Structural basis for Mg2+-dependent autoinhibition and stepwise gating in the Arabidopsis MRS2-1 Mg2+ channel
Abstract
Magnesium ion (Mg2+) is essential for plant growth and metabolism, and plant MRS2/MGT channels, which belong to the diverse metal ion transporter (MIT) superfamily, mediate Mg2+ transport for Mg2+ homeostasis, yet the structural basis of Mg2+ transport by plant MRS2/MGT channels remains poorly understood. Here, we determined cryo-EM structures of Arabidopsis thaliana MRS2-1 (AtMRS2-1) in the presence and absence of Mg2+, together with whole-cell patch-clamp recordings and molecular dynamics simulations to investigate its gating mechanism. In the Mg2+-bound structure, AtMRS2-1 adopts a closed conformation with multiple cytoplasmic Mg2+-binding sites. These sites are distinct from those reported in other bacterial and eukaryotic MIT-superfamily members, indicating divergence in cytoplasmic Mg2+ sensing within the MIT superfamily. Patch-clamp recordings showed that these cytoplasmic sites contribute to cytoplasmic Mg2+-dependent autoinhibition. Under Mg2+-free conditions, AtMRS2-1 adopts two distinct conformations. State II exhibits an expanded pore and likely corresponds to the open state. By contrast, State I displays partial expansion of the cytoplasmic domain while the pore remains closed, suggesting an intermediate state along the activation pathway. In State I, Arg225 in one subunit interacts with Tyr354 and Thr358 in the adjacent subunit, and disruption of these interactions impairs channel activity. Together, our results support a stepwise gating model in which dissociation of cytoplasmic Mg2+ drives cytoplasmic-domain rearrangement, leading to an intermediate state consistent with a preopen state before full pore opening. These findings provide a structural framework for Mg2+ sensing, autoinhibition, and stepwise gating in a plant MRS2 channel, while extending the comparative framework for MIT-superfamily gating mechanisms.
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