{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/45599"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/45599","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Mechanistic studies of ABC transporters","abstract":"ATP-binding cassette (ABC) transporters are a superfamily of primary membrane transporters that are abundant yet highly diverse in structures and functions. These proteins power the active transport of a large variety of substrates across biological membranes, by harnessing the energy of ATP binding and hydrolysis and coupling to their conformational changes, which facilitate the substrate translocation with an alternating-access mechanism. The architecture of ABC transporters contains two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs) locating at the cytoplasmic side of the membrane. While the TMDs provide the pathway for substrate translocation and are highly variable among different subfamilies, the NBDs provide the power source of the active transport and are highly conserved. Since ATP binding/hydrolysis takes place remotely from the membrane, the transport mechanism is an allosteric control of the TMD conformations through nucleotide and/or substrate binding. In isolated NBDs, the conformational changes induced by ATP binding and hydrolysis have been shown to be the dimer formation and dissociation, since the nucleotide binding site is locating at the dimer interface. However the nature of conformational changes in the context of full transporters is still under debate, despite the crystal structures of several ABC transporters have been resolved in different conformations. Multiple contradicting mechanistic models have been proposed based on biochemical and structural characterizations of different ABC transporters. The major issues of the controversy include, whether a common mechanism can be applied to all ABC transporter, and if not, what is shared by different mechanisms and what are the distinguishing features. On the other hand, although different NBD conformations have been captured in crystal structures, the static pictures obtained through crystallography only provide characterization the two end states but no little information about the transitions between the two. That is, how the NBD dimer separates after hydrolysis, and how do separated NBDs form dimer upon ATP binding, have not yet been described explicitly. Due to the lack of information, the mode of action of the NBDs is also disagreed among different mechanistic models. Specifically, whether the hydrolysis and conformational changes in NBDs should take place symmetrically or alternatingly, and how the TMDs correspond to the conformational changes in the NBDs, are keys to address the controversies among different proposed mechanisms. Using molecular dynamics simulations of isolated NBDs and full ABC transporters, I have investigated the nature of conformational changes in ABC transporters. Firstly, the effect of ATP hydrolysis is studied in an isolated NBD dimer. It is found that the dimer opening does not require hydrolysis at both NBDs, and the hydrolysis reaction itself is able to trigger the opening without the dissociation of the hydrolysis products. The key factors determining the formation of NBD dimers was identified through simulations of isolated NBD dimers with altered chemical properties at several conserved residues. Specifically, a strictly conserved H-bond at the dimer interface which ruptures upon ATP hydrolysis, and long-range electrostatic interactions are both required for the maintenance of a stable ATP-bound dimeric NBD conformation. The dynamics of the transporters appear to be different in different types of ABC transporter, suggesting likely distinct transport mechanisms. The coupling mechanisms between the NBDs and TMDs are also different among subfamilies, possibly contributing to the variations between their transport mechanisms. Nevertheless, a conserved NBD-TMD coupling mechanism is identified in one subfamily of ABC transporters, that several conserved motifs connect the NBD-TMD interface and form rigid bodies even under different conformational states. Finally, formations of transient water-conducting states have been identified in the simulations of full ABC transporters, which only occurs when transport-relevant conformational transitions are taking place, suggesting that the alternating-access mechanism of transport might only apply to the substrate, and the transporter can be leaky for smaller species including water and ions during the transitions between major conformational states.","abstract_html":"ATP-binding cassette (ABC) transporters are a superfamily of primary membrane transporters that are abundant yet highly diverse in structures and functions. These proteins power the active transport of a large variety of substrates across biological membranes, by harnessing the energy of ATP binding and hydrolysis and coupling to their conformational changes, which facilitate the substrate translocation with an alternating-access mechanism. The architecture of ABC transporters contains two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs) locating at the cytoplasmic side of the membrane. While the TMDs provide the pathway for substrate translocation and are highly variable among different subfamilies, the NBDs provide the power source of the active transport and are highly conserved. Since ATP binding/hydrolysis takes place remotely from the membrane, the transport mechanism is an allosteric control of the TMD conformations through nucleotide and/or substrate binding. In isolated NBDs, the conformational changes induced by ATP binding and hydrolysis have been shown to be the dimer formation and dissociation, since the nucleotide binding site is locating at the dimer interface. However the nature of conformational changes in the context of full transporters is still under debate, despite the crystal structures of several ABC transporters have been resolved in different conformations. Multiple contradicting mechanistic models have been proposed based on biochemical and structural characterizations of different ABC transporters. The major issues of the controversy include, whether a common mechanism can be applied to all ABC transporter, and if not, what is shared by different mechanisms and what are the distinguishing features. On the other hand, although different NBD conformations have been captured in crystal structures, the static pictures obtained through crystallography only provide characterization the two end states but no little information about the transitions between the two. That is, how