Abstract:Most pathogenic bacteria require ferrous iron (Fe 2+ ) in order to sustain infection within hosts. The ferrous iron transport (Feo) system is the most highly-conserved prokaryotic transporter of Fe 2+ , but its mechanism remains to be fully characterized. Most Feo systems are composed of two proteins: FeoA, a soluble SH3-like accessory protein and FeoB, a membrane protein that translocates Fe 2+ across a lipid bilayer. Some bacterial feo operons encode FeoC, a third soluble, winged-helix protein that remains e… Show more
“…80-100 kDa) polytopic transmembrane protein that contains an N-terminal soluble G-protein-like domain termed NFeoB(12, 14, 18). The roles of FeoA and FeoC remain somewhat enigmatic; however, these proteins have been shown to interact with NFeoB in vitro(19) , FeoA appears to regulate GTP hydrolysis in vitro(17) , and some FeoCs bind oxygen-sensitive [Fe-S] clusters, presumably for regulatory purposes(20). In vivo , several observations indicate that both proteins interact with FeoB and are required for Feo-dependent iron uptake in Vibrio cholerae ( Vc )(21, 22), the pathogenic bacterium responsible for the diarrheal disease cholera.…”
Ferrous iron (Fe2+) is required for the growth and virulence of many pathogenic bacteria, includingVibrio cholerae(Vc), the causative agent of the disease cholera. For this bacterium, Feo is the primary system that transports Fe2+into the cytosol. FeoB, the main component of this system, is regulated by a soluble cytosolic domain termed NFeoB. Recent reanalysis has shown that NFeoBs can be classified as either GTP-specific or NTP-promiscuous, but the structural and mechanistic bases for these differences were not known. To explore this intriguing property of FeoB, we solved the X-ray crystal structures ofVcNFeoB in both the apo and GDP-bound forms. Surprisingly, this promiscuous NTPase displayed a canonical NFeoB G-protein fold like GTP-specific NFeoBs. Using structural bioinformatics, we hypothesized that residues surrounding the nucleobase could be important for both nucleotide affinity and specificity. We then solved the X-ray crystal structures of N150TVcNFeoB in the apo and GDP-bound forms to reveal H-bonding differences surround the guanine nucleobase. Interestingly, isothermal titration calorimetry revealed similar binding thermodynamics of the WT and N150T proteins to guanine nucleotides, while the behavior in the presence of adenine nucleotides was dramatically different. AlphaFold models ofVcNFeoB in the presence of ADP and ATP showed important conformational changes that contribute to nucleotide specificity among FeoBs. Combined, these results provide a structural framework for understanding FeoB nucleotide promiscuity, which could be an adaptive measure utilized by pathogens to ensure adequate levels of intracellular iron across multiple metabolic landscapes.
“…80-100 kDa) polytopic transmembrane protein that contains an N-terminal soluble G-protein-like domain termed NFeoB(12, 14, 18). The roles of FeoA and FeoC remain somewhat enigmatic; however, these proteins have been shown to interact with NFeoB in vitro(19) , FeoA appears to regulate GTP hydrolysis in vitro(17) , and some FeoCs bind oxygen-sensitive [Fe-S] clusters, presumably for regulatory purposes(20). In vivo , several observations indicate that both proteins interact with FeoB and are required for Feo-dependent iron uptake in Vibrio cholerae ( Vc )(21, 22), the pathogenic bacterium responsible for the diarrheal disease cholera.…”
Ferrous iron (Fe2+) is required for the growth and virulence of many pathogenic bacteria, includingVibrio cholerae(Vc), the causative agent of the disease cholera. For this bacterium, Feo is the primary system that transports Fe2+into the cytosol. FeoB, the main component of this system, is regulated by a soluble cytosolic domain termed NFeoB. Recent reanalysis has shown that NFeoBs can be classified as either GTP-specific or NTP-promiscuous, but the structural and mechanistic bases for these differences were not known. To explore this intriguing property of FeoB, we solved the X-ray crystal structures ofVcNFeoB in both the apo and GDP-bound forms. Surprisingly, this promiscuous NTPase displayed a canonical NFeoB G-protein fold like GTP-specific NFeoBs. Using structural bioinformatics, we hypothesized that residues surrounding the nucleobase could be important for both nucleotide affinity and specificity. We then solved the X-ray crystal structures of N150TVcNFeoB in the apo and GDP-bound forms to reveal H-bonding differences surround the guanine nucleobase. Interestingly, isothermal titration calorimetry revealed similar binding thermodynamics of the WT and N150T proteins to guanine nucleotides, while the behavior in the presence of adenine nucleotides was dramatically different. AlphaFold models ofVcNFeoB in the presence of ADP and ATP showed important conformational changes that contribute to nucleotide specificity among FeoBs. Combined, these results provide a structural framework for understanding FeoB nucleotide promiscuity, which could be an adaptive measure utilized by pathogens to ensure adequate levels of intracellular iron across multiple metabolic landscapes.
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