Exploring the structure and function of Thermotoga maritima CorA reveals the mechanism of gating and ion selectivity in Co2+/Mg2+ transport.

The CorA family of divalent cation transporters utilizes Mg2+ and Co2+ as primary substrates. The molecular mechanism of its function, including ion selectivity and gating, has not been fully characterized. Recently we reported a new structure of a CorA homologue from Methanocaldococcus jannaschii, which provided novel structural details that offered the conception of a unique gating mechanism involving conversion of an open hydrophilic gate into a closed hydrophobic one. In the present study we report functional evidence for this novel gating mechanism in the Thermotoga maritima CorA together with an improved crystal structure of this CorA to 2.7 Å (1 Å=0.1 nm) resolution. The latter reveals the organization of the selectivity filter to be similar to that of M. jannaschii CorA and also the previously unknown organization of the second signature motif of the CorA family. The proposed gating is achieved by a helical rotation upon the binding of a metal ion substrate to the regulatory binding sites. Additionally, our data suggest that the preference of this CorA for Co2+ over Mg2+ is controlled by the presence of threonine side chains in the channel. Finally, the roles of the intracellular metal-binding sites have been assigned to increased thermostability and regulation of the gating. These mechanisms most likely apply to the entire CorA family as they are regulated by the highly conserved amino acids.

Structural insights into the mechanisms of Mg2+ uptake, transport, and gating by CorA.

Despite the importance of Mg(2+) for numerous cellular activities, the mechanisms underlying its import and homeostasis are poorly understood. The CorA family is ubiquitous and is primarily responsible for Mg(2+) transport. However, the key questions-such as, the ion selectivity, the transport pathway, and the gating mechanism-have remained unanswered for this protein family. We present a 3.2 Å resolution structure of the archaeal CorA from Methanocaldococcus jannaschii, which is a unique complete structure of a CorA protein and reveals the organization of the selectivity filter, which is composed of the signature motif of this family. The structure reveals that polar residues facing the channel coordinate a partially hydrated Mg(2+) during the transport. Based on these findings, we propose a unique gating mechanism involving a helical turn upon the binding of Mg(2+) to the regulatory intracellular binding sites, and thus converting a polar ion passage into a narrow hydrophobic pore. Because the amino acids involved in the uptake, transport, and gating are all conserved within the entire CorA family, we believe this mechanism is general for the whole family including the eukaryotic homologs.

A cost-effective method for simultaneous homo-oligomeric size determination and monodispersity conditions for membrane proteins.

The use of blue native polyacrylamide gel electrophoresis (BN-PAGE) has been reported in the literature to retain both water-soluble and membrane protein complexes in their native hetero-oligomeric state and to determine the molecular weight of membrane proteins. However, membrane proteins show abnormal mobility when compared with water-soluble markers. Although one could use membrane proteins as markers or apply a conversion factor to the observed molecular weight to account for the bound Coomassie blue dye, when one just wants to assess homo-oligomeric size, these methods appear to be too time-consuming or might not be generally applicable. Here, during detergent screening studies to identify the best detergent for achieving a monodisperse sample, we observed that under certain conditions membrane proteins tend to form ladders of increasing oligomeric size. Although the ladders themselves contain no indication of which band represents the correct oligomeric size, they provide a scale that can be compared with a single band, representing the native homo-oligomeric size, obtained in other conditions of the screen. We show that this approach works for three membrane proteins: CorA (42 kDa), aquaporin Z (25 kDa), and small hydrophobic (SH) protein from respiratory syncytial virus (8 kDa). In addition, polydispersity results and identification of the most suitable detergent correlate optimally not only with size exclusion chromatography (SEC) but also with results from sedimentation velocity and equilibrium experiments. Because it involves minute quantities of sample and detergent, this method can be used in high-throughput approaches as a low-cost technique.