Characterization of the molecular properties of KtrC, a second RCK domain that regulates a Ktr channel in Bacillus subtilis

Detalhes bibliográficos
Autor(a) principal: Rocha, R
Data de Publicação: 2019
Outros Autores: Teixeira-Duarte, CM, JMP, J, Morais-Cabral, JH
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
Texto Completo: https://hdl.handle.net/10216/127799
Resumo: RCK (regulating conductance of K+) domains are common regulatory domains that control the activity of eukaryotic and prokaryotic K+ channels and transporters. In bacteria these domains play roles in osmoregulation, regulation of turgor and membrane potential and in pH homeostasis. Whole-genome sequencing unveiled RCK gene redundancy, however the biological role of this redundancy is not well understood. In Bacillus subtilis, there are two closely related RCK domain proteins (KtrA and KtrC) that regulate the activity of the Ktr cation channels. KtrA has been well characterized but little is known about KtrC. We have characterized the structural and biochemical proprieties of KtrC and conclude that KtrC binds ATP and ADP, just like KtrA. However, in solution KtrC exist in a dynamic equilibrium between octamers and non-octameric species that is dependent on the bound ligand, with ATP destabilizing the octameric ring relative to ADP. Accordingly, KtrC-ADP crystal structures reveal closed octameric rings similar to those in KtrA, while KtrC-ATP adopts an open assembly with RCK domains forming a super-helix. In addition, both KtrC-ATP and -ADP octamers are stabilized by the signaling molecule cyclic-di-AMP, which binds to KtrC with high affinity. In contrast, c-di-AMP binds with 100-fold lower affinity to KtrA. Despite these differences we show with an E. coli complementation assay that KtrC and KtrA are interchangeable and able to form functional transporters with both KtrB and KtrD. The distinctive properties of KtrC, in particular ligand-dependent assembly/disassembly, suggest that this protein has a specific physiological role that is distinct from KtrA.
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spelling Characterization of the molecular properties of KtrC, a second RCK domain that regulates a Ktr channel in Bacillus subtilisAdenosine Diphosphate / chemistryAdenosine Diphosphate / metabolismAdenosine Triphosphate / chemistryAdenosine Triphosphate / metabolismAmino Acid MotifsBacillus subtilis / chemistryBacillus subtilis / metabolismBacterial Proteins / chemistryBacterial Proteins / geneticsBacterial Proteins / metabolismBinding SitesCation Transport Proteins / chemistryCation Transport Proteins / geneticsCation Transport Proteins / metabolismCations, MonovalentCloning, MolecularCrystallography, X-RayDinucleoside Phosphates / chemistryDinucleoside Phosphates / metabolismEscherichia coli / geneticsEscherichia coli / metabolismGene ExpressionGenetic Complementation TestGenetic Vectors / chemistryGenetic Vectors / metabolismIon TransportModels, MolecularPotassium / chemistryPotassium / metabolismProtein BindingProtein Conformation, alpha-HelicalProtein Conformation, beta-StrandProtein Interaction Domains and MotifsProtein Isoforms / chemistryProtein Isoforms / geneticsProtein Isoforms / metabolismProtein MultimerizationRecombinant Proteins / chemistryRecombinant Proteins / geneticsRecombinant Proteins / metabolismRCK (regulating conductance of K+) domains are common regulatory domains that control the activity of eukaryotic and prokaryotic K+ channels and transporters. In bacteria these domains play roles in osmoregulation, regulation of turgor and membrane potential and in pH homeostasis. Whole-genome sequencing unveiled RCK gene redundancy, however the biological role of this redundancy is not well understood. In Bacillus subtilis, there are two closely related RCK domain proteins (KtrA and KtrC) that regulate the activity of the Ktr cation channels. KtrA has been well characterized but little is known about KtrC. We have characterized the structural and biochemical proprieties of KtrC and conclude that KtrC binds ATP and ADP, just like KtrA. However, in solution KtrC exist in a dynamic equilibrium between octamers