Mycobacterium tuberculosis is thought to undergo transformation into its non-replicating persistence state under the influence of hypoxia or nitric oxide (NO). This transformation is thought to be mediated via two sensor histidine kinases, DosS and DosT, each of which contains two GAF domains that are responsible for detecting oxygen tension. In this study we determined the crystal structures of the first GAF domain (GAF-A) of DosS, which shows an interaction with a heme. A b-type heme was embedded in a hydrophobic cavity of the GAF-A domain and was roughly perpendicular to the -sheet of the GAF domain. The heme iron was liganded by His-149 at the proximal heme axial position. The iron, in the oxidized form, was sixcoordinated with a water molecule at the distal position. Upon reduction, the iron, in ferrous form, was five-coordinated, and when the GAF domain was exposed to atmospheric O 2 , the ferrous form was oxidized to generate the Met form rather than a ferrous O 2 -bound form. Because the heme is isolated inside the GAF domain, its accessibility is restricted. However, a defined hydrogen bond network found at the heme site could accelerate the electron transferability and would explain why DosS was unable to bind O 2 . Flavin nucleotides were shown to reduce the heme iron of DosS while NADH was unable to do so. These results suggest that DosS is a redox sensor and detects hypoxic conditions by its reduction.Mycobacterium tuberculosis is still one of the most dreaded pathogens in existence, and one of the reasons for its success as a pathogen lies in its ability to persist for years within its host. One-third of the world population is estimated to carry M. tuberculosis in the dormant form (1), and while in this state, the pathogen is insensitive to most available chemotherapy. M. tuberculosis has been shown to undergo a metabolic transformation to its non-replicating persistence state under the influence of environmental stimuli such as hypoxia or nitric oxide (2). Recent studies have implied that CO is also an environmental trigger of mycobacterial persistence (3, 4).A two-component regulatory system mediates the genetic response to oxygen limitation and NO exposure in M. tuberculosis (5). The regulatory system consists of two sensor proteins, DosS and DosT and the cognate response regulator DosR (6, 7). DosS and DosR are also known as DevS and DevR (6). DosS and DosT are histidine kinases that undergo autophosphorylation in response to an environmental change, and subsequently transduce the signal to DosR (a transcriptional regulator). DosS and DosT each contain two GAF domains at the N-terminal sensory domain and an HATPase (histidine kinase-like ATPase) domain at its C terminus (8). Hypoxia sensing by DosS or DosT is presumably carried out through the GAF domains.GAF domains are small molecule binding domains found in many proteins from various organisms and are known to play important roles as regulatory elements. Many GAF-containing proteins have two GAF domains in tandem and the two domains have s...
Background: GST domains have been found in diverse proteins involved in translational systems. Results: Four GST domains from human methionyl-tRNA synthetase, glutaminyl-prolyl-tRNA synthetase, ARS-interacting multifunctional protein (AIMP) 2, and AIMP3 are complexed in an ordered fashion. Conclusion: Four components in the human multisynthetase complex are assembled through a GST domain tetrameric complex. Significance: GST domain assemblies act as scaffolds for the formation of multicomponent protein complexes.
The DevS histidine kinase of Mycobacterium smegmatis contains tandem GAF domains (GAF-A and GAF-B) in its N-terminal sensory domain. The heme iron of DevS is in the ferrous state when purified and is resistant to autooxidation from a ferrous to a ferric state in the presence of O 2 . The redox property of the heme and the results of sequence comparison analysis indicate that DevS of M. smegmatis is more closely related to DosT of Mycobacterium tuberculosis than DevS of M. tuberculosis. The binding of O 2 to the deoxyferrous heme led to a decrease in the autokinase activity of DevS, whereas NO binding did not. The regulation of DevS autokinase activity in response to O 2 and NO was not observed in the DevS derivatives lacking its heme, indicating that the ligand-binding state of the heme plays an important role in the regulation of DevS kinase activity. The redox state of the quinone/quinol pool of the respiratory electron transport chain appears not to be implicated in the regulation of DevS activity. Neither cyclic GMP (cGMP) nor cAMP affected DevS autokinase activity, excluding the possibility that the cyclic nucleotides serve as the effector molecules to modulate DevS kinase activity. The three-dimensional structure of the putative GAF-B domain revealed that it has a GAF folding structure without cyclic nucleotide binding capacity.Mycobacterium smegmatis is a nonpathogenic and fast-growing mycobacterium whose adaptive response to a gradual decrease in oxygen tension and exposure to NO is similar to that of Mycobacterium tuberculosis (7). This adaptive capability of mycobacteria has been suggested to allow them to persist in a latent state in the immune-competent host, especially in the case of M. tuberculosis (14,24,35,52). The hypoxic conditions within granulomas, although this is controversial (1), and the NO synthesized by activated macrophages have been proposed to serve as possible signals for the transition of mycobacteria to the nonreplicating, latent state (35, 50, 52).The DevSR (DosSR) two-component system plays a crucial role in the adaptation of mycobacteria to hypoxic and NO conditions. Approximately 48 genes of M. tuberculosis were reported to be induced under hypoxic conditions, as well as on exposure to NO. The upregulation of these genes is mediated by the DevSR system (35, 38, 39, 52). The DevSR two-component system consists of the DevS histidine kinase (HK) and its cognate response regulator (39, 40). In addition to DevS, the DosT HK was found to cross talk with DevR and to be functional in M. tuberculosis (39,40). DevS and DosT show high sequence similarity to each other over the length of their primary structures. The N-terminal sensory domains of DevS and DosT contain two putative GAF domains. The first GAF domain (GAF-A) serves as a heme-binding domain, while the function of the second one (GAF-B) remains to be revealed (18,22,43,46). It was recently demonstrated that either the binding of O 2 to the ferrous form of hemes of both DevS and DosT or the oxidation of Fe 2ϩ within the heme to...
AIMP2/p38 is a multifunctional tumor suppressor that normally resides in the cytosol as a scaffold protein of the multi-tRNA synthetase complex (MSC). One of the tumorsuppressive functions of AIMP2 is to facilitate ubiquitinmediated degradation of FUSE-binding protein (FBP, FUBP1), a transcriptional activator of c-Myc. However, the mechanism by which AIMP2 functions within this pathway and its significance in tumorigenesis are uncertain. Here, we report that Smurf2 is responsible for AIMP2-mediated ubiquitination of FBP, and a mutation in AIMP2 that inhibited its nuclear interaction with Smurf2 enhanced cellular transformation and tumorigenesis in vivo. Treatment of HeLa cells with TGFb resulted in the phosphorylation of AIMP2 on S156, a residue that is exposed on the embedded GST domain of AIMP2. We further found that phospho-AIMP2 dissociated from the MSC and translocated to the nucleus, where it bound to Smurf2, enhancing ubiquitination of FBP. AIMP2 also inhibited nuclear export of Smurf2 to sustain TGFb signaling. Collectively, these findings present a novel tumor-suppressive interaction between AIMP2 and Smurf2 and suggest that the disruption of this interaction can lead to oncogenic transformation.
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