Investigating the roles of the conserved Cu 2+ -binding residues on Brucella FtrA in producing conformational stability and functionality.

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.