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

Tereza Skalova,Alzbeta Lengalova,Jan Dohnalek,Karl Harlos,Peter Mihalcin,Petr Kolenko,Martin Stranava,Jan Blaha,Toru Shimizu,Markéta Martínková

doi:10.1074/jbc.ra119.011574

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.

Highlights

Ampicillin was obtained from P-lab (Prague, Czech Republic)
Tyr-15 mutants of the isolated AfGcHK globin domain were prepared by In-Fusion cloning (Clontech Laboratories)
Samples for analysis were prepared by dissolving the appropriate AfGcHK protein in 50 mM Tris-HCl, 150 mM NaCl, pH 8.0, to a final concentration of 10 ␮M

Summary

Results

A new crystal structure of the globin (sensing) domain of AfGcHK in the presence of imidazole was solved at a resolution of 1.8 Å (Fig. 1 and Table 1). The bulky imidazole molecule IMD203 binds to the heme distal side, pushing away the side chain of Tyr-45, which forms a hydrogen bond to the cyanide ligand in the dimeric structure [21]. The spectra of the Y15F and Y15W mutants were identical to those for the WT protein independently of the coordination and redox state of the heme-iron center, indicating that replacing Tyr-15 with an aromatic amino acid (Phe or Trp) does not greatly change the heme binding environment of the full-length protein or the isolated globin domain (Table 3 and Fig. S3). Autoxidation was slower in the corresponding isolated globin domain mutants (autoxidation rate Ͼ10 minϪ1), so mixed spectra of the Fe(III)-OHϪ and Fe(II)-O2 forms were observed (Fig. S3) Note that both the Fe(II)-O2 form of the full-length AfGcHK enzyme and its autoxidation product, the Fe(III)-OHϪ form, are catalytically fully active [20] (Table S1). ␮M 18.9 Ϯ 2.3a No enzyme activity 28.5 Ϯ 3.9 No enzyme activity 18.7 Ϯ 3.1 kcat minϪ1 1.08 Ϯ 0.03a

Discussion

Summary

Experimental procedures