Globin-coupled histidine kinase Dotaz Zobrazit nápovědu
AfGcHK is a globin-coupled histidine kinase that is one component of a two-component signal transduction system. The catalytic activity of this heme-based oxygen sensor is due to its C-terminal kinase domain and is strongly stimulated by the binding of O2 or CO to the heme Fe(II) complex in the N-terminal oxygen sensing domain. Hydrogen sulfide (H2S) is an important gaseous signaling molecule and can serve as a heme axial ligand, but its interactions with heme-based oxygen sensors have not been studied as extensively as those of O2, CO, and NO. To address this knowledge gap, we investigated the effects of H2S binding on the heme coordination structure and catalytic activity of wild-type AfGcHK and mutants in which residues at the putative O2-binding site (Tyr45) or the heme distal side (Leu68) were substituted. Adding Na2S to the initial OH-bound 6-coordinate Fe(III) low-spin complexes transformed them into SH-bound 6-coordinate Fe(III) low-spin complexes. The Leu68 mutants also formed a small proportion of verdoheme under these conditions. Conversely, when the heme-based oxygen sensor EcDOS was treated with Na2S, the initially formed Fe(III)-SH heme complex was quickly converted into Fe(II) and Fe(II)-O2 complexes. Interestingly, the autophosphorylation activity of the heme Fe(III)-SH complex was not significantly different from the maximal enzyme activity of AfGcHK (containing the heme Fe(III)-OH complex), whereas in the case of EcDOS the changes in coordination caused by Na2S treatment led to remarkable increases in catalytic activity.
- MeSH
- biokatalýza účinky léků MeSH
- fosforylace účinky léků MeSH
- hem chemie metabolismus MeSH
- histidinkinasa chemie genetika metabolismus MeSH
- kinetika MeSH
- kyslík chemie metabolismus MeSH
- molekulární struktura MeSH
- mutageneze cílená MeSH
- Myxococcales enzymologie MeSH
- sulfan chemie farmakologie MeSH
- Publikační typ
- časopisecké články MeSH
An emerging class of novel heme-based oxygen sensors containing a globin fold binds and senses environmental O2 via a heme iron complex. Structure-function relationships of oxygen sensors containing a heme-bound globin fold are different from those containing heme-bound PAS and GAF folds. It is thus worth reconsidering from an evolutionary perspective how heme-bound proteins with a globin fold similar to that of hemoglobin and myoglobin could act as O2 sensors. Here, we summarize the molecular mechanisms of heme-based oxygen sensors containing a globin fold in an effort to shed light on the O2-sensing properties and O2-stimulated catalytic enhancement observed for these proteins.
- MeSH
- Azotobacter vinelandii enzymologie MeSH
- Bordetella pertussis enzymologie MeSH
- chemotaxe MeSH
- Escherichia coli enzymologie MeSH
- globiny chemie MeSH
- hem chemie MeSH
- hemoglobiny chemie MeSH
- katalytická doména MeSH
- katalýza MeSH
- kyslík chemie MeSH
- lyasy štěpící vazby P-O chemie MeSH
- molekulární evoluce MeSH
- molekulární sekvence - údaje MeSH
- myoglobin chemie MeSH
- proteinkinasy chemie MeSH
- proteiny z Escherichia coli chemie MeSH
- regulace genové exprese enzymů * MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
The heme-based oxygen sensor protein AfGcHK is a globin-coupled histidine kinase in the soil bacterium Anaeromyxobacter sp. Fw109-5. Its C-terminal functional domain exhibits autophosphorylation activity induced by oxygen binding to the heme-Fe(II) complex located in the oxygen-sensing N-terminal globin domain. A detailed understanding of the signal transduction mechanisms in heme-containing sensor proteins remains elusive. Here, we investigated the role of the globin domain's dimerization interface in signal transduction in AfGcHK. We present a crystal structure of a monomeric imidazole-bound AfGcHK globin domain at 1.8 Å resolution, revealing that the helices of the WT globin dimer are under tension and suggesting that Tyr-15 plays a role in both this tension and the globin domain's dimerization. Biophysical experiments revealed that whereas the isolated WT globin domain is dimeric in solution, the Y15A and Y15G variants in which Tyr-15 is replaced with Ala or Gly, respectively, are monomeric. Additionally, we found that although the dimerization of the full-length protein is preserved via the kinase domain dimerization interface in all variants, full-length AfGcHK variants bearing the Y15A or Y15G substitutions lack enzymatic activity. The combined structural and biophysical results presented here indicate that Tyr-15 plays a key role in the dimerization of the globin domain of AfGcHK and that globin domain dimerization is essential for internal signal transduction and autophosphorylation in this protein. These findings provide critical insights into the signal transduction mechanism of the histidine kinase AfGcHK from Anaeromyxobacter.
