Probing the Roles of the Two Different Dimers Mediated by the Receiver Domain of the Response Regulator PhoB

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Abstract

Structural analysis of the Escherichia coli response regulator transcription factor PhoB indicates that the protein dimerizes in two different orientations that are both mediated by the receiver domain. The two dimers exhibit 2-fold rotational symmetry: one involves the α4-β5-α5 surface and the other involves the α1/α5 surface. The α4-β5-α5 dimer is observed when the protein is crystallized in the presence of the phosphoryl analog BeF3-, while the α1/α5 dimer is observed in its absence. From these studies, a model of the inactive and active states of PhoB has been proposed that involves the formation of two distinct dimers. In order to gain further insight into the roles of these dimers, we have engineered a series of mutations in PhoB intended to perturb each of them selectively. Our results indicate that perturbation of the α4-β5-α5 surface disrupts phosphorylation-dependent dimerization and DNA binding as well as PhoB-mediated transcriptional activation of phoA, while perturbations to the α1/α5 surface do not. Furthermore, experiments with a GCN4 leucine zipper/PhoB chimera protein indicate that PhoB is activated through an intermolecular mechanism. Together, these results support a model of activation of PhoB in which phosphorylation promotes dimerization via the α4-β5-α5 face, which enhances DNA binding and thus the ability of PhoB to regulate transcription.

Introduction

Bacteria are routinely challenged by rapidly changing environments. The ability to monitor their surroundings and alter their physiology in response to environmental changes is critical to competitiveness and survival. One strategy that bacteria employ to accomplish this adaptation is regulation via two-component signal transduction systems.1, 2, 3 Two-component systems (TCSs) abound in prokaryotes, while they are only minimally present in most eukaryotes. In fact, many eukaryotes, including all animals, have no TCS proteins encoded within their genomes. TCSs regulate a wide variety of physiological functions, including motility, metabolic function, quorum sensing, and virulence in many important human pathogens.4, 5, 6, 7 The absence of these systems from humans, and their importance in many aspects of bacterial physiology, make TCSs attractive targets for development of novel antimicrobial therapeutics.8, 9, 10, 11, 12

The typical TCS consists of two conserved proteins, a histidine protein kinase (HK) and a response regulator (RR) that function together in a phosphotransfer pathway.1, 3, 13 The HK monitors the environment for specific cues and regulates the phosphorylation level of the RR in a stimulus-dependent manner. RRs are typically multi-domain proteins consisting of a conserved receiver (REC) domain and a variable effector domain.14 The role of the REC domain is to regulate the activity of the associated effector domain in a phosphorylation-dependent manner and the role of the effector domain is to mediate the response.

REC domains are highly versatile signaling modules that are capable of regulating the activities of a diverse array of effector domains.14 Although a variety of different types of effector domains have been identified in RRs, the commonest type is a DNA-binding effector domain.14, 15 RRs with DNA-binding effector domains can be sub-classified on the basis of the specific fold of the DNA-binding domain. The most populated of these subfamilies is the OmpR/PhoB subfamily,14 named for its best known members and characterized by a winged helix-turn-helix DNA-binding domain.16, 17, 18

As is true for all RRs, the REC domain of OmpR/PhoB RRs regulates the activity of the protein by mediating distinct inter- and/or intramolecular interactions in the inactive and active states.19, 20, 21, 22, 23 Activation of RR transcription factors typically involves dimerization or higher-order oligomerization.3, 14, 20,23, 24, 25, 26, 27, 28, 29, 30 In several RRs, the REC domain has been shown to mediate the formation of more than one type of dimer. For example, the REC domain of NtrC1, a transcription factor from Aquifex aeolicus, mediates the formation of two types of symmetric dimers. Each dimer differentially influences higher-order oligomerization of the molecule, an essential component of transcriptional activation. Domain arrangements in the inactive state dimer prevent oligomerization via the AAA+ ATPase domain, while formation of the active state dimer relieves this inhibition.20 PleD, a diguanylate cyclase from Caulobacter crescentus, also forms distinct dimers in active and inactive states. Catalysis of cyclic di-GMP synthesis occurs only when the active sites of two PleD protomers are positioned in a specific orientation relative to each other, an orientation that is dependent on how the REC domains dimerize.30 The REC domain of Escherichia coli PhoB also forms two types of dimers.23, 31

PhoB is the central regulator in the phosphate assimilation pathway in E. coli. Together with the HK PhoR, PhoB regulates expression of the Pho regulon, a set of ∼ 40 genes that function in either the uptake or metabolism of a variety of different sources of phosphate.32, 33, 34, 35 Under conditions of phosphate limitation, PhoB is phosphorylated; this enhances its affinity for DNA and allows it to regulate transcription of the Pho regulon. Despite extensive previous characterization of PhoB and structural information for both isolated REC and DNA-binding domains in various states,23, 31,36, 37, 38, 39 many fundamental questions regarding the molecular mechanisms involved in activation remain unanswered.

