, which cluster with the fast autodephosphorylation rates, versus transcriptional response regulators, which cluster with the slow autodephosphorylation rates, may simply reflect differences in interactions with their paired sensor kinases.
Exploiting the rules of response regulator autodephosphorylation
Construction of constitutively active or dominant negative small GTPases, based on knowledge of key positions applicable to most family members, has found widespread utility in dissecting signalling networks (Campbell et al., 2005). Similarly, if there are simple rules for how variable residues control autodephosphorylation rate, then this information could be used to manipulate the phosphoryl group stability of any response regulator via site-directed mutagenesis, which might be a useful tool for the in vivo investigation of two-component signalling systems. Although the absolute magnitude of autodephosphorylation rate associated with a particular substitution depends on the context of other active site residues and the response regulator backbone, the relative impact of a specific amino acid was fairly consistent. Therefore, the experimental data reported here could be used to make preliminary predictions about the consequences of certain amino acid substitutions. Comparing the rates of mutants with ‘89’ substitutions in the context of a fixed amino acid at ‘59’ (Table S2) yields a consensus order for autodephosphorylation rate from fastest to slowest of Glu > Leu,Lys > Arg,His > Tyr and is consistent for both CheY and Spo0F even though residue ‘89’ occupies different spatial positions in the crystal structures of these two response regulators (Fig. 3). The order of residues at position ‘59’ is less evident, because all CheY single substitutions tested at this position had little effect on rate. However, by comparing the consequences of residues at ‘59’ while keeping a fixed residue at ‘89’ (Table S3), a rank of Glu > Asn > Met,Lys was revealed. Finally, there are six examples of the same ‘59’/‘89’ combinations in both CheY and Spo0F. Five of the six exhibit the same rank order in autodephosphorylation rate: ‘59’N-‘89’E, ‘59’N-‘89’L > ‘59’N-‘89’H, ‘59’N-‘89’Y > ‘59’K-‘89’Y (Table S4).
The utility of manipulating response regulator autodephosphorylation rates for in vivo studies will depend on whether alteration of positions ‘59’ and ‘89’ affects other functions of the particular response regulator that has been modified, including interaction with other proteins in the signalling network. Changing positions ‘59’ or ‘89’ appears to affect response regulator function in some cases and not in others: First, with regard to sensor kinases, positions ‘59’ and ‘89’ are hypothesized to be among the response regulator residues important for specific recognition of a paired sensor kinase (Hoch and Varughese, 2001; Li et al., 2003). We observed little or no impairment of phosphotransfer from CheA-P or KinA-P to mutant CheY or Spo0F proteins in vitro (see Experimental procedures), but our assay method was relatively insensitive to phosphotransfer kinetics. Second, with regard to output proteins, many CheY mutants with single substitutions at positions 59 or 89 can be phosphorylated and interact productively with the FliM flagellar switch protein in vivo, as inferred from ability to support clockwise flagellar rotation (Silversmith et al., 2001; 2003; data not shown). However, the five CheY mutants in this study with substitutions at both positions 59 and 89 showed predominantly counterclockwise flagellar rotation (data not shown), consistent with reduced phosphorylation and/or function. Several Spo0F mutants with substitutions at positions ‘59’ or ‘89’ efficiently exchange phosphoryl groups with the downstream protein Spo0B in vitro (Tzeng and Hoch, 1997; Zapf et al., 1998). However, Ala substitutions at position ‘59’ or ‘89’ of Spo0F prevent sporulation in vivo, a defect that has been attributed to reduced phosphorylation by KinA (Tzeng and Hoch, 1997; Zapf et al., 1998). Third, with regard to phosphatases, changing CheY Glu89 prevents CheZ-stimulated dephosphorylation, but not binding to CheZ (Silversmith et al., 2001; Silversmith et al., 2003). In Spo0F, Ala substitutions at positions ‘59’ or ‘89’ reduce RapB-mediated stimulation of dephosphorylation fivefold to 10-fold compared with wild-type (Tzeng et al., 1998). If positions ‘59’ and ‘89’ are important for phosphatase-stimulated dephosphorylation of other response regulators, then alteration of these residues could be a useful strategy to increase response regulator phosphorylation in vivo for those systems that include phosphatases. In summary, our study suggests candidates for engineering altered autodephosphorylation rates, but their practical utility will have to be evaluated on a case-by-case basis.