- MeSH
- bakteriální proteiny chemie metabolismus MeSH
- fosforylace MeSH
- globiny chemie metabolismus MeSH
- histidinkinasa chemie metabolismus MeSH
- konformace proteinů, alfa-helix MeSH
- konformace proteinů MeSH
- krystalografie rentgenová MeSH
- molekulární modely MeSH
- multimerizace proteinu MeSH
- Myxococcales chemie metabolismus MeSH
- proteinové domény MeSH
- signální transdukce MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The heme-based oxygen sensor histidine kinase AfGcHK is part of a two-component signal transduction system in bacteria. O2 binding to the Fe(II) heme complex of its N-terminal globin domain strongly stimulates autophosphorylation at His183 in its C-terminal kinase domain. The 6-coordinate heme Fe(III)-OH- and -CN- complexes of AfGcHK are also active, but the 5-coordinate heme Fe(II) complex and the heme-free apo-form are inactive. Here, we determined the crystal structures of the isolated dimeric globin domains of the active Fe(III)-CN- and inactive 5-coordinate Fe(II) forms, revealing striking structural differences on the heme-proximal side of the globin domain. Using hydrogen/deuterium exchange coupled with mass spectrometry to characterize the conformations of the active and inactive forms of full-length AfGcHK in solution, we investigated the intramolecular signal transduction mechanisms. Major differences between the active and inactive forms were observed on the heme-proximal side (helix H5), at the dimerization interface (helices H6 and H7 and loop L7) of the globin domain and in the ATP-binding site (helices H9 and H11) of the kinase domain. Moreover, separation of the sensor and kinase domains, which deactivates catalysis, increased the solvent exposure of the globin domain-dimerization interface (helix H6) as well as the flexibility and solvent exposure of helix H11. Together, these results suggest that structural changes at the heme-proximal side, the globin domain-dimerization interface, and the ATP-binding site are important in the signal transduction mechanism of AfGcHK. We conclude that AfGcHK functions as an ensemble of molecules sampling at least two conformational states.
- MeSH
- bakteriální proteiny chemie metabolismus MeSH
- fosforylace MeSH
- hem chemie MeSH
- histidinkinasa chemie metabolismus MeSH
- hmotnostní spektrometrie MeSH
- krystalografie rentgenová MeSH
- kvarterní struktura proteinů MeSH
- kyslík metabolismus MeSH
- molekulární modely MeSH
- Myxococcales metabolismus MeSH
- oxidace-redukce MeSH
- proteinové domény MeSH
- signální transdukce MeSH
- vodík-deuteriová výměna MeSH
- železité sloučeniny chemie MeSH
- železnaté sloučeniny chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The nonradioactive method, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of Phos-tag (Phos-tag electrophoresis), is used to evaluate a kinase autophosphorylation and/or phosphotransfer reaction from a kinase/ATP to its protein substrate. This method outperforms radioisotope methods using [32P]ATP for detecting trace amounts of phosphorylated protein in fresh protein preparations. Phos-tag electrophoresis has been used to perform detailed analyses of the kinase activity of a heme-based oxygen sensor-specifically, a globin-coupled histidine kinase from the soil bacterium Anaeromyxobacter sp. Fw109-5 (AfGcHK).
