Structural Analysis of the Domain Interface in DrrB, a Response Regulator of the OmpR/PhoB Subfamily

Abstract

The N-terminal regulatory domains of bacterial response regulator proteins catalyze phosphoryl transfer and function as phosphorylation-dependent regulatory switches to control the output activities of C-terminal effector domains. Structures of numerous isolated regulatory and effector domains have been determined. However, a detailed understanding of regulatory interactions among these domains has been limited by the relative paucity of structural data for intact multidomain response regulator proteins. The first multidomain structures determined, those of transcription factor NarL and methylesterase CheB, both revealed extensive interdomain interfaces. The regulatory domains obstruct access to the functional sites of the effector domains, indicating a regulatory mechanism based on inhibition. In contrast, the recently determined structure of the OmpR/PhoB homologue DrrD revealed no significant interdomain interface, suggesting that the domains are tethered by a flexible linker and lack a fixed orientation relative to each other. To address the generality of this feature, we have determined the 1.8-Å resolution crystal structure of Thermotoga maritima DrrB, providing a second structure of a multidomain response regulator of the OmpR/PhoB subfamily. The structure reveals an extensive domain interface of 751 Å 2 and therefore differs greatly from that observed in DrrD. Residues that are crucial players in defining the activation state of the regulatory domain contribute to this interface, implying that conformational changes associated with phosphorylation will influence these intramolecular contacts. The DrrB and DrrD structures are suggestive of different signaling mechanisms, with intramolecular communication between N-and C-terminal domains making substantially different contributions to effector domain regulation in individual members of the OmpR/PhoB family.The use of modular phosphorylation-regulated switch domains to control effector protein activity is a prevalent theme in both eukaryotic and prokaryotic signal transduction pathways. In eukaryotes, the modular switch is often a small GTPase of the ras superfamily, in which a single phosphate moiety of a bound guanine nucleotide (GTP versus GDP) dictates the activation state of the protein and its intermolecular associations with downstream targets (for a review, see reference 43). In prokaryotes, the switch is typically the regulatory domain of a multidomain response regulator protein that is paired with a histidine protein kinase within a twocomponent signaling system (for reviews, see references 34 and 45). Phosphorylation of a specific aspartyl residue in the regulatory domain of the response regulator protein results in activation of an attached effector domain and generation of the output response of the signaling pathway. In both families of switch proteins, phosphorylation-induced conformational changes alter molecular surfaces that are exploited for proteinprotein interactions specific to the unphosphorylated or phosphorylated states. This provid...

Structure of the DNA-Binding Domain of the Response Regulator PhoP from Mycobacterium tuberculosis,

“…The N -terminal β-sheet is proposed to play a role in interacting with the N-terminal domain, thus playing an important role in transmitting the signal of phosphorylation to the C-terminal domain for regulation of the DNA-binding activity (24). In the structure of PhoPC, the N-terminal β-sheet forms the hexamer interface by interacting with the N-terminal end of helix α6, the transactivation loop, and one side of α8 of the upstream molecule.…”

“…However, phosphorylation of PhoB from E. coli induces dimerization (22) and increases its affinity for DNA (23); AcrA from E. coli also dimerizes upon phosphorylation (17). Crystal structures of full-length DrrB (24) and DrrD (25) from Thermotoga maritima indicate that in the unphosphorylated state the DNA recognition helix is freely exposed to the solvent, which would make it readily available for DNA binding. However, in the structure of PrrA from MTB, the recognition helix is involved in interactions with the regulatory domain, although in solution there may be an equilibrium with an open form (26).…”

Structural Analysis and Solution Studies of the Activated Regulatory Domain of the Response Regulator ArcA: A Symmetric Dimer Mediated by the α4-β5-α5 Face

“…This has been supported by subsequent biochemical and biophysical studies. [29][30][31][32] In contrast, the crystal structures of two full-length RRs of the OmpR/PhoB subfamily from Thermotoga maritima, DrrD 33 and DrrB, 34 showed their recognition helices to be completely exposed, making them readily available for DNA binding. The structural data, together with several biochemical studies that point to the significance of dimerization for members of the OmpR/PhoB subfamily, [34][35][36][37][38] are suggestive of a different mechanism for transcriptional regulation.…”

“…Interestingly, in the NtrC/ DctD subfamily, which lacks conservation in the α4-β5-α5 face, different members have been shown to use distinctly different surfaces of the regulatory domain for multimerization. 61 Another notable feature of the OmpR/PhoB subfamily is that in the inactive state the DNA recognition helix of the effector domain is freely exposed to the solvent 33,34 making it readily available for DNA binding in contrast to other RRs in which the functional regions of the effector domains are occluded by the unphosphorylated regulatory domains. 27,28 Taking into account these marked differences, it is hypothesized that members of the OmpR/PhoB subfamily use a common mechanism of regulation by dimerization.…”

“…A multiple sequence alignment of the regulatory domains of all members of this subfamily from E. coli, 20 in addition to subfamily members from other organisms for which structures of the regulatory domain are available, 33,34,49,50 revealed that the residues involved in key dimer interface interactions are highly conserved within this subfamily (Fig. 5).…”