A GAF Domain in the Hypoxia/NO-inducible Mycobacterium tuberculosis DosS Protein Binds Haem

Sardiwal, Sunita; Kendall, Sharon L.; Movahedzadeh, Farahnaz; Rison, Stuart C.G.; Stoker, Neil G.; Djordjević, Snežana

doi:10.1016/j.jmb.2005.09.011

Cited by 122 publications

(128 citation statements)

References 44 publications

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“…For DosS, the sharp Soret (409 nm), ␣ (575 nm), and ␤ (536 nm) bands, in addition to another weak band at Ϸ640 nm, are indicative of a hexa-coordinated, high-spin heme protein [supporting information (SI) Fig. 8] and is in support of a previous study (12). For DosT, the Soret (412 nm), ␣ (575 nm), Author contributions: A.K., R.P.P., J.R.L., and A.J.C.S.…”

Section: Mtb Doss and Dost Are Heme Proteinssupporting

confidence: 81%

Mycobacterium tuberculosis DosS is a redox sensor and DosT is a hypoxia sensor

Kumar¹,

Toledo

Patel

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

297

375

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A fundamental challenge to the study of oxidative stress responses of Mycobacterium tuberculosis (Mtb) is to understand how the protective host molecules are sensed and relayed to control bacilli gene expression. The genetic response of Mtb to hypoxia and NO is controlled by the sensor kinases DosS and DosT and the response regulator DosR through activation of the dormancy/NO (Dos) regulon. However, the regulatory ligands of DosS and DosT and the mechanism of signal sensing were unknown. Here, we show that both DosS and DosT bind heme as a prosthetic group and that DosS is rapidly autooxidized to attain the met (Fe 3؉ ) form, whereas DosT exists in the O 2-bound (oxy) form. EPR and UV-visible spectroscopy analysis showed that O 2, NO, and CO are ligands of DosS and DosT. Importantly, we demonstrate that the oxidation or ligation state of the heme iron modulates DosS and DosT autokinase activity and that ferrous DosS, and deoxy DosT, show significantly increased autokinase activity compared with met DosS and oxy DosT. Our data provide direct proof that DosS functions as a redox sensor, whereas DosT functions as a hypoxia sensor, and that O 2, NO, and CO are modulatory ligands of DosS and DosT. Finally, we identified a third potential dormancy signal, CO, that induces the Mtb Dos regulon. We conclude that Mtb has evolved finely tuned redox and hypoxia-mediated sensing strategies for detecting O 2, NO, and CO. Data presented here establish a paradigm for understanding the mechanism of bacilli persistence.carbon monoxide ͉ dormancy ͉ nitric oxide ͉ oxygen ͉ persistence T uberculosis (TB) is a major global health burden, and current estimates suggest that one-third of the world's population (Ϸ2 billion) is latently infected with TB (1). Latency is important largely because persistent Mycobacterium tuberculosis (Mtb) are in a state of ''drug unresponsiveness'' wherein the bacilli are resistant to existing antimycobacterial drugs. A major question in the TB field is: ''what are the mechanisms that allow Mtb to persist in human tissues for decades without replicating, to then abruptly resume growth and cause disease?'' Addressing that question is essential to the development of effective therapeutic intervention strategies. Recent evidence implicates NO as an environmental trigger of mycobacterial persistence (2-5). The latter findings are particularly interesting in light of the fact that inducible NO synthase (iNOS) and therefore NO production is crucial for protection of mice against Mtb (6), and that human macrophages in Mtb-infected tissues express iNOS (2, 7). Another factor associated with latent TB is hypoxia (8). The role of oxygen tension in TB is receiving wide attention, especially because it was demonstrated that rapid withdrawal of oxygen is lethal to Mtb, whereas a gradual depletion allows time for adaptation and bacterial survival (8). Interestingly, a significant overlap exists between the gene expression profiles of Mtb cells treated with NO and that of bacilli cultured under hypoxic conditions (3-5). ...

