A common dimerization interface in bacterial response regulators KdpE and TorR

The full-length, two-domain response regulator RegX3 from Mycobacterium tuberculosis is a dimer stabilized by three-dimensional domain swapping. Dimerization is known to occur in the OmpR/PhoB subfamily of response regulators upon activation but has previously only been structurally characterized for isolated receiver domains. The RegX3 dimer has a bipartite intermolecular interface, which buries 2357 Å 2 per monomer. The two parts of the interface are between the two receiver domains (dimerization interface) and between a composite receiver domain and the effector domain of the second molecule (interdomain interface). The structure provides support for the importance of threonine and tyrosine residues in the signal transduction mechanism. These residues occur in an active-like conformation stabilized by lanthanum ions. In solution, RegX3 exists as both a monomer and a dimer in a concentration-dependent equilibrium. The dimer in solution differs from the active form observed in the crystal, resembling instead the model of the inactive full-length response regulator PhoB.Tuberculosis is a global threat to human health, with onethird of the world's population infected with the causative agent of tuberculosis, Mycobacterium tuberculosis. With the emergence of multidrug-resistant strains of M. tuberculosis, novel therapeutic agents are urgently required. This will be best achieved by developing a better understanding of the molecular biology of this pathogen. In this context, signaling systems play an important role in the continued survival of the organism and are central to the way M. tuberculosis responds to environmental stress, especially that generated by the host immune system.The predominant signaling system for adaptive gene expression changes in bacteria is called a two-component system (TCS).3 TCS are found in eubacteria, archaea, and eukarya; however, they are rare in eukarya. As the name implies, the system typically consists of two proteins: a sensor histidine kinase (SK) and a response regulator (RR) (1). SKs are usually membrane-anchored proteins with a characteristic core consisting of a histidine-containing dimerization domain and a catalytic domain with ATPase activity. In response to extracellular conditions, and in some cases intracellular conditions, the SK autophosphorylates at a histidine residue within the dimerization domain. This autophosphorylation occurs upon dimerization through a phosphate transfer from the catalytic domain (annotated as a HATPase_c domain by the SMART server (2, 3)) of the adjacent protomer to the conserved histidine in the dimerization domain (HisKA domain). The activated SK may then function as a phosphate donor to a universally conserved aspartic acid residue in the RR. RRs typically consist of two domains, a receiver and an effector domain, although a large family of 54 activators also contain a central AAAϩ domain. The N-terminal receiver domain acts as the phosphoacceptor, whereas the C-terminal effector domain is usually a DNA-binding domain involved in the tra...

Section: Discussionsupporting

confidence: 82%

Section: Discussionmentioning

confidence: 99%

The Structure of a Full-length Response Regulator from Mycobacterium tuberculosis in a Stabilized Three-dimensional Domain-swapped, Activated State

King-Scott

Nowak

Mylonas

et al. 2007

“…This observation is probably of limited relevance here because the FixJ family binds palindromic DNA, whereas the OmpR/ PhoB family binds to (minimally) a tandem repeat of DNA (47), and dimer formation is dependent upon DNA binding. Dimers of isolated receiver domains (both activated and inactivated) with diad symmetry are observed, for example, in PhoB (48) and TorR (49), although the nature of the dimer interface differs in the two cases. In the crystals of PrrA, there are also two molecules per asymmetric unit, but the molecules pack head-to-tail with the principle interactions being between activation and effector domains of adjacent molecules.…”

Section: Resultsmentioning

confidence: 99%

“…In the crystals of PrrA, there are also two molecules per asymmetric unit, but the molecules pack head-to-tail with the principle interactions being between activation and effector domains of adjacent molecules. It has been postulated that dimerization could be part of the activation process, in part because the residues at the dimer interface observed in the structure of the receiver domains of TorR and KdpE are highly conserved in the OmpR/PhoB family (49). They are also largely conserved in PrrA; however, the small angle x-ray scattering data, either for inactivated or for activated PrrA, show no evidence of dimer formation.…”

Section: Resultsmentioning

confidence: 99%

The Structural Basis of Signal Transduction for the Response Regulator PrrA from Mycobacterium tuberculosis