and non-octameric species that is dependent on the bound ligand, with ATP destabilizing the octameric ring relative to ADP. Accordingly, KtrC-ADP crystal structures reveal closed octameric rings similar to those in KtrA, while KtrC-ATP adopts an open assembly with RCK domains forming a super-helix. In addition, both KtrC-ATP and -ADP octamers are stabilized by the signaling molecule cyclic-di-AMP, which binds to KtrC with high affinity. In contrast, c-di-AMP binds with 100-fold lower affinity to KtrA. Despite these differences we show with an E. coli complementation assay that KtrC and KtrA are interchangeable and able to form functional transporters with both KtrB and KtrD. The distinctive properties of KtrC, in particular ligand-dependent assembly/disassembly, suggest that this protein has a specific physiological role that is distinct from KtrA.Academic Press20192019-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfapplication/vnd.openxmlformats-officedocument.wordprocessingml.documenthttps://hdl.handle.net/10216/127799eng1047-847710.1016/j.jsb.2019.02.002Rocha, RTeixeira-Duarte, CMJMP, JMorais-Cabral, JHinfo:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2023-11-29T15:41:32Zoai:repositorio-aberto.up.pt:10216/127799Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T00:29:49.244297Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse
dc.title.none.fl_str_mv Characterization of the molecular properties of KtrC, a second RCK domain that regulates a Ktr channel in Bacillus subtilis
title Characterization of the molecular properties of KtrC, a second RCK domain that regulates a Ktr channel in Bacillus subtilis
spellingShingle Characterization of the molecular properties of KtrC, a second RCK domain that regulates a Ktr channel in Bacillus subtilis
Rocha, R
Adenosine Diphosphate / chemistry
Adenosine Diphosphate / metabolism
Adenosine Triphosphate / chemistry
Adenosine Triphosphate / metabolism
Amino Acid Motifs
Bacillus subtilis / chemistry
Bacillus subtilis / metabolism
Bacterial Proteins / chemistry
Bacterial Proteins / genetics
Bacterial Proteins / metabolism
Binding Sites
Cation Transport Proteins / chemistry
Cation Transport Proteins / genetics
Cation Transport Proteins / metabolism
Cations, Monovalent
Cloning, Molecular
Crystallography, X-Ray
Dinucleoside Phosphates / chemistry
Dinucleoside Phosphates / metabolism
Escherichia coli / genetics
Escherichia coli / metabolism
Gene Expression
Genetic Complementation Test
Genetic Vectors / chemistry
Genetic Vectors / metabolism
Ion Transport
Models, Molecular
Potassium / chemistry
Potassium / metabolism
Protein Binding
Protein Conformation, alpha-Helical
Protein Conformation, beta-Strand
Protein Interaction Domains and Motifs
Protein Isoforms / chemistry
Protein Isoforms / genetics
Protein Isoforms / metabolism
Protein Multimerization
Recombinant Proteins / chemistry
Recombinant Proteins / genetics
Recombinant Proteins / metabolism
title_short Characterization of the molecular properties of KtrC, a second RCK domain that regulates a Ktr channel in Bacillus subtilis
title_full Characterization of the molecular properties of KtrC, a second RCK domain that regulates a Ktr channel in Bacillus subtilis
title_fullStr Characterization of the molecular properties of KtrC, a second RCK domain that regulates a Ktr channel in Bacillus subtilis
title_full_unstemmed Characterization of the molecular properties of KtrC, a second RCK domain that regulates a Ktr channel in Bacillus subtilis
title_sort Characterization of the molecular properties of KtrC, a second RCK domain that regulates a Ktr channel in Bacillus subtilis
author Rocha, R
author_facet Rocha, R
Teixeira-Duarte, CM
JMP, J
Morais-Cabral, JH
author_role author
author2 Teixeira-Duarte, CM
JMP, J
Morais-Cabral, JH
author2_role author
author
author
dc.contributor.author.fl_str_mv Rocha, R
Teixeira-Duarte, CM
JMP, J
Morais-Cabral, JH
dc.subject.por.fl_str_mv Adenosine Diphosphate / chemistry
Adenosine Diphosphate / metabolism
Adenosine Triphosphate / chemistry
Adenosine Triphosphate / metabolism
Amino Acid Motifs
Bacillus subtilis / chemistry
Bacillus subtilis / metabolism
Bacterial Proteