Structural basis for CorA channel gating using conformation-specific synthetic antigen binders and single-particle cryo-EM.

Abstract

The magnesium ion (Mg2+) is an abundant divalent cation in living cells, and plays a significant role in many biological processes, including energy metabolism, protein production, nucleotide replication and transcription, and muscle contraction. In prokaryotes CorA is the primary channel for Mg2+ uptake, and functionally complements the mitochondrial magnesium channel, Mrs2, whose dysfunction leads to cell death. Here we employ two different conformation-specific synthetic antigen binders (sABs) and single-particle cryo-EM to solve the structures of nanodisc-reconstituted T. maritima CorA in desaturating amounts of Mg2+ and in the complete absence of magnesium to resolutions of 3.1-3.4Å. We generated these sABs using a phage display library and competitive selections. We show CorA is nearly continuously variable in conformation when magnesium is even slightly depleted, but that discrete conformers can be isolated from our cryo-EM datasets due to our sAB-based approach. The resolutions in our dataset greatly improve on earlier cryo-EM studies, and these high-resolution structures represent the first ones in lipid-filled nanodiscs. These data show two major conformational changes as magnesium is depleted from regulatory cytoplasmic binding sites. One, the rearrangement of the cytoplasmic N-terminal regulatory region due to the absence of bound Mg2+, driven both by charge-based repulsion and compensating interactions. Two, concerted molecular motions driven by electrostatic interactions, leading to dilation of the pore and ostensibly channel activation. We also describe a conformational ensemble, with a population size inversely proportional to environmental [Mg2+], where both apparently inactive and active conformers present in equilibrium. Our new model shows CorA exists as metastable states in solution, with a stochastic relationship between open and closed channels, rather than a traditional deterministic mechanism. We believe our study provides a new template for understanding magnesium homeostasis in homologous systems.