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

Abstract

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.

Isolation and Proteomic Analysis of Extracellular Vesicles from Lactobacillus salivarius SNK-6

“…Among these, substrate-binding protein (SBP) is an important component of transport systems and functions in substrate recognition [ 26 ]. SBP-dependent ABC transporters differ among organisms and target a variety of substances, including metal ions, amino acids, peptides, and sugars, which are essential for the growth and survival of bacteria in different environments [ 27 ]. The LsEVs analyzed in this study were rich in proteins that bind to metal ions and amino acids as substrates of MetQ/NlpA family ABC transporter substrate-binding proteins, which are reportedly beneficial for colonization and survival of L. salivarius SNK-6 in vivo.…”