Investigating the roles of the conserved Cu²⁺-binding residues on Brucella FtrA in producing conformational stability...

Brucella is a zoonotic pathogen requiring iron for its survival and acquires this metal through the expression of several high-affinity uptake systems. Of these, the newly discovered ferrous iron transporter, FtrABCD, is proposed to take part in ferrous iron uptake. Sequence homology shows that, FtrA, the proposed periplasmic ferrous-binding component, is a P19-type protein (a periplasmic protein from C. jejuni which shows Cu²⁺ dependent iron affinity). Previous structural and biochemical studies on other P19 systems have established a Cu²⁺ dependent Mn²⁺ affinity as well as formation of homodimers for these systems. The Cu²⁺ coordinating amino acids from these proteins are conserved in Brucella FtrA, hinting towards similar properties. However, there has been no experimental evidence, till date, establishing metal affinities and the possibility of dimer formation by Brucella FtrA. Using wild-type FtrA and Cu²⁺-binding mutants (H65A, E67A, H118A, and H151A) we investigated the metal affinities, folding stabilities, dimer forming abilities, and the molecular basis of the Cu²⁺ dependence for this P19-type protein employing homology modeling, analytical gel filtration, calorimetric, and spectroscopic methods. The data reported here confirm a Cu²⁺-dependent, low-μM Mn²⁺ (Fe²⁺ mimic) affinity for the wild-type FtrA. In addition, our data clearly show the loss of Mn²⁺ affinity, and the formation of less stable protein conformations as a result of mutating these conserved Cu²⁺-binding residues, indicating the important roles these residues play in producing a native and functional fold of Brucella FtrA.