Abstract
The Bacillus subtilis protein YybT (or GdpP) and its homologs were recently established as stress signaling proteins that exert their biological effect by degrading the bacterial messenger cyclic di-AMP. YybT homologs contain a small Per-ARNT-Sim (PAS) domain (~80 amino acids) that can bind b-type heme with 1:1 stoichiometry despite the small size of the domain and the lack of a conserved heme iron-coordinating residue. We determined the solution structure of the PAS domain of GtYybT from Geobacillus thermodenitrificans by NMR spectroscopy to further probe its function. The solution structure confirms that PASGtYybT adopts the characteristic PAS fold composed of a five-stranded antiparallel β sheet and a few short α-helices. One α-helix and three central β-strands of PASGtYybT are noticeably shorter than those of the typical PAS domains. Despite the small size of the protein domain, a hydrophobic pocket is formed by the side chains of nonpolar residues stemming from the β-strands and α-helices. A set of residues in the vicinity of the pocket and in the C-terminal region at the dimeric interface exhibits perturbed NMR parameters in the presence of heme or zinc protoporphyrin. Together, the results unveil a compact PAS domain with a potential ligand-binding pocket and reinforce the view that the PASYybT domains function as regulatory domains in the modulation of cellular cyclic di-AMP concentration.
Highlights
The Bacillus subtilis protein YybT and its homologs were recently established as stress signaling proteins that exert their biological effect by degrading the bacterial messenger cyclic di-AMP
LlYybT-encoding llmg_1816 gene rendered Lactococcus lactis more tolerant to acid stress [1], the biological function of YybT proteins remained obscure until the discovery that the DHH/ DHHA1 domain of BsYybT from Bacillus subtilis possesses phosphodiesterase activity toward cyclic di-AMP (c-di-AMP)4 [2], a newly discovered messenger molecule found in Grampositive bacteria [3, 4]
Results obtained from the NMR titration with reconstituted heme and zinc protoporphyrin IX (ZnPPIX) suggest that the hydrophobic pocket is potentially involved in the binding of heme
Summary
The Bacillus subtilis protein YybT (or GdpP) and its homologs were recently established as stress signaling proteins that exert their biological effect by degrading the bacterial messenger cyclic di-AMP. YybT homologs contain a small PerARNT-Sim (PAS) domain (ϳ80 amino acids) that can bind b-type heme with 1:1 stoichiometry despite the small size of the domain and the lack of a conserved heme iron-coordinating residue. Early studies showed that the disruption of the LlYybT-encoding llmg_1816 gene rendered Lactococcus lactis more tolerant to acid stress [1], the biological function of YybT proteins remained obscure until the discovery that the DHH/ DHHA1 domain of BsYybT from Bacillus subtilis possesses phosphodiesterase activity toward cyclic di-AMP (c-di-AMP)4 [2], a newly discovered messenger molecule found in Grampositive bacteria [3, 4]. To further probe the structure and function of the PASYybT domain, we determined the high-resolution NMR structure of the PAS domain (GtYybT) of the YybT protein from the thermophilic microorganism G. thermodenitrificans to reveal that the small PASGtYybT domain adopts a PAS fold with a putative hydrophobic ligand-binding site surrounded by shortened strands and loops. Results obtained from the NMR titration with reconstituted heme and zinc protoporphyrin IX (ZnPPIX) suggest that the hydrophobic pocket is potentially involved in the binding of heme
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