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.

布鲁氏菌是一种人畜共患病原体，其生存需要铁，并通过几个高亲和力摄取系统的表达获得这种金属。其中，新发现的二价铁转运蛋白 FtrABCD 被认为参与二价铁的吸收。序列同源性表明，FtrA（所提出的周质亚铁结合成分）是一种 P19 型蛋白（来自空肠弯曲菌的周质蛋白，显示出 Cu ²⁺依赖性铁亲和力）。先前对其他 P19 系统的结构和生化研究已经建立了 Cu ²⁺依赖性 Mn ²⁺亲和力以及这些系统的同二聚体的形成。这些蛋白质中的 Cu ²⁺配位氨基酸在布鲁氏菌 FtrA中是保守的，这暗示着相似的特性。然而，迄今为止，还没有实验证据证明布鲁氏菌FtrA 与金属的亲和力以及形成二聚体的可能性。使用野生型 FtrA 和 Cu ²⁺结合突变体（H65A、E67A、H118A 和 H151A），我们研究了该 P19 型的金属亲和力、折叠稳定性、二聚体形成能力以及 Cu ²⁺依赖性的分子基础采用同源建模、分析凝胶过滤、量热和光谱方法对蛋白质进行分析。此处报告的数据证实了对野生型 FtrA 的 Cu ²⁺依赖性、低μM Mn ²⁺ （Fe ²⁺模拟物）亲和力。 此外，我们的数据清楚地显示了 Mn ²⁺亲和力的丧失，以及由于这些保守的 Cu ²⁺结合残基突变而形成不太稳定的蛋白质构象，表明这些残基在产生天然和功能性蛋白质中发挥着重要作用。布鲁氏菌FtrA 折叠。