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

Abstract

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.

Keywords: bacterial protein kinase; cell signaling; crystal structure; dimerization interface; globin; heme; heme-based oxygen sensor; histidine kinase; signal transduction; two component system.

Figure 1.

A, structure of the monomeric imidazole-bound AfGcHK globin domain (yellow chain, PDB code 6OTD) and B, a comparison of the imidazole-bound AfGcHK globin domain (yellow) to its dimeric cyanide-bound counterpart (cyan and dark blue; PDB code 5OHE, chains G and H). The rectangular boxes in the left-hand images enclose the heme-binding pockets, which are shown in expanded form in the right-hand images. The Tyr-15 residues (two in the dimeric complex and one in the monomer) and the two bound imidazole molecules (in the monomeric case only) are shown in ball-and-stick form. Helices H1, H6, and H7, which form the globin domain dimerization interface, are labeled. The chain termini are labeled N and C. The kinase domain of the full-length protein (not shown in the figure) would be located in front of the C terminus of helix H7.

Figure 2.

Changes in the conformations of helices H1, H6, and H7 (which form the AfGcHK globin domain dimerization interface) after the break of the dimer. The monomer (6OTD) is shown in yellow, the corresponding parts of the dimer (5OHE) are shown in cyan, and the surface of the remaining part of the monomer is shown in gray.

Figure 3.

Oligomeric states of the WT and Tyr-15 mutant forms of the full-length AfGcHK protein and the isolated globin domain. A, size exclusion chromatography results for the full-length WT protein (red line) and its Y15A (green line), Y15F (blue line), Y15G (orange line), and Y15W (pink line) mutants at a concentration of 10 μm in 20 mm Tris-HCl, 150 mm NaCl, pH 8.0 (for further details, see “Experimental procedures”). B, size exclusion chromatography results for the isolated globin domain of WT AfGcHK (red line) and its Y15A (green line), Y15F (blue line), Y15G (orange line), and Y15W (pink line) mutants with overlaid calibration profiles of the standards BSA, ovalbumin and myoglobin (black lines) at 10 μm in 20 mm Tris-HCl, 150 mm NaCl, pH 8.0 (for further details, see “Experimental procedures”).

Figure 4.

Schematic depiction of the deactivation of AfGcHK autophosphorylation by the disruption of globin domain dimerization induced by the Y15A mutation. When globin domain dimerization is prevented, the protein loses its capacity for signal transduction, making it enzymatically inactive.