Crystal structure of the CorA Mg2+ transporter

Abstract

The magnesium ion, Mg 2+ , is essential for myriad biochemical processes and remains the only major biological ion whose transport mechanisms remain unknown. The CorA family of magnesium transporters is the primary Mg 2+ uptake system of most prokaryotes 1-3 and a functional homologue of the eukaryotic mitochondrial magnesium transporter 4 . Here we determine crystal structures of the full-length Thermotoga maritima CorA in an apparent closed state and its isolated cytoplasmic domain at 3.9 Å and 1.85Å resolution, respectively. The transporter is a funnel-shaped homopentamer with two transmembrane helices per monomer. The channel is formed by an inner group of five helices and putatively gated by bulky hydrophobic residues. The large cytoplasmic domain forms a funnel whose wide mouth points into the cell and whose walls are formed by five long helices that are extensions of the transmembrane helices. The cytoplasmic neck of the pore is surrounded, on the outside of the funnel, by a ring of highly conserved positively charged residues. Two negatively charged helices in the cytoplasmic domain extend back towards the membrane on the outside of the funnel and abut the ring of positive charge. An apparent Mg 2+ ion was bound between monomers at a conserved site in the cytoplasmic domain, suggesting a mechanism to link gating of the pore to the intra-cellular concentration of Mg 2+ . The CorA magnesium transporter is a homopentamer with fivefold symmetry about a central pore and can be divided into three parts (Fig. 1). A carboxy-terminal transmembrane domain comprises two transmembrane helices from each monomer (Fig. 2). The middle portion resembles a funnel, narrow at the entrance ( 5 Å) and wide at the mouth ( 20Å), that is formed largely by a long -helix extension of the inner transmembrane helix. Finally, a large cytoplasmic domain lies exterior to the funnel.The cytoplasmic domain of CorA is a seven-stranded parallel/antiparallel -sheet ( 2 1 3 7 6 5 4 ) sandwiched between two sets of -helices ( 1, 2, 3) and ( 4, 5, 6) ( Fig. 1). The domain fold is unlike all other known structures of ion channels or transporters and constitutes a new protein fold (see Supplementary Information). This domain, solved in its soluble form at 1.85 Å resolution ( Supplementary Fig. S1), is linked to the transmembrane helices by the long 7 helix (residues 251-312), termed the stalk helix. The stalk helix kinks as it enters the membrane, extends through the membrane, forms the first transmembrane helix (TM1; residues 293-312) and harbours the 'YGMNF' signature sequence of CorA (residues 311-315) 5,6 ( Fig. 2 and Supplementary Fig. S2). The five TM1 helices (residues 293-312) form the pore. After a short extracellular seven-amino-acid loop, the TM2 helix (residues 326-345) returns back to the cytoplasm and ends in a highly conserved C-terminal KKKKWL motif (Fig. 3). In the current structure, neither the extracellular loop nor the final two amino acids could be resolved.The cytoplasmic domain shows the lowest sequence conservati...

Structure and electrostatic property of cytoplasmic domain of ZntB transporter

“…12,25 The motif is located between two transmembrane ahelices on the outer surface of the membrane and it tends to be disordered in the crystal structures. [1][2][3] The mechanism of the ion selectivity for ZntB as a cation exporter has never been clear and it is not obvious why the metal ion needs to cross the entire pore to be selected for passage. While possible Mg 2þ gating mechanisms through the transmembrane were discussed in the Tm-CorA structures, 1-3 N314 residue at the external entrance to the pore and a pair of hydrophobic rings formed by M291 and L294, respectively, seemed to play important roles in selecting ion size and possible ion dehydration/rehydration steps.…”

“…The reported CorA intracellular domain dimers are likely to be artifacts of extensive truncation of stalk helix a6, in which the coiled-coil subdomain was dissociated. 1,3 A Vp-ZntB construct truncated at N220, corresponding to the truncated Tm-CorA (1-266), 1 would have dramatically reduced the monomer-monomer interface (a loss of $1000 Å 2 buried area) and is not expected to form a pentamer.…”

“…In general, the TmCorA pore is too constricted to allow for ion permeation, which is in line with the supposition that the structure is in a closed state. 1 On the other hand, the Vp-ZntB full-length model is more open with only a few regions reducing the pore to <5 Å [ Fig. 4(A)].…”

“…The three recently determined full-length Thermatoga maritima CorA (Tm-CorA) lower resolution structures (2.9, 3.7, and 3.9 Å) have revealed a striking homopentamer with two transmembrane a-helical spanners per monomer and a large funnel-shaped intracellular assembly. [1][2][3] However, two structures of cytoplasmic domains of Tm-CorA (1.85 Å) 1 and Archaeoglobus fulgidus CorA (Af-CorA) (2.9 Å) 3 showed dimeric arrangements of the CorA subunits. The observed conformational differences between the full-length TmCorA pentamer and the Af-CorA intracellular domain dimer were used to propose a close/open CorA translocation cycle model.…”

“…Analysis of metal binding sites suggested a possible gating mechanism linking the pore to the intracellular concentration of Mg 2þ . 1 Other divalent cations, such as Co 2þ and Ca 2þ can also be accommodated by CorA, 2,3 but it is not clear how binding the metal ions can influence transport through the pore. In one study, a putative Cl À anion was found near the metal-binding sites and was suggested to be important in stabilizing the conformation of the N-terminal subdomain.…”