In silico and in vitro characterization of ten putative RsbR-like proteins in Saprospira grandis

Kim, Sun Ae
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[Honolulu] : [University of Hawaii at Manoa], [May 2012]
Globin-coupled sensors (GCSs) are heme-binding proteins that consist of an N-terminal sensor globin domain and a C-terminal transducer domain. Their functions are varied depending on the C-terminal domain and can be classified into two groups such as aerotactic and gene regulation groups. The gene regulating group is further divided into DNA-binding, 2nd messenger and protein-protein interaction group. So far, aerotactic transducers: HemAT-Hs from Halobacterium salinarum and HemAT-Bs from Bacillus subtilis are well characterized in a molecular level and a cellular level. In addition, GCSs from the 2nd messenger group within the gene regulating group such as BpeGreg (Bordetella pertussis), EcGreg (E. coli), CvGreg (Chromobacterium violaceum) and AvGreg (Azotobacter vinelandii) are recently characterized as well. Recently, genome sequencing of Saporospira grandis has been completed and revealed in-depth genomic information that related to interesting traits of this organism such as gliding motility, production of rhapidosome and ixotrophic nutrient obtaining mechanisms. Moreover, the initial machine annotation showed that there are ten putative GCSs which contain C-terminal STAS domains. Based on the machine annotation results, we have ten genes that were predicted to be GCSs and have a C-terminal STAS (Sulfur transporter and anti-sigma antagonist) domain (Table 1.1). The putative function of these proteins is an anti-sigma factor antagonist like SpoIIAA, RsbR and RsbS in B. subtilis which involves in nutritional, physical and environmental stress responses. The protein named RsbR1 (SGRA_3210) within S. grandis was first discovered and its neighbor genes rsbS,-T,-V,-X and rsbU shared similarities with rsbR and its adjacent genes (rsbS,-T,-U,-V,-W, sigB, rsbX) in B. subtilis. Also, the structure of RsbR consists of the N-terminal non-heme globin domain and the C-terminal STAS domain. All these genes and their encoded proteins are related to activate more than 150 stress response genes upon physical stresses through releasing a sigma factor B. Based on these similarities, we postulate RsbR1 and nine other proteins are globin-coupled sensors that are involved in the stress response. In this study, in silico and in vitro characterization of ten GCSs were carried out on both the N-terminal globin and the C-terminal STAS domains. Multiple alignments of the globin domains showed that only SGRA_0571 (RsbR2), SGRA_3210 (RsbR1) and SGRA_3852 (RsbR3) aligned their histidine residue with the proximal histidine residue in known globin domains. Two phosphorylation sites from RsbR and its paralogs in B. subtilis were aligned with nine GCSs except SGRA_3852 and YtvA which is known not to have any phosphorylation site, instead it has a GTP binding motif. Two amino acid residues (D and G) from the GTP binding motif (DXXG) are all aligned in all GCSs. The 3D structure alignments of the globin domain of Geobactor sulfurreducens and RsbR1, RsbR2 and RsbR3 showed around 23% identity but the proximal histidine, where it held the heme, were all aligned with the histidine residue of three GCSs. For in vitro characterization, all ten genes were cloned, expressed and purified. The purified proteins were dialyzed and carried UV spectroscopic analysis. Only three GCSs (RsbR1, RsbR2 and RsbR3) exhibited oxygenated myoglobin like peaks. This shows that these proteins are able to bind oxygen. RsbR1 was undergone for crystallization. Initial crystallization screening showed some red crystals. Initially we found ten GCS genes from the annotation of the genome. In silico results predicted them all as GCSs. Multiple alignments of the globin domains and STAS domains of ten GCSs showed only RsbR1, RsbR2 and RsbR3 had the conserved proximal histidine residues which were vital for heme-binding. The STAS domains of ten GCSs were shared high homology with the STAS domains of RsbR and its paralogs from B. subtilis including two conserved phosphorylation sites and possible GTP-binding motifs. However, RsbR3 did not show the conserved phophrylation sites. All ten GCS genes were cloned, expressed and purified. RsbR1, RsbR2 and RsbR3 displayed oxygenated myoglobin-like absorption spectra and they were the only proteins that were validated as GCSs.
M.S. University of Hawaii at Manoa 2012.
Includes bibliographical references.
microbiology, Saprospira grandis
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