Biochemical studies have established a correlation between activation and dimerization;24,25 however, the specific roles of the two dimers are not clear. Although the intact form of PhoB has not been crystallized, structural information is available for the isolated REC domain of PhoB in both the presence and the absence of the non-covalent phosphoryl analog BeF3-.23, 31 In the presence of BeF3-, PhoB dimerizes with 2-fold symmetry via the α4-β5-α5 face, while a symmetric dimer mediated by the α1/α5 face is observed in its absence. In these two dimers, Thr83 and Tyr102, which undergo a conserved repositioning upon activation,3, 40 are in rotameric conformations associated with the active state in the α4-β5-α5 dimer,23 and in rotameric conformations associated with the inactive state in the α1/α5 dimer.31 This, coupled with the fact that the α4-β5-α5 dimer forms only in the presence of the phosphoryl analog BeF3-,23 suggests that the α4-β5-α5 dimer is the active state dimer. However, structural studies of two constitutively active PhoB mutant proteins have been interpreted as evidence that the α1/α5 dimer is the active state dimer.39

To ascribe activities to the two dimers and to gain further understanding of the role of dimerization in activation of PhoB, we have characterized proteins with single- and double-site substitutions in several of the residues that stabilize the two different dimers. Perturbation of the α4-β5-α5 surface disrupts phosphorylation-dependent DNA binding and the ability of PhoB to activate transcription, while perturbation of the α1/α5 surface does not. In addition, experiments carried out with a chimeric form of PhoB, in which the REC domain was replaced by a leucine zipper dimerization domain, suggest that dimerization alone is sufficient to activate the protein for transcriptional regulation. These results are consistent with the model of the active state proposed on the basis of structural analyses,22, 23 and suggest that dimerization is a critical feature for activation of PhoB and presumably for all members of the OmpR/PhoB subfamily.

Section snippets

Rationale for mutagenesis

Specific mutations were made to PhoB with the goal of separately disrupting either the α4-β5-α5 dimer (Fig. 1a) or the α1/α5 dimer (Fig. 1b). The α4-β5-α5 dimer of PhoB is stabilized by a few hydrophobic interactions and an extensive network of salt bridges (Fig. 1c). Residues involved in these interactions are highly and exclusively conserved within the OmpR/PhoB subfamily28 and similar interfaces are observed in the structures of all other OmpR/PhoB family members crystallized in an active

Amino acid substitutions to the α4-β5-α5 surface disrupt PhoB activation

To gain a more complete understanding of the roles of the two dimers in the activity of PhoB, we engineered and characterized PhoB proteins containing a set of amino acid substitutions intended to selectively perturb each of the two dimer interfaces. Our data indicate that inactive unphosphorylated PhoB exists primarily as a monomer in equilibrium with a small amount of α1/α5 dimer and that active phosphorylated PhoB exists primarily as an α4-β5-α5 dimer. Both in vitro and in vivo analysis

Mutations at the α4-β5-α5 dimer interface have different impacts on transcriptional activation

In addition to targeting the D101–R115 salt bridge, mutations were made to the residues that form the other three salt bridges that stabilize the α4-β5-α5 dimer: R91–E111, E96–K117, and D100–R122. In each case, the transcriptional activity of the compensatory double-residue mutant protein was greater than that of either protein containing a single-residue substitution affecting the same salt bridge. These results are consistent with our model. However, the relative activity of these mutants

Cloning and molecular biology

For transcriptional analysis, the various PhoB constructs were cloned into the low copy number plasmid pMLB1120.215 with protein expression driven by the lac promoter. For purification, the T7 expression systems encoded in either pJES30764 or pET-21b (Novagen, Madison, WI) were used. Native proteins were used for transcription analysis; however, for purification, PhoBF20D and PhoBR91E/E111R were expressed with a His6 tag and a thrombin cleavage site. Plasmids used for expression of the various

Acknowledgements

We thank J. Gao and T. Wu for assistance in plasmid construction and protein purification, and J. Guhaniyogi and Y. Tao for synthesis of phosphoramidate. This work was supported by a grant from the National Institutes of Health (R37GM047958). A.M.S. is an investigator of the Howard Hughes Medical Institute.

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