Unusual heme-binding PAS domain from YybT family proteins

Abstract

YybT family proteins (COG3887) are functionally unknown proteins that are widely distributed among the firmicutes, including the human pathogens Staphylococcus aureus and Listeria monocytogenes. Recent studies suggested that YybT family proteins are crucial for the in vivo survival of bacterial pathogens during host infection. YybT family proteins contain an N-terminal domain that shares minimum sequence homology with Per-ARNT-Sim (PAS) domains. Despite the lack of an apparent residue for heme coordination, the putative PAS domains of BsYybT and GtYybT, two representative members of the YybT family proteins from Bacillus subtilis and Geobacillus thermodenitrificans, respectively, are found to bind b-type heme with 1:1 stoichiometry. Heme binding suppresses the catalytic activity of the DHH/DHHA1 phosphodiesterase domain and the degenerate GGDEF domain. Absorption spectroscopic studies indicate that YybT proteins do not form stable oxyferrous complexes due to the rapid oxidation of the ferrous iron upon O(2) binding. The ferrous heme, however, forms a hexacoordinated complex with carbon monoxide (CO) and a pentacoordinated complex with nitric oxide (NO). The coordination of NO, but not CO, to the heme stimulates the phosphodiesterase activity. These results suggest that YybT family proteins function as stress-signaling proteins for monitoring cellular heme or the NO level by using a heme-binding PAS domain that features an unconventional heme coordination environment.

FIG. 1.

Domain organization of YybT family proteins (COG3887). The residue numbers are shown to indicate the domain boundaries of BsYybT.

FIG. 2.

Absorption spectra of recombinant YybT proteins expressed from E. coli. (A) Absorption spectrum of BsYybT_84-303. (B) Absorption spectra of the three GtYybT protein constructs. (C) Identification of type b heme by the pyridine hemochrome method.

FIG. 3.

Sequence and modeling analysis of heme binding. (A) Sequence alignment of the PAS domains of YybT family proteins with the heme-binding PAS domain of BjFixL and EcDOS. Residue numbering is based on BsYybT. The position of residue Phe¹⁰⁷ is highlighted by the triangle, and the conserved histidine residues for heme coordination in BjFixL and EcDOS are boxed. The predicted secondary structure for PAS_BsYybT is shown on the top, and the secondary structure for BjFixL is shown at the bottom. Sequences were aligned by using the program ClustalW, and the figure was generated by using the program ESPript. (BsPAS is from Bacillus subtilis; BaPAS is from Bacillus anthracis; GtPAS is from Geobacillus thermodenitrificans; LlPAS is from Lactooccus lactis; LmPAS is from Listeria monocytogene; SaPAS is from Staphylococcus aureus; SmPAS is from Streptococcus mutans). (B) Overlaid structures of the PAS domains of EcDOS (black structure) and BjFixL (gray structure) with the heme and the corresponding residue of Phe¹⁰⁷ shown as sticks. (C) Absorption spectra of the wild-type GtYybT_55-162 and F107A mutant.

FIG. 4.

UV-vis absorption spectra of apo-BsYybT_84-659 and reconstituted BsYybT_84-659 and GtYybT_55-162. The proteins were reconstituted with ferric heme in the absence of reducing agent. The spectra were taken in 50 mM Tris (pH 8.0), 150 mM NaCl at 23°C.

FIG. 5.

Absorption spectra of BsYybT_84-659 and its adducts_. (A) Spectra of BsYybT_84-659 in its ferric, ferrous, Fe(II)-CO, and Fe(II)-NO forms. (B) Spectra of BsYybT_84-659 in its Fe(II)-CN⁻ and Fe(III)-CN⁻ forms.

FIG. 6.

Regulation of the activities of the catalytic domains by the PAS domain. (A) The effect of heme binding and ligand coordination on PDE activity. (B) Dependence of the PDE activity of ferrous BsYybT_84-659 on the concentration of NO donor. (C) Steady-state kinetic measurement of PDE activity of ferric BsYybT_84-659 in the presence and absence of KCN. (D) Effect of heme binding on ATP hydrolysis catalyzed by the GGDEF domain of BsYybT_84-659.