Locking the Asymmetric open Conformation of MG2+ Channel Cora with a Synthetic Antibody Fragment

Mg2+ is the most abundant divalent ion in living cells. Cells have adapted sophisticated mechanisms to regulate, store and transport Mg2+ across the membrane. In prokaryotes, the major uptake pathway for Mg2+ is the ion channel CorA. X-ray structures of closed state CorA at high Mg2+ concentrations reveal a symmetric homopentameric assembly where Mg2+ ions are bound in between the cytoplasmic domains inter-locking the channel. Recent data obtained by single particle cryo-EM suggest that at low Mg2+ concentrations, the open state CorA adopts an asymmetric conformation in which one of the cytoplasmic subunits ‘sways away’ from the axis of conductance pathway.Here, we have identified high affinity synthetic antibody fragments (sAB) using a novel phage display protocol. sAB C12 was generated against nanodiscs-incorporated CorA under free Mg2+ conditions. Interestingly, even in the presence of Mg2+ the stoichiometry of sAB C12 binding to CorA remains at 1:5 (Fab:CorA), consistent with the asymmetric nature of the open state. Further, the crystal structure of the soluble domain of CorA in complex with sAB C12 shows that the sAB binds to a region of CorA not accessible in the closed state of the channel. Based on these results, additional analysis of C12-CorA interaction using biochemical, CW-EPR, and oocyte TEVC experiments confirm that the channel is locked in the open, conductive state. It is expected that sAB C12 complex can be used to gain further insights into this unique asymmetric conformation and to elucidate the molecular basis of Mg2+ permeation and gating in CorA.