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Section: Mtb Doss and Dost Are Heme Proteinssupporting

confidence: 81%

Mycobacterium tuberculosis DosS is a redox sensor and DosT is a hypoxia sensor

Kumar¹,

Toledo

Patel

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

297

375

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“…NO (Airgas), 15 18 O 2 (99% 13 C; ICON Stable Isotopes) adducts were obtained after removal of excess dithionite from the reduced samples by ultrafiltration by injecting the gases in septum-sealed capillaries. These procedures were performed in a glove box with a controlled atmosphere of less than 1 ppm O 2 (Omni-Lab System; Vacuum Atmospheres Co.).…”

Section: Site-directed Mutagenesis and Cloning Of The Doss Distalmentioning

confidence: 99%

“…GAF-A has a heme prosthetic group, although the function of the GAF-B domain, which has no heme, remains unclear. DosS also has a kinase core with an ATP binding domain and a histidine phosphate-accepting domain that upon autophosphorylation initiates the signaling cascade (18). The crystal structure of the DosS GAF-A domain identifies His-149 as the proximal iron ligand and Tyr-171 as a highly conserved residue close to the heme ( Fig.…”

mentioning

confidence: 99%

Distal Hydrogen-bonding Interactions in Ligand Sensing and Signaling by Mycobacterium tuberculosis DosS

Basudhar

Madrona

Yukl

et al. 2016

Journal of Biological Chemistry

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Mycobacterium tuberculosis DosS is critical for the induction of M. tuberculosis dormancy genes in response to nitric oxide (NO), carbon monoxide (CO), or hypoxia. These environmental stimuli, which are sensed by the DosS heme group, result in autophosphorylation of a DosS His residue, followed by phosphotransfer to an Asp residue of the response regulator DosR. To clarify the mechanism of gaseous ligand recognition and signaling, we investigated the hydrogen-bonding interactions of the iron-bound CO and NO ligands by site-directed mutagenesis of Glu-87 and His-89. Autophosphorylation assays and molecular dynamics simulations suggest that Glu-87 has an important role in ligand recognition, whereas His-89 is essential for signal transduction to the kinase domain, a process for which Arg-204 is important. Mutation of Glu-87 to Ala or Gly rendered the protein constitutively active as a kinase, but with lower autophosphorylation activity than the wild-type in the Fe(II) and the Fe(II)-CO states, whereas the E87D mutant had little kinase activity except for the Fe(II)-NO complex. The H89R mutant exhibited attenuated autophosphorylation activity, although the H89A and R204A mutants were inactive as kinases, emphasizing the importance of these residues in communication to the kinase core. Resonance Raman spectroscopy of the wild-type and H89A mutant indicates the mutation does not alter the heme coordination number, spin state, or porphyrin deformation state, but it suggests that interdomain interactions are disrupted by the mutation. Overall, these results confirm the importance of the distal hydrogen-bonding network in ligand recognition and communication to the kinase domain and reveal the sensitivity of the system to subtle differences in the binding of gaseous ligands.Tuberculosis, an airborne disease caused by Mycobacterium tuberculosis, is believed to have infected nearly 1 out of 3 people worldwide (1). Like any infection, tuberculosis leads to a host immune response resulting in recruitment of lymphocytes and macrophages (2). This process is associated with high levels of NO and CO produced by inducible nitric-oxide synthase and heme oxygenase-1 (HO-1), respectively, as part of the defense mechanism of macrophages (3-5). The ability of M. tuberculosis to successfully survive within the host for years in a clinically undetectable dormant state known as non-replicating persistence (NRP), 3 in which it is resistant to most of the currently available treatments, makes it of paramount importance to elucidate this defense strategy (6). The mechanism of NRP is not fully understood; however, hypoxia (7, 8), high CO and NO levels (5, 9), nutrient deprivation (10), and the pH of the microenvironment (11) are among the factors leading to NRP. It has been shown that NRP can initiate changes in energy metabolism and cellular signaling pathways that lead to M. tuberculosis growth arrest. Decreased cellular activity is believed to be the main reason for the long term treatment protocols required to eradicate the bacteria. Mo...

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“…The architecture of these HKs can be divided into an N-terminal sensor core and a C-terminal kinase core (KC) (see Fig. 1A) (10). There are two GAF domains in the sensor cores; the first of these, GAF-A, has a heme group that functions to sense oxygen or redox status (11,12), whereas the function of the second, GAF-B, is not yet known (13 thought to be constitutively active when the sensor core is removed (8,13).…”

mentioning

confidence: 99%

“…There are two GAF domains in the sensor cores; the first of these, GAF-A, has a heme group that functions to sense oxygen or redox status (11,12), whereas the function of the second, GAF-B, is not yet known (13 thought to be constitutively active when the sensor core is removed (8,13). KC consists of an ATP binding domain (ABD) and a dimerization and histidine phospho-accepting (DHp) domain, which contains the phosphate-accepting histidine (10). Because the DHp domains of DosS and DosT belong to the HisKA_3 family, they are known as HisKA domains.…”

mentioning

confidence: 99%