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

Abstract

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...

Molecular strategies for phosphorylation-mediated regulation of response regulator activity

“…Receiver domain structures recently determined by structural genomics initiatives have substantially increased the number of structures for which physiological data are lacking. No physiological significance has yet been ascribed to the several domain swapped receiver domain dimers that have been observed [3,4], likely promoted by the high protein concentrations used in crystallization. Protein concentration can also bias conformational equilibrium by promoting dimerization.…”

“…As many current RR structures contain only the truncated receiver domains, the function and distribution of this extended α5 linker remain to be defined. In addition to the above dimer conformations, a small number of structures display a domain-swapped configuration [3,4] or a dimer interface involving regions other than the α4-β5-α5 face. These alternative interfaces, currently of unknown physiological relevance, further illustrate RR plasticity.…”

Bacterial Two-Component Systems: Structures and Signaling Mechanisms

“…OmpR/PhoB subfamily RRs have been extensively studied in recent years. In addition to many isolated receiver and effector domain structures, there are six full-length RR structures available, DrrB [47] and DrrD [48] from T. maritima, and RegX3 [49], PrrA [35], MtrA [50], and PhoP [34] from M. tuberculosis. All structures are in the unphosphorylated forms.…”

Adaptation to Environmental Stimuli within the Host: Two-Component Signal Transduction Systems of Mycobacterium tuberculosis

“…Regulation by RegX3 is direct and is initiated through recognition of a loosely conserved, inverted-repeat element present in the promoter regions of these genes (Table 3) (49). Structural analysis of RegX3 dimers supports the ability of this protein to recognize an inverted-repeat element when the protein is in an active state (80). In the presence of elevated P i levels, SenX3 acts as a RegX3 phosphatase, preventing the accumulation of phosphorylated RegX3 and subsequent activation of downstream gene targets (49).…”