Structural and thermodynamic insights into a novel Mg2+–citrate‐binding protein from the ABC transporter superfamily

More than one third of proteins require metal ions to accomplish their functions, making them obligatory for the growth and survival of microorganisms in varying environmental niches. In prokaryotes, besides their involvement in various cellular and physiological processes, metal ions stimulate the uptake of citrate molecules. Citrate is a source of carbon and energy and is reported to be transported by secondary transporters. In Gram‐positive bacteria, citrate molecules are transported in complex with divalent metal ions, whereas in Gram‐negative bacteria they are translocated by Na+/citrate symporters. In this study, the presence of a novel divalent‐metal‐ion‐complexed citrate‐uptake system that belongs to the primary active ABC transporter superfamily is reported. For uptake, the metal‐ion‐complexed citrate molecules are sequestered by substrate‐binding proteins (SBPs) and transferred to transmembrane domains for their transport. This study reports crystal structures of an Mg2+–citrate‐binding protein (MctA) from the Gram‐negative thermophilic bacterium Thermus thermophilus HB8 in both apo and holo forms in the resolution range 1.63–2.50 Å. Despite binding various divalent metal ions, MctA possesses the coordination geometry to bind its physiological metal ion, Mg2+. The results also suggest an extended subclassification of cluster D SBPs, which are known to bind and transport divalent‐metal‐ion‐complexed citrate molecules. Comparative assessment of the open and closed conformations of the wild‐type and mutant MctA proteins suggests a gating mechanism of ligand entry following an `asymmetric domain movement' of the N‐terminal domain for substrate binding. MctA is an Mg2+‐complexed citrate‐binding protein from a Gram‐negative bacterium that belongs to the ABC transporter superfamily. Comparison of the crystal structures of wild‐type and mutant MctA proteins suggest a gating mechanism of substrate entry following an `asymmetric domain movement' mechanism of substrate binding.