Structure-Function Studies of CorA-Mrs2-Alr1 Superfamily of Magnesium Channels: Bacterial CorA and Yeast Alr1

Magnesium (Mg2+) plays an important role in many essential cellular processes, and its concentration in the cell is tightly controlled by the interplay between various Mg2+ transporters and channels. The ubiquitous CorA-Mrs2-Alr1 superfamily is the most widespread and the best studied family of Mg2+ channels to date. Nevertheless, the transport and homeostasis of Mg2+ in both prokaryotic and eukaryotic cells remain poorly understood. This thesis details the investigation of the structure-function relationship of the bacterial CorA and yeast Alr1 using biophysical, biochemical and bioinformatic approaches. Members of the CorA-Mrs2-Alr1 superfamily contain a canonical GMN motif located at the entrance to the ion translocation pore, which has been proposed to function as the Mg2+ selectivity filter. Using anomalous x-ray diffraction, we show that the CorA substrates, cobalt and nickel, which have similar physicochemical properties to Mg2+, are able to bind at the GMN motif in a manner similar to Mg2+. The CorA non-substrate samarium only binds at the periplasmic loops of CorA, while cobalthexaammine, a CorA inhibitor and an inert structural analogue of a hexahydrated Mg2+, binds at the periplasmic loops and slightly above the GMN motif. Altogether, our results suggest that the GMN motif selects for Mg2+ out of an indiscriminate pool of cations at the periplasmic loops region based on the ion’s size and the geometry of its first hydration shell. Moreover, we suggest a knock-on mechanism for ion transduction through CorA, with the cobalthexammine binding site slightly above the GMN motif marking the knock-on site for hexahydrated Mg2+. Alr1 structurally differs from other members of the CorA-Mrs2-Alr1 superfamily by a long N-terminal extension preceding the CorA domain. Bioinformatic analysis indicates that the N-terminal extension is mostly unstructured and contains multiple molecular recognition and phosphorylation sites, suggesting that it may participate in protein-protein interactions. Furthermore, we experimentally characterized the first 377 amino acids of Alr1 as intrinsically disordered. Truncation of up to 297 amino acids from the N-terminus resulted in no effect on Alr1 activity. We hypothesize that the intrinsically disordered region of the Alr1 protein may serve a function unrelated to Mg2+ uptake through Alr1, like protein regulation.Ph.D.2020-11-19 00:00:0