Mg²⁺ Channel Selectivity probed by EPR

Summary

The functionally important extracellular loop is not resolved in crystal structures of a putative bacterial Mg²⁺ channel CorA. In this issue, Dalmas et al. use EPR to determine a structural model for this conserved loop, providing new insight into the ion selectivity.

With concentrations as high as 25 mM, magnesium (Mg²⁺) is the most abundant divalent cation in cells. Chemically, Mg²⁺ prefers the hexa-coordination and forms complexes with octahedral geometry. Moreover, its preponderance of oxygen as its ligand offers unique chemical and structural platforms that are utilized in structure-function biology: Mg²⁺ is woven into many enzymatic reactions in nearly all metabolic pathways. In particular, it acts as a cofactor for every reaction that generates energy from ATP hydrolysis. Also, Mg²⁺ is alloyed into various biological structures such as chlorophyll, RNA/DNA, and proteins and stabilizes their biologically active forms.

Although small fluctuations of Mg²⁺ concentration in the cell may be tolerated, a balance of Mg²⁺ within a certain range is vital for healthy cellular functions. Lipid membranes are impermeable to divalent cations. Therefore, cell membranes require transmembrane channels or transporters that allow Mg²⁺ to pass through in a controlled fashion. Up to now, many Mg²⁺ channels/transporters have been identified and crystal structures of some of them have been solved (Moomaw and Maguire, 2008).

The CorA protein, widespread in bacterial species, is a putative bacterial Mg²⁺ channel. It is a homopentamer and the overall fold of two CorA monomers within the pentameric structure is schematically shown in Figure 1. The structure can be divided into three sections: extracellular loops, the pore, and the cytoplasmic funnel. The funnel, fenced primarily by stalk helices, is quite unique and not seen in any other protein structures. When new channel structures were determined (Eshaghi et al., 2006; Lunin et al., 2006; Payandeh and Pai, 2006) the anticipation was to get an answer for the first and foremost question about the channel: How does this protein selectively transport Mg²⁺ among others ions? For CorA the selective filter may be located in the extracellular loop region because mutations in this conserved loop result in important functional effects. Unfortunately, this region is not resolved in the crystal structure. Thus, the mechanism of the ion selection still remains unknown.

Figure 1.

A structural model for Thermotoga maritima CorA in the membrane. Two monomers in the homopentameric arrangement are shown.

In a paper published in this issue of Structure Perozo and coworkers (Dalmas et al., 2010) present the first glimpse of the putative Mg²⁺ selectivity filter in CorA. The authors used a highly innovative method combining electron paramagnetic resonance (EPR) spectroscopy and theory to develop a structural model of the putative selectivity filter.

Perozo and coworkers use site-directed spin labeling (SDSL)(Altenbach et al., 1990) in which native residues are replaced one by one with cysteines and the nitroxide probe is attached to thus engineered cysteine residue. Accessibility of a water soluble paramagnetic species Ni(II)-EDDA to the nitroxide is measured with an EPR relaxation method for each position in the loop. In parallel, an initial structural model was derived from the Rosetta ab initio modeling. Finally, the structure is refined to fit the Ni(II)-EDDA accessibility data. Additionally, the authors find that the nitroxides attached at position 316 show strong spin-spin coupling (Rabenstein and Shin, 1995), indicating that residue 316 may project towards the center of the pentameric ring. Putting together the structure shows a negatively charged nest in which a hydrated Mg²⁺ ion can sit nicely. Perhaps, the nest is designed to accept only hydrated Mg²⁺, and discriminate against other divalent cations with different water coordination. Additionally, the electron negativity would aid the selection of divalent cations versus monovalent ones by concentrating the former near the nest. Further, the backbone and side chains are expected to be dynamic. Therefore, the structural plasticity and the induced fit upon binding might also play a role in discerning Mg²⁺ from other ions.

In K⁺ channels, the permeant ion enters the selectivity filter in a naked form after shedding the bound water molecules. In the filter the bare ion is stabilized by the tight interactions with carbonyls from the surrounding amino acids (Doyle et al., 1998). However, for Mg²⁺, the mechanism of ion selection may be entirely different. Energetically, dehydration of divalent cations is 5-6 times more difficult than that of monovalent cations. For Mg²⁺, a hexa-coordination on all vertices of an octahedron by protein oxygens in replacement of water molecules is geometrically difficult to satisfy in the selectivity filter, although partial replacement of water molecules is a possibility. Therefore, it is most likely that divalent cations enter into the selectivity filter as fully or partially hydrated forms and pass through the pore as hydrated. Therefore, selection of Mg²⁺ ion by the negatively charged nest could be entirely different from the case with K⁺ and the structural model of the selective filter of CorA presented here by Perozo and coworkers may have general implications for many channels/transporters for divalent cations.

The available CorA crystal structure determined in a detergent shows the pore is too narrow to allow hydrated Mg²⁺ to enter and pass. This ‘closed’ conformation is validated by the Perozo’s extensive EPR work in the lipid membrane, which paves the way to study the expected conformational changes with channel opening using EPR.

In the Perozo’s paper the authors used an innovative EPR method to determine the structure of the crystallographically unresolved extracellular loop of CorA, shedding a new light on the ion selectivity for divalent cations. Crystal structures often miss functionally important structural motifs. Thus, this new complementary EPR approach to crystallography may have the wide applicability in structure-functional studies of membrane proteins.