Characterizing Structural Changes of the Magnesium channel, CorA: Implications for Gating

CorA is the primary magnesium channel in prokaryotes and the most extensively studied member of the CorA/Mrs2/Alr1 superfamily of magnesium channels that extends into eukaryotes. Existing crystallographic structures define the closed state of CorA and provide a structural basis for its structure-function relationships. However, detailed mechanisms of gating and ion permeation remain poorly understood. In this thesis, a combination of biochemical and biophysical methods were used to understand the molecular details involved in CorA gating. Specifically, dynamic structural elements involved in CorA gating were identified through limited proteolysis and mass spectrometry. Cysteine cross-linking along with mutagenesis assays show that the formation of an inter-protomer salt-bridge at the membrane-cytosol interface may be a key component of the activation mechanism. This ties in with the earlier limited proteolysis and mass spectrometry results indicating that the channel exposes its membrane-cytosol interface during gating. Finally, we report the first structural elucidation of the CorA soluble domain in a pentameric arrangement. The structure reveals significantly increased closure of the soluble domain funnel compared to the regular closed state, suggesting that the membrane domain can impose constraints on the soluble domain to resist the closing motion of the funnel triggered by Mg2+ binding. A model is proposed based on this observation, which may explain the mechanics underlying channel gating. Altogether, these studies have furthered the understanding of the molecular basis of CorA gating, particularly on the communication mechanism linking structural rearrangements in the soluble regulatory domain and the motion of the membrane domain. Furthermore, the results from this thesis agree with a sequential gating mechanism in which the channel undergoes asymmetric conformational changes.Ph.D