the NBD dimer separates after hydrolysis, and how do separated NBDs form dimer upon ATP binding, have not yet been described explicitly. Due to the lack of information, the mode of action of the NBDs is also disagreed among different mechanistic models. Specifically, whether the hydrolysis and conformational changes in NBDs should take place symmetrically or alternatingly, and how the TMDs correspond to the conformational changes in the NBDs, are keys to address the controversies among different proposed mechanisms. Using molecular dynamics simulations of isolated NBDs and full ABC transporters, I have investigated the nature of conformational changes in ABC transporters. Firstly, the effect of ATP hydrolysis is studied in an isolated NBD dimer. It is found that the dimer opening does not require hydrolysis at both NBDs, and the hydrolysis reaction itself is able to trigger the opening without the dissociation of the hydrolysis products. The key factors determining the formation of NBD dimers was identified through simulations of isolated NBD dimers with altered chemical properties at several conserved residues. Specifically, a strictly conserved H-bond at the dimer interface which ruptures upon ATP hydrolysis, and long-range electrostatic interactions are both required for the maintenance of a stable ATP-bound dimeric NBD conformation. The dynamics of the transporters appear to be different in different types of ABC transporter, suggesting likely distinct transport mechanisms. The coupling mechanisms between the NBDs and TMDs are also different among subfamilies, possibly contributing to the variations between their transport mechanisms. Nevertheless, a conserved NBD-TMD coupling mechanism is identified in one subfamily of ABC transporters, that several conserved motifs connect the NBD-TMD interface and form rigid bodies even under different conformational states. Finally, formations of transient water-conducting states have been identified in the simulations of full ABC transporters, which only occurs when transport-relevant conformational transitions are taking place, suggesting that the alternating-access mechanism of transport might only apply to the substrate, and the transporter can be leaky for smaller species including water and ions during the transitions between major conformational states.","abstract_has_math":false,"creators":["Wen, Po-Chao"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Biophysics & Computnl Biology","degree_department":null,"school":null,"contributors":["Tajkhorshid, Emad","Grosman, Claudio F.","Gruebele, Martin","Luthey-Schulten, Zaida A."],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2013,"date_issued":"2013-08-22T16:49:10Z","date_published":"2013-08-22T16:49:10Z","updated_at":"2026-07-22T22:25:36Z","subjects":["ATP-binding cassette (ABC) transporters transporters","Molecular dynamics","Membrane transport","Protein dynamics","Conformational change","Maltose transporter","P-Glycoprotein"],"languages":["en"],"rights":["Copyright 2013 Po-Chao Wen"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/2142/45599","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Tajkhorshid, Emad","Grosman, Claudio F.","Gruebele, Martin","Luthey-Schulten, Zaida A."]},{"key":"dc:creator","label":"Author","values":["Wen, Po-Chao"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2013-08-22T16:49:10Z","2015-08-22T10:00:51Z","2013-08"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biophysics & Computnl Biology"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois at Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["ATP-binding cassette (ABC) transporters transporters","Molecular dynamics","Membrane transport","Protein dynamics","Conformational change","Maltose transporter","P-Glycoprotein"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 2013 Po-Chao Wen"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/2142/45599"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["ATP-binding cassette (ABC) transporters are a superfamily of primary membrane transporters that are abundant yet highly diverse in structures and functions. These proteins power the active transport of a large variety of substrates across biological membranes, by harnessing the energy of ATP binding and hydrolysis and coupling to their conformational changes, which facilitate the substrate translocation with an alternating-access mechanism. The architecture of ABC transporters contains two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs) locating at the cytoplasmic side of the membrane. While the TMDs provide the pathway for substrate translocation and are highly variable among different subfamilies, the NBDs provide the power source of the active transport and are highly conserved. Since ATP binding/hydrolysis takes place remotely from the membrane, the transport mechanism is an allosteric control of the TMD conformations through nucleotide and/or substrate binding. In isolated NBDs, the conformational changes induced by ATP binding and hydrolysis have been shown to be the dimer formation and dissociation, since the nucleotide binding site is locating at the dimer interface. However the nature of conformational changes in the context of full transporters is still under debate, despite the crystal structures of several ABC transporters have been resolved in different conformations. Multiple contradicting mechanistic models have been proposed based on biochemical and structural characterizations of different ABC transporters. The major issues of the controversy include, whether a common mechanism can be applied to all ABC transporter, and if not, what is shared by different mechanisms and what are the distinguishing features. On the other hand, although different NBD conformations have been captured in crystal structures, the static pictures obtained through crystallography only provide characterization the two end states but no little information about the transitions between the two. That is, how the NBD dimer separates after hydrolysis, and how do separated NBDs form dimer upon ATP binding, have not yet been described explicitly. Due to the lack of information, the mode of action of the NBDs is also disagreed among different mechanistic models. Specifically, whether the hydrolysis and conformational changes in NBDs should take place symmetrically or alternatingly, and how the TMDs correspond to the conformational changes in the NBDs, are keys to address the controversies among different proposed mechanisms. Using molecular dynamics simulations of isolated NBDs and full ABC transporters, I have investigated the nature of