8slb

Publication Abstract from PubMed

CorA, the primary magnesium ion channel in prokaryotes and archaea, is a prototypical homopentameric ion channel that undergoes ion-dependent conformational transitions. CorA adopts five-fold symmetric non-conductive states in the presence of high concentrations of Mg(2+), and highly asymmetric flexible states in its complete absence. However, the latter were of insufficient resolution to be thoroughly characterized. In order to gain additional insights into the relationship between asymmetry and channel activation, we exploited phage display selection strategies to generate conformation-specific synthetic antibodies (sABs) against CorA in the absence of Mg(2+). Two sABs from these selections, C12 and C18, showed different degrees of Mg(2+)-sensitivity. Through structural, biochemical, and biophysical characterization, we found the sABs are both conformation-specific but probe different features of the channel under open-like conditions. C18 is highly specific to the Mg(2+)-depleted state of CorA and through negative-stain electron microscopy (ns-EM), we show sAB binding reflects the asymmetric arrangement of CorA protomers in Mg(2+)-depleted conditions. We used X-ray crystallography to determine a structure at 2.0 A resolution of sAB C12 bound to the soluble N-terminal regulatory domain of CorA. The structure shows C12 is a competitive inhibitor of regulatory magnesium binding through its interaction with the divalent cation sensing site. We subsequently exploited this relationship to capture and visualize asymmetric CorA states in different [Mg(2+)] using ns-EM. We additionally utilized these sABs to provide insights into the energy landscape that governs the ion-dependent conformational transitions of CorA.

Conformation-specific Synthetic Antibodies Discriminate Multiple Functional States of the Ion Channel CorA.,Erramilli SK, Dominik PK, Deneka D, Tokarz P, Kim SS, Reddy BG, Skrobek BM, Dalmas O, Perozo E, Kossiakoff AA J Mol Biol. 2023 Sep 1;435(17):168192. doi: 10.1016/j.jmb.2023.168192. Epub 2023 , Jul 1. PMID:37394032^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.