The findings of this study have further potential application. There has been substantial interest recently in engineering bacterial regulatory networks with particular properties, essentially creating programmable cellular behaviour in which a specific input results in a desired output (Kaern et al., 2003; Kobayashi et al., 2004; Anderson et al., 2006; Voigt, 2006). A biosensor circuit can be envisioned in which an unstable output is mediated by response regulator activation. The availability of interchangeable receiver domain modules with a range of different characteristic phosphoryl group half-lives would facilitate construction of circuits with outputs of different duration following a given stimulus. In order for autodephosphorylation to control signal decay, the circuit would not contain a separate phosphatase. Our studies suggest mutants can be found that will interact sufficiently well with a sensor kinase and output protein to create a functioning circuit.
Finally, it is simple with current technology to identify genes encoding response regulators. However, it is often difficult or impossible to ascertain through bioinformatic means the specific output that is controlled by a particular response regulator. Complete elucidation of the features that control response regulator autodephosphorylation rate may make it possible to infer the timescale (seconds, minutes or hours) of signal transduction (and hence the timescale of the controlled biological process) from response regulator amino acid sequence alone. In turn, such a predictive capability would permit exploration of the sensory capacity of an organism in relation to its ecological niche or physiological characteristics (e.g. does a slow-growing species process information at a slow rate?).
Factors other than positions ‘59’ and ‘89’ must contribute to phosphoryl group stability
Comparisons of autodephosphorylation rates between CheY or Spo0F mutant proteins created in this study and response regulators with matching amino acids at positions ‘59’ and ‘89’ showed some cases of remarkably good agreement (e.g. less than twofold rate difference between E. coli CheY 59NE-89EH and R. sphaeroides CheB1; Table S5). At the other extreme, there was a 160-fold difference between the autodephosphorylation rates of T. maritima DrrA and the matched E. coli CheY 59NM-89EK mutant. Such discrepancies suggest that the amino acid combinations at positions ‘59’ and ‘89’ do not solely account for the intrinsic dephosphorylation rate of response regulators. The data in Table 1 suggest the potential contributions of position ‘88’ are another obvious area for future investigation. However, many of the rate discrepancies noted in Table S5 exist between response regulators that are also matched at position ‘88’. Therefore, other as yet unidentified factors that influence autodephosphorylation rate must exist.
Some differences in rate may plausibly be attributed to differences in the positioning of residues ‘59’ and ‘89’ with respect to the active site. One example is His‘89’, which has been proposed to affect phosphoryl group stability by sterically inhibiting access of the nucleophilic water molecule in FixJ (Birck et al., 1999; Fig. 3B), or possibly serving as a base to activate the water molecule in NtrC (Hastings et al., 2003). Similarly, the different spatial orientations of position ‘89’ observed in CheY·BeF3- (Lee et al., 2001) and Spo0F·BeF3- (Gardino et al., 2003; Varughese et al., 2006; Fig. 3) may explain the range in rates (threefold to 22-fold) observed for the nine pairs of CheY and Spo0F proteins with matched amino acids at positions ‘59’ and ‘89’ (Table S5). In contrast, no such obvious differences in structure are observed between CheY·BeF3- (Lee et al., 2001), Arc·BeF3- (Toro-Roman et al., 2005) and PhoB·BeF3- (Bachhawat et al., 2005; Fig. 3A), yet CheY mutants autodephosphorylate over 100 times and over 10 times faster than the respective ArcA and PhoB proteins they were designed to mimic (Table S5). However, subtle differences between response regulator structures that alter the precise geometry of active site side-chains or backbone amides could conceivably affect the energetics of interactions with the phosphoryl group in the transition state and hence translate into a large effect on reaction rates. The eventual ability to predict absolute dephosphorylation rate, rather than relative rate, would enhance the potential applications described above.