conformational changes in ABC transporters. Firstly, the effect of ATP hydrolysis is studied in an isolated NBD dimer. It is found that the dimer opening does not require hydrolysis at both NBDs, and the hydrolysis reaction itself is able to trigger the opening without the dissociation of the hydrolysis products. The key factors determining the formation of NBD dimers was identified through simulations of isolated NBD dimers with altered chemical properties at several conserved residues. Specifically, a strictly conserved H-bond at the dimer interface which ruptures upon ATP hydrolysis, and long-range electrostatic interactions are both required for the maintenance of a stable ATP-bound dimeric NBD conformation. The dynamics of the transporters appear to be different in different types of ABC transporter, suggesting likely distinct transport mechanisms. The coupling mechanisms between the NBDs and TMDs are also different among subfamilies, possibly contributing to the variations between their transport mechanisms. Nevertheless, a conserved NBD-TMD coupling mechanism is identified in one subfamily of ABC transporters, that several conserved motifs connect the NBD-TMD interface and form rigid bodies even under different conformational states. Finally, formations of transient water-conducting states have been identified in the simulations of full ABC transporters, which only occurs when transport-relevant conformational transitions are taking place, suggesting that the alternating-access mechanism of transport might only apply to the substrate, and the transporter can be leaky for smaller species including water and ions during the transitions between major conformational states.","Item withdrawn by Mark Zulauf (zulauf@illinois.edu) on 2013-06-27T19:26:02Z Item was in collections: University of Illinois Theses & Dissertations (ID: 1) No. of bitstreams: 1 Po-Chao_Wen.pdf: 86145251 bytes, checksum: 54ca03b9a2f2eaac3c33f640a0f322cd (MD5)","Made available in DSpace on 2013-08-22T16:49:10Z (GMT). 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These proteins power the active transport of a large variety of substrates across biological membranes, by harnessing the energy of ATP binding and hydrolysis and coupling to their conformational changes, which facilitate the substrate translocation with an alternating-access mechanism. The architecture of ABC transporters contains two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs) locating at the cytoplasmic side of the membrane. While the TMDs provide the pathway for substrate translocation and are highly variable among different subfamilies, the NBDs provide the power source of the active transport and are highly conserved. Since ATP binding/hydrolysis takes place remotely from the membrane, the transport mechanism is an allosteric control of the TMD conformations through nucleotide and/or substrate binding. In isolated NBDs, the conformational changes induced by ATP binding and hydrolysis have been shown to be the dimer formation and dissociation, since the nucleotide binding site is locating at the dimer interface. However the nature of conformational changes in the context of full transporters is still under debate, despite the crystal structures of several ABC transporters have been resolved in different conformations. Multiple contradicting mechanistic models have been proposed based on biochemical and structural characterizations of different ABC transporters. The major issues of the controversy include, whether a common mechanism can be applied to all ABC transporter, and if not, what is shared by different mechanisms and what are the distinguishing features. On the other hand, although different NBD conformations have been captured in crystal structures, the static pictures obtained through crystallography only provide characterization the two end states but no little information about the transitions between the two. That is, how the NBD dimer separates after hydrolysis, and how do separated NBDs form dimer upon ATP binding, have not yet been described explicitly. Due to the lack of information, the mode of action of the NBDs is also disagreed among different mechanistic models. Specifically, whether the hydrolysis and conformational changes in NBDs should take place symmetrically or alternatingly, and how the TMDs correspond to the conformational changes in the NBDs, are keys to address the controversies among different proposed mechanisms. Using molecular dynamics simulations of isolated NBDs and full ABC transporters, I have investigated the nature of conformational changes in ABC transporters. Firstly, the effect of ATP hydrolysis is studied in an isolated NBD dimer. It is found that the dimer opening does not require hydrolysis at both NBDs, and the hydrolysis reaction itself is able to trigger the opening without the dissociation of the hydrolysis products. The key factors determining the formation of NBD dimers was identified through simulations of isolated NBD dimers with altered chemical properties at several conserved residues. Specifically, a strictly conserved H-bond at the dimer interface which ruptures upon ATP hydrolysis, and long-range electrostatic interactions are both required for the maintenance of a stable ATP-bound dimeric NBD conformation. The dynamics of the transporters appear to be different in different types of ABC transporter, suggesting likely distinct transport mechanisms. The coupling mechanisms between the NBDs and TMDs are also different among subfamilies, possibly contributing to the variations between their transport mechanisms. Nevertheless, a conserved NBD-TMD coupling mechanism is identified in one subfamily of ABC transporters, that several conserved motifs connect the NBD-TMD interface and form rigid bodies even under different conformational states. Finally, formations of transient water-conducting states have been identified in the simulations of full ABC transporters, which only occurs when transport-relevant conformational transitions are taking place, suggesting that the alternating-access mechanism of transport might only apply to the substrate, and the transporter can be leaky for smaller species including water and ions during the transitions between major conformational states.","Item withdrawn by Mark Zulauf (zulauf@illinois.edu) on 2013-06-27T19:26:02Z Item was in collections: University of Illinois Theses & Dissertations (ID: 1) No. of bitstreams: 1 Po-Chao_Wen.pdf: 86145251 bytes, checksum: 54ca03b9a2f2eaac3c33f640a0f322cd (MD5)","Made available in DSpace on 2013-08-22T16:49:10Z (GMT). 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