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

Abstract

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.

Cryo-EM Structures of the Magnesium Channel CorA Reveal Symmetry Break upon Gating

“…This would, as a consequence, randomly trigger the inward movement of subunit B (or, alternatively, subunit E). Our proposed mechanism is therefore incompatible with previous proposals that have assumed concerted movements of the five subunits, leading to a symmetric open state (Chakrabarti et al, 2010; Dalmas et al, 2014b; Nordin et al, 2013). …”

“…2C). These putative permeating Mg 2+ correlate well with the equivalent sites observed in the available crystal structures of Mg 2+ -bound CorA (Eshaghi et al, 2006; Nordin et al, 2013; Pfoh et al, 2012). …”

Loss of cytosolic Mg2+ binding sites in the Thermotoga maritima CorA Mg2+ channel is not sufficient for channel opening

“…A divalent cation occurs at about the same position in other published Tm CorA structures as well (14–16). It has been proposed that this Mg 2+ ion could be (partially) hydrated as its location is about 4 Å away from the carbonyl groups of G312 and the side chains of N314 of the YGMNF motif within the pore (15, 16). Similar distances were observed in RR-CorA.…”

“…It was reported that the Tm CorA D253K mutant was more stable than the wild-type protein due to salt bridge formation with D89 (10). The same scenario is very likely by mutating only D89 to K which could then form a salt bond with D253 (16). While it is also possible that support for the pentameric assembly derives from crystal contacts and nonspecifically bound Mg 2+ because both proteins were crystallized in 0.2 M Mg 2+ , we do not think this is likely.…”

“…A Mg 2+ is located between D89 of the α3 helix of one monomer and D253 of the α7 stalk helix of the neighboring monomer in all Tm CorA structures (M1 binding site) (8–10, 14). An additional Mg 2+ ion interaction within each monomer has been observed in vicinity of L12, E88, D175 and D253 in some structures (M2 binding site) (9–10, 14–16). …”