Diguanylate cyclase-containing globin coupled sensors (GCSs) are oxygen-sensing proteins which catalyze conversion of two guanosine triphosphate (GTP) molecules into bis(3’-5’)-cyclic dimeric guanosine monophosphate (c-di-GMP) via a diguanylate cyclase (DGC) domain. While the GCS proteins are ubiquitous across bacteria, the mechanism by which the DGC domain is activated upon O2 binding to the globin domain has not been elucidated. c-di-GMP is a bacterial second messenger that modulates bacterial biofilm formation, which are communities of bacteria attached to surfaces in an excreted polymeric matrix. Biofilms allow bacteria to resist antibiotics and the host’s natural defenses, linking c-di-GMP signaling pathways with infections. To determine the mechanism of O2-dependent diguanylate cyclase activation in GCS proteins, full-length GCS proteins and isolated globin domains from Bordetella pertussis and Pectobacterium carotovorum ssp. carotovorum have been characterized. Specifically, the crystal structure of the globin domain from Bordetella pertussis (BpeGlobin) has been solved, affording important structural information about the environment of the heme pocket. In order to determine specific interactions occurring within the heme pocket, Fourier-transform infrared spectroscopy (FTIR) was performed on BpeGlobin and PccGlobin at different pH values, and on full length BpeGReg mutants Y43F and S68A. These experiments confirmed the findings of the crystal structure and further verified the presence of a conserved water within the heme distal pocket. To explore the role of the conserved proximal histidine in transmitting the ligand binding signal, BpeGReg(H99G) and PccGCS(H112G) mutants were characterized via UV/Vis spectroscopy and analytical gel filtration. Of note, all tested mutations to the proximal histidine resulted in a shift in oligomeric state to the tetramer, suggesting its role in determining oligomerization. Further studies will include FTIR of the histidine mutants as well as kinetic assays of all mutants. These experiments will contribute to a more comprehensive characterization of the heme pocket residues and their roles in propagating signals from the globin domain to the catalytic domain.

Elucidating the roles of heme pocket residues in bacterial oxygen sensors

255th National Meeting (2018)

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