Recognition of ferric catecholates by FepA

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Escherichia coli FepA is an outer membrane transporter responsible for importing specific ferric siderophores.
• The transporter exhibits selectivity for certain catecholate siderophores over others, but the molecular basis for this discrimination is not fully understood.

Purpose of the Study:

• To investigate the molecular determinants of ligand recognition and transport selectivity by E. coli FepA.
• To elucidate the roles of specific amino acid residues and structural domains in the FepA-siderophore interaction.

Main Methods:

• Direct binding and competition experiments were used to assess FepA's affinity for various siderophores.
• Alanine scanning mutagenesis was employed to probe the contribution of aromatic residues to ligand binding.
• N-domain loop deletions and fluorescence/radioisotopic assays were utilized to study ligand release kinetics and transport mechanisms.

Main Results:

• FepA demonstrated selective adsorption of catecholate ferric siderophores, with Fe-enterobactin (FeEnt) binding with higher affinity than synthetic analogs like Fe-TRENCAM.
• Mutagenesis revealed that aromatic residues within the FepA beta-barrel loops significantly influence FeEnt binding affinity (Kd) and transport efficiency (Km).
• N-domain loop deletions drastically impaired transport (Km) with less impact on binding (Kd), suggesting a distinct role in ligand translocation.

Conclusions:

• The beta-barrel loops of FepA harbor the primary determinants for recognizing and initially binding ferric siderophores.
• N-terminal domains play a critical role in facilitating the transport of the bound ligand across the membrane.
• FepA's selectivity for specific siderophores is established at the adsorption stage, mediated by interactions with aromatic residues.