A urea channel from Bacillus cereus reveals a novel hexameric structure

Area of Science:

• Biochemistry
• Structural Biology
• Microbiology

Background:

• Bacteria utilize urea as a nitrogen source, with breakdown products ammonia and bicarbonate neutralizing stomach acidity, particularly by pathogens like Helicobacter pylori.
• Urea uptake in H. pylori is facilitated by the UreI protein, a member of the urea amide channel (UAC) family.

Purpose of the Study:

• To elucidate the structure and function of UACBc, a UAC homolog from Bacillus cereus.
• To investigate the substrate specificity and oligomeric state of UACBc.

Main Methods:

• Purification and characterization of the UACBc channel.
• Circular Dichroism (CD) and Infrared (IR) spectroscopy for structural analysis.
• Site-directed fluorescent labeling to determine transmembrane topology.
• Cross-linking and size-exclusion chromatography to assess oligomeric state.
• Cryo-electron microscopy (cryo-EM) of 2D crystals to obtain a high-resolution projection map.
• Bioinformatic analyses for structural modeling and functional site identification.

Main Results:

• UACBc demonstrated permeability to urea and other small amides.
• Spectroscopic analyses indicated a predominantly alpha-helical structure oriented perpendicular to the membrane.
• Site-directed labeling confirmed seven transmembrane (TM) helices and a cytoplasmic C-terminus.
• UACBc exists primarily as a hexamer in detergent solution.
• Cryo-EM revealed a planar hexameric ring structure with six TM helices forming a central pore and an additional peripheral helix per protomer.
• Bioinformatic analysis tentatively assigned TM regions to density features, identifying potential channel-lining residues.

Conclusions:

• UACBc forms a hexameric urea amide channel with a distinct transmembrane helical arrangement.
• The structural model provides insights into the molecular basis of urea and small amide transport.
• This study enhances understanding of urea transport mechanisms in bacteria, relevant to pathogen survival and nutrient acquisition.