Internal Ion Pairs Control Transport Through TonB-Dependent Siderophore Receptors

Area of Science:

• Microbiology
• Structural Biology
• Biochemistry

Background:

• TonB-dependent receptors (TBDRs), such as FepA and FhuA, facilitate the transport of essential nutrients like ferric siderophores across the Gram-negative bacterial outer membrane.
• The uptake mechanism involves conformational changes within the N-terminal luminal domain (NTLD) situated inside the C-terminal β-barrel (CTβB) channel.

Purpose of the Study:

• To investigate the role of conserved electrostatic interactions at the NTLD-CTβB interface in TBDR function.
• To determine the mechanistic importance of specific ion pairs in mediating ferric siderophore transport.

Main Methods:

• Systematic alanine substitutions were engineered in FepA and FhuA to disrupt conserved ion pairs at four identified sites.
• Mutant functionality was assessed using siderophore nutrition assays, colicin susceptibility tests, and fluorescence spectroscopic measurements for uptake and binding affinity.

Main Results:

• Disruption of electrostatic bonds at site-2 significantly reduced or abolished ferric siderophore uptake and colicin susceptibility.
• Site-2 mutations did not affect the binding affinity of FepA for ferric enterobactin or FhuA for ferrichrome.
• Eliminating interactions at other sites (1, 3, 4) did not impede ferric enterobactin uptake.
• Site-2 mutations increased susceptibility to proteolysis, indicating NTLD destabilization.

Conclusions:

• The evolutionarily conserved ion pairs at site-2 are essential for the conformational changes required for ferric siderophore transport through the CTβB into the periplasm.
• These electrostatic interactions stabilize the NTLD within the CTβB channel, playing a critical role in TBDR-mediated nutrient uptake.