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).

• Klíčová slova
• Autophosphorylation reaction, Heme-based oxygen sensors, Kinase activity, Ligand binding, Ligand-dependent kinase activity, Phos-tag electrophoresis, Phosphotransfer reaction,
• MeSH
• adenosintrifosfát metabolismus   MeSH
• Bacteria metabolismus   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• hem * metabolismus   MeSH
• kyslík metabolismus   MeSH
• ligandy MeSH
• proteiny * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 1,3-bis(bis(pyridin-2-ylmethyl)amino)propan-2-ol MeSH Prohlížeč
• adenosintrifosfát MeSH
• hem * MeSH
• kyslík MeSH
• ligandy MeSH
• proteiny * 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.

• Klíčová slova
• bacterial protein kinase, cell signaling, crystal structure, dimerization interface, globin, heme, heme-based oxygen sensor, histidine kinase, signal transduction, two component system,
• 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
• Názvy látek
• bakteriální proteiny MeSH
• globiny MeSH
• histidinkinasa 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.

• Klíčová slova
• bacterial protein kinase, crystal structure, globin, heme-containing oxygen sensor, histidine kinase, hydrogen-deuterium exchange, signal transduction, two component signal transduction system,
• 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
• Názvy látek
• bakteriální proteiny MeSH
• hem MeSH
• histidinkinasa MeSH
• kyslík MeSH
• železité sloučeniny MeSH
• železnaté sloučeniny MeSH