Nowak

Panjikar

Konarev

et al. 2006

The structure of the two-domain response regulator PrrA from Mycobacterium tuberculosis shows a compact structure in the crystal with a well defined interdomain interface. The interface, which does not include the interdomain linker, makes the recognition helix and the trans-activation loop of the effector domain inaccessible for interaction with DNA. Part of the interface involves hydrogen-bonding interactions of a tyrosine residue in the receiver domain that is believed to be involved in signal transduction, which, if disrupted, would destabilize the interdomain interface, allowing a more extended conformation of the molecule, which would in turn allow access to the recognition helix. In solution, there is evidence for an equilibrium between compact and extended forms of the protein that is far toward the compact form when the protein is inactivated but moves toward a more extended form when activated by the cognate sensor kinase PrrB.The use of regulatory systems to sense and respond to changing environmental conditions is an intrinsic feature that enables bacteria to survive and adapt to a variety of external challenges. Two-component signaling (TCS) 2 systems are the principle mechanism used by bacteria to perform this task (1). A typical TCS consists of a sensor histidine kinase and a response regulator (RR). Histidine kinases are usually membrane-anchored proteins with a characteristic core consisting of a histidine-containing dimerization domain and a catalytic domain. In response to extracellular, and in a few cases, intracellular, conditions, the histidine kinase autophosphorylates at a histidine residue, usually in the dimerization domain, by phosphotransfer from the catalytic (ATPase) domain of the adjacent protomer. It then acts as a phosphodonor to a universally conserved aspartic acid residue in the response regulator. RRs typically consist of two domains with the N-terminal (receiver) domain being the phosphoacceptor domain and the C-terminal domain being the effector. The effector domain is, in most cases, DNA binding and is involved in transcriptional regulation of the genes necessary to respond to the sensed environment. Phosphorylation of the aspartic acid residue located in the receiver domain activates the effector domain in a manner that is still incompletely understood, not least because there is little structural information on full-length response regulators and no structural information on activated full-length (i.e. multidomain) response regulators.TCSs have been identified as potential antibacterial targets because they play a key role in controlling cellular processes (2). The lack of these systems in higher eukaryotes makes them potentially selective and unique antibacterial drug targets, There is, however, little biochemical information available on the TCSs of pathogenic bacteria such as Mycobacterium tuberculosis (MtB). The H37Rv strain of MtB has 12 putative TCSs including the recently discovered Rv3220-Rv1626 pair (3) as well as five putative orphan response regulator and se...

“…However, in vitro phosphorylation experiments with PleD resulted in an exiguous increase of DGC activity, possibly due to suboptimal assay conditions or to low stability of the phosphorylated form (4). For this reason we tested activation of PleD by beryllium fluoride (BeF 3 ) a molecular mimic of a phosphoryl group that has been widely used for biochemical and structural studies of bacterial response regulators (12)(13)(14)(15)(16)(17)(18). As shown in Fig.…”

Section: Activation Of the Pled Diguanylate Cyclase By Berylliummentioning

confidence: 99%

Activation of the Diguanylate Cyclase PleD by Phosphorylation-mediated Dimerization

Paul

Abel

Wassmann

et al. 2007

172

161

Diguanylate cyclases (DGCs) are key enzymes of second messenger signaling in bacteria. Their activity is responsible for the condensation of two GTP molecules into the signaling compound cyclic di-GMP. Despite their importance and abundance in bacteria, catalytic and regulatory mechanisms of this class of enzymes are poorly understood. In particular, it is not clear if oligomerization is required for catalysis and if it represents a level for activity control. To address this question we perform in vitro and in vivo analysis of the Caulobacter crescentus diguanylate cyclase PleD. PleD is a member of the response regulator family with two N-terminal receiver domains and a C-terminal diguanylate cyclase output domain. PleD is activated by phosphorylation but the structural changes inflicted upon activation of PleD are unknown. We show that PleD can be specifically activated by beryllium fluoride in vitro, resulting in dimerization and c-di-GMP synthesis. Cross-linking and fractionation experiments demonstrated that the DGC activity of PleD is contained entirely within the dimer fraction, confirming that the dimer represents the enzymatically active state of PleD. In contrast to the catalytic activity, allosteric feedback regulation of PleD is not affected by the activation status of the protein, indicating that activation by dimerization and product inhibition represent independent layers of DGC control. Finally, we present evidence that dimerization also serves to sequester activated PleD to the differentiating Caulobacter cell pole, implicating protein oligomerization in spatial control and providing a molecular explanation for the coupling of PleD activation and subcellular localization.