Coordination and redox state–dependent structural changes of the heme-based oxygen sensor AfGcHK associated with intraprotein signal transduction

Abstract

The heme-based oxygen sensor histidine kinase GcHK is part of a two-component signal transduction system in bacteria. O binding to the Fe(II) heme complex of its N-terminal globin domain strongly stimulates autophosphorylation at His in its C-terminal kinase domain. The 6-coordinate heme Fe(III)-OH and -CN complexes of GcHK 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 GcHK 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 ofGcHK. We conclude that GcHK functions as an ensemble of molecules sampling at least two conformational states.

Structural Insight into H‐NOX Gas Sensing and Cognate Signaling Protein Regulation

“…Moreover, there was no direct interaction between the H‐NOX and the kinase DHp and CA domains in both the absence and presence of NO, indicating that the NO‐dependent inhibition occurs through allosteric regulation rather than direct steric hindrance of the active site. Similar results have been observed in a hemoglobin‐based oxygen sensor kinase, Af GCHK . Although having a different sensor domain, Af GCHK exhibited increased H/D exchange behavior when kinase activity was inhibited.…”

“…Similar results have been observed in ah emoglobin-based oxygen sensork inase, AfGCHK. [77,78] Although havingadifferent sensor domain, AfGCHK exhibited increased H/D exchange behavior when kinase activity wasi nhibited.I na ddition, no direct interaction between the hemoglobin and the kinase domain was observed, indicating that allostericr egulation could be the general mechanism for heme-containing sensork inase. These results enabled the proposal of the first model that links H-NOX conformational changes to kinase inhibition.…”

π-Helix controls activity of oxygen-sensing diguanylate cyclases

“…Signal transduction following ligand binding to GCS proteins has been postulated to occur through conformational shifts in the globin domain that are propagated through the middle domain to reach the output domain [23,24]. The cross-linking and globin titration experiments described in this work demonstrate that GCS proteins adopt conformations that allow the globin domain to make interactions with both the middle and output domains of two DGC-containing GCS proteins, PccGCS and BpeGReg (Figures 3 and 5).…”

“…As the αG/αH loop in the globin was observed to become rigid after gas ligand binding to the heme within the histidine kinase-containing GCS from Anaeromyxobacter sp. Fw109-5 [23], this same increase in rigidity of the αG/αH helices loop may occur in BpeGReg, which could result in signal transduction through a π-helical shift in the middle domain and increased DGC activity. Cross-links also show that PccGCS globin αF helix (contains proximal histidine that shifts due to gaseous ligand binding) interacts with the π-helical αB helix of the middle domain, suggesting that the middle domain π-helix is involved in signal transduction pathways in both BpeGReg and PccGCS.…”

“…Sensor globin domains from HemAT-Bs (MCP-GCS from B. subtilis), Af GcHK (kinase-GCS from Anaeromyxobacter sp. FW109-5), EcDosC (DGC-GCS from E. coli), and BpeGReg (DGC-GCS from Bordetella pertussis) [21] have been crystallized as dimers in different ligation states (Fe II , Fe II -CO, Fe III -CN, Fe II -O 2 , Fe III -H 2 O) and have highlighted some conformational changes within the globin domain that might be involved in signal transduction [22][23][24]. These include displacement of the globin monomers relative to each other, changes in helix flexibility (as evidenced by B factors), and subtle changes in heme pocket conformation.…”