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.

From the Department of Biochemistry Faculty of Science Charles University Hlavova 2030 8 Prague 2 128 43 Czech Republic

the Department of Solid State Engineering Faculty of Nuclear Sciences and Physical Engineering Czech Technical University Prague Brehova 7 115 19 Praha 1 Czech Republic

the Institute of Biotechnology of the Czech Academy of Sciences v v i Biocev 252 50 Vestec Czech Republic and

the Institute of Microbiology of the Czech Academy of Sciences v v i Biocev 252 50 Vestec Czech Republic

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Monitoring the Kinase Activity of Heme-Based Oxygen Sensors and Its Dependence on O2 and Other Ligands Using Phos-Tag Electrophoresis

Hydrogen/Deuterium Exchange Mass Spectrometry of Heme-Based Oxygen Sensor Proteins

Disruption of the dimerization interface of the sensing domain in the dimeric heme-based oxygen sensor AfGcHK abolishes bacterial signal transduction

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PDB
5OHE, 5OHF, 2W31, 3DGE, 4ZVA, 4ZVB