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

Determining conditions optimal for host growth, maximal protein yield, and lysis buffer composition is of critical importance for the efficient purification of soluble and well-folded recombinant proteins suitable for functional and/or structural studies. Small-scale optimization of conditions for protein production and stability saves time, labor, and costs. Here we describe a protocol for quick protein production and solubility screen using TissueLyser II system from Qiagen enabling simultaneous processing of 96 protein samples, with application to recombinant proteins encompassing two intracellular domains of ethylene-recognizing sensor histidine kinase ETHYLENE RESPONSE1 (ETR1) from Arabidopsis thaliana. We demonstrate that conditions for expression and cell lysis found in our small-scale screen allow successful large-scale production of pure and functional domains of sensor histidine kinase, providing a strategy potentially transferable to other similar catalytic domains.

• MeSH
• Escherichia coli genetika   metabolismus   MeSH
• exprese genu * MeSH
• fosforylace MeSH
• histidinkinasa chemie   genetika   izolace a purifikace   metabolismus   MeSH
• rekombinantní proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• rozpustnost MeSH
• rychlé screeningové testy * 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

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

The oxygen sensor histidine kinase AfGcHK from the bacterium Anaeromyxobacter sp. Fw 109-5 forms a two-component signal transduction system together with its cognate response regulator (RR). The binding of oxygen to the heme iron of its N-terminal sensor domain causes the C-terminal kinase domain of AfGcHK to autophosphorylate at His183 and then transfer this phosphate to Asp52 or Asp169 of the RR protein. Analytical ultracentrifugation revealed that AfGcHK and the RR protein form a complex with 2:1 stoichiometry. Hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) suggested that the most flexible part of the whole AfGcHK protein is a loop that connects the two domains and that the heme distal side of AfGcHK, which is responsible for oxygen binding, is the only flexible part of the sensor domain. HDX-MS studies on the AfGcHK:RR complex also showed that the N-side of the H9 helix in the dimerization domain of the AfGcHK kinase domain interacts with the helix H1 and the β-strand B2 area of the RR protein's Rec1 domain, and that the C-side of the H8 helix region in the dimerization domain of the AfGcHK protein interacts mostly with the helix H5 and β-strand B6 area of the Rec1 domain. The Rec1 domain containing the phosphorylable Asp52 of the RR protein probably has a significantly higher affinity for AfGcHK than the Rec2 domain. We speculate that phosphorylation at Asp52 changes the overall structure of RR such that the Rec2 area containing the second phosphorylation site (Asp169) can also interact with AfGcHK. Proteins 2016; 84:1375-1389. © 2016 Wiley Periodicals, Inc.

• MeSH
• Aeromonas salmonicida genetika   metabolismus   MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• Escherichia coli genetika   metabolismus   MeSH
• fosforylace MeSH
• hem chemie   metabolismus   MeSH
• histidin chemie   metabolismus   MeSH
• histidinkinasa chemie   genetika   metabolismus   MeSH
• klonování DNA MeSH
• kyselina aspartová chemie   metabolismus   MeSH
• kyslík chemie   metabolismus   MeSH
• Myxococcales chemie   enzymologie   MeSH
• proteinové domény MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• sekundární struktura proteinů MeSH
• signální transdukce * MeSH
• strukturní homologie proteinů MeSH
• vodík-deuteriová výměna MeSH
• železo chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH