Abstract
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.
Highlights
The heme-based oxygen sensor histidine kinase AfGcHK is part of a two-component signal transduction system in bacteria
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
Ibility and solvent exposure of helix H11. 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
Summary
We first determined the X-ray crystal structures of isolated AfGcHK globin domains bearing heme in the Fe(III)-CNϪ form (active state) and containing heme Fe(II) (inactive state) complexes and compared them with HDX–MS data for the full-length AfGcHK protein. Crystal structure of the isolated heme Fe(III)-CN؊ globin domain (PDB code 5OHE). The crystal structure of the isolated globin domain in the presence of KCN was solved at a resolution of 1.85 Å. Overall structure—The asymmetric unit of the crystal consists of four dimers (Fig. 2A) formed by eight protein chains (AB, CD, EF, and GH) positioned in a disphenoidal arrangement. The crystal structure confirms that the domain adopts the characteristic globin fold of GCSs (Fig. 2B). Globin domain of AfGcHK with cyanide, partially reduced (the dithionite-soaked crystal)
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