Structural Determinants of Vibrio cholerae FeoB Nucleotide Promiscuity

Area of Science:

• Microbiology
• Structural Biology
• Biochemistry

Background:

• Ferrous iron (Fe2+) is essential for pathogenic bacteria like Vibrio cholerae (Vc).
• The Feo system, particularly FeoB, is crucial for intracellular Fe2+ transport in Vc.
• The regulatory cytosolic domain, NFeoB, exhibits varying nucleotide specificity (GTP-specific vs. NTP-promiscuous), but the reasons were unclear.

Purpose of the Study:

• To elucidate the structural and mechanistic basis for the nucleotide specificity differences observed in NFeoBs.
• To investigate the structural properties of VcNFeoB and its nucleotide binding characteristics.

Main Methods:

• X-ray crystallography was used to determine the structures of VcNFeoB in apo and GDP-bound states.
• Structural bioinformatics and mutagenesis (N150T) were employed to identify key residues.
• Isothermal titration calorimetry (ITC) assessed nucleotide binding thermodynamics.
• AlphaFold modeling predicted conformational changes upon nucleotide binding.

Main Results:

• VcNFeoB, despite being promiscuous, possesses a canonical G-protein fold similar to GTP-specific NFeoBs.
• Mutagenesis (N150T) revealed altered hydrogen bonding around the guanine nucleobase.
• ITC showed distinct binding behaviors with adenine nucleotides for the mutant compared to wild-type.
• AlphaFold models indicated conformational changes related to nucleotide specificity.

Conclusions:

• A structural framework for FeoB nucleotide promiscuity is provided.
• This promiscuity may be an adaptive strategy for pathogens to maintain intracellular iron levels under diverse metabolic conditions.