Investigation of the Role of Electrostatic Charge in Activation of the Escherichia coli Response Regulator CheY

Signal transduction in prokaryotes is frequently accomplished by means of two-component regulatory systems (reviewed in reference 42). In a typical manifestation of this scheme, the detection of environmental stimuli by the unique external portion of a transmembrane sensor kinase regulates the autophosphorylation of the conserved internal portion. Transfer of phosphoryl groups from a His residue in the sensor kinase to an Asp residue in the conserved regulatory domain of a response regulator protein regulates the output function of the response regulator (e.g., DNA binding). Electrostatic charge has long been suspected to play an important role in the activation of response regulators. The highly conserved features of a typical response regulator active site include three negatively charged Asp residues and one positively charged Lys residue (43). The three acidic residues coordinate a divalent cation, typically Mg2+, which is required for phosphorylation and dephosphorylation of the response regulator (28). The usual mechanism of reversible functional activation involves the introduction of a negatively charged phosphoryl group (—PO32−) into the active site, resulting in the repositioning of various charged moieties. In particular, the phosphorus atom of the phosphoryl group forms a covalent bond with a carboxylate oxygen of one of the conserved Asp residues, and the three oxygen atoms of the phosphoryl group interact noncovalently with the divalent cation, with the conserved Lys residue, and with a highly conserved Ser or Thr residue (5, 10, 24, 25). Alternatively, many response regulators can be partially and irreversibly activated in a sensor kinase-independent manner by mutations that cause various charge changes in the vicinity of the active site (7, 15, 21, 23, 30, 40). The active but nonphosphorylated mutant proteins presumably mimic key features of the charge distribution in their phosphorylated wild-type counterparts. One of the best-characterized response regulators is Escherichia coli CheY. The phosphorylation state of CheY determines the direction of rotation of the flagellar motors and hence the swimming behavior of the cell. Phosphorylated CheY (CheY-P) results in clockwise (CW) flagellar rotation, whereas counterclockwise (CCW) flagellar rotation occurs in the absence of CheY-P. Chemotaxis depends on coordinating rapid activation and inactivation of CheY in response to changes in external chemical concentrations experienced by the cell as it swims through its environment. Replacement of conserved Asp13 with Arg or Lys but no other amino acids among 15 tested resulted in phosphorylation-independent activation of CheY, implying that a positive charge is the critical characteristic leading to the activation of CheY13DK and CheY13DR (7, 8, 32). The phosphorylation-mediated mechanism of activation of a wild-type response regulator outlined above does not offer an obvious explanation for the constitutive activities of these two mutant proteins. There is evidence that the mechanism of response regulator activation tolerates some variations in the positions of charged moieties, increasing the likelihood that activated structures may be attained by mutation. In particular, (i) replacement of Asp13 with either Arg or Lys activates CheY (7, 8), (ii) mutant FixJ (31) and CheY (2) response regulators lacking the primary Asp phosphorylation site are partially activated by phosphorylation at an adjacent Ser residue, (iii) an engineered —CH2-S-CH2-PO32− side chain at position 57 (cf. —CH2-CO-O-PO32− in wild-type CheY-P) partially activates CheY (16), and (iv) replacement of the aspartyl phosphate side chain with —CH2-S-S-PO32− activates the CheB response regulator (33) but not CheY (36). In order to (i) explore more fully the role of electrostatic charge in response regulator activation, (ii) gain insight into the mechanism underlying the activities of constitutive CheY13DK and CheY13DR mutants, and (iii) identify additional constitutively active CheY mutants, we undertook a large-scale mutagenesis to perturb the distribution of electrostatic charge in the vicinity of the CheY active site. Using the X-ray crystal structure of CheY · Mg2+ (41), we identified the surface residues closest to the site of phosphorylation (Asp57), arbitrarily truncating the list at 13 residues, which represents ∼10% of the amino acids in CheY. Val11, Ser56, and Met85 were excluded from consideration due to their location in the hydrophobic core of CheY, and Gly65 was not analyzed because it has no side chain to participate in structural rearrangements. Each of the chosen 13 residues was then changed to a charged amino acid (Asp, Glu, Arg, His, and Lys), and the phenotypes of the resulting CheY mutants were evaluated.