Insights into ion selectivity and translocation in the bacterial magnesium channel CorA provided by anomalous diffraction experiments

Natasha Kruglyak^1,4, Roland Pfoh^1,4, Pedram Mehrabi^2,4, Emil F. Pai^1,2,3,4

¹ Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada

²Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada

³ Department of Molecular and Medical Genetics, University of Toronto, Toronto,

Ontario, Canada

⁴ Campbell Family Cancer Research Institute/Ontario Cancer Institute, Toronto, Ontario, Canada

natasha.kruglyak@mail.utoronto.ca

Magnesium (Mg²⁺) is the most abundant divalent cation in eukaryotic and prokaryotic cells, having numerous important physiological functions [1]. However, of the four main biological cations, the transport and homeostasis of magnesium remains the least understood. Members of the ubiquitous CorA family of Mg²⁺ channels contain a canonical Gly-Met-Asn (₃₁₂GMN₃₁₄) signature motif at the extracellular mouth of the permeation pathway, which has been proposed to form part of the Mg²⁺ selectivity filter of these channels [2-4]. In this study, we use anomalous x-ray diffraction to examine the binding at the selectivity filter of two transported substrates of CorA, cobalt (Co²⁺) and nickel (Ni²⁺), the non-substrate samarium (Sm³⁺) as well as the known CorA inhibitor cobalthexammine, which is also an inert structural analogue of hexahydrated Mg²⁺[5]. Our results indicate that while Co²⁺ and Ni²⁺ are able to bind at the ₃₁₂GMN₃₁₄ motif coordinating with G₃₁₂ and N₃₁₄, Sm³⁺ is excluded from the selectivity filter. Cobalthexammine is also able to bind at the mouth of the CorA pore, but at a position slightly peripheral to the ₃₁₂GMN₃₁₄ motif, suggesting that CorA allows binding of a hexahydrated Mg²⁺, but only in a position that does not allow further penetration into the selectivity filter, explaining the inhibitory effect of cobalthexammine. Moreover, we suggest that our results strongly support a knock-on mechanism for ion transduction through the CorA pore, where a Mg²⁺ bound at G₃₁₂ and N₃₁₄ is pushed down the permeation pathway by an incoming Mg²⁺ bound at N₃₁₄ (Figure 1).

Figure 1. Co²⁺ and cobalthexammine binding at the GMN motif of CorA. The position of the cobalthexammine is superimposed onto the structure obtained with Co²⁺. Only four protomers and the anomalous density for Co²⁺ (cyan mesh) are shown for clarity. The arrow indicates the direction of ion translocation.

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