{"id":{"repo_id":"toronto-retro","oai_identifier":"oai:utoronto.scholaris.ca:1807/102908"},"canonical_url":"https://search.dev.ndltd.org/etd/toronto-retro/oai:utoronto.scholaris.ca:1807/102908","repository":{"repo_id":"toronto-retro","name":"University of Toronto","base_url":"https://utoronto.scholaris.ca/server/oai/request"},"display":{"title":"Structure-Function Studies of CorA-Mrs2-Alr1 Superfamily of Magnesium Channels: Bacterial CorA and Yeast Alr1","abstract":"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.","abstract_html":"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.","abstract_has_math":false,"creators":["Kruglyak, Natalya"],"institution":null,"degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":"Biochemistry","school":null,"contributors":[],"advisors":["Pai, Emil F"],"committee_chairs":[],"committee_members":[],"year":2018,"date_issued":"2018-11","date_published":"2018-11","updated_at":"2026-07-27T21:28:22Z","subjects":["Intrinsically disordered proteins","Ion channels","Magnesium channels","Membrane proteins","Structural biology","X-ray crystallography"],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/1807/102908","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Pai, Emil F"]},{"key":"dc:contributor.department","label":"Department","values":["Biochemistry"]},{"key":"dc:creator","label":"Author","values":["Kruglyak, Natalya"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2018-11"]},{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2020-11-19T05:01:43Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2020-11-19T05:01:43Z"]},{"key":"dc:date.issued","label":"Date","values":["2018-11"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Intrinsically disordered proteins","Ion channels","Magnesium channels","Membrane proteins","Structural biology","X-ray crystallography"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://hdl.handle.net/1807/102908"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["Ph.D."]},{"key":"dc:title","label":"Title","values":["Structure-Function Studies of CorA-Mrs2-Alr1 Superfamily of Magnesium Channels: Bacterial CorA and Yeast Alr1"]}]}],"canonical_facts":{"dc:contributor.advisor":["Pai, Emil F"],"dc:contributor.department":["Biochemistry"],"dc:creator":["Kruglyak, Natalya"],"dc:date":["2018-11"],"dc:date.accessioned":["2020-11-19T05:01:43Z"],"dc:date.available":["2020-11-19T05:01:43Z"],"dc:date.issued":["2018-11"],"dc:description.abstract":["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."],"dc:description.degree":["Ph.D."],"dc:identifier.uri":["http://hdl.handle.net/1807/102908"],"dc:subject":["Intrinsically disordered proteins","Ion channels","Magnesium channels","Membrane proteins","Structural biology","X-ray crystallography"],"dc:title":["Structure-Function Studies of CorA-Mrs2-Alr1 Superfamily of Magnesium Channels: Bacterial CorA and Yeast Alr1"],"dc:type":["Thesis"]},"updated_at":"2026-07-27T21:28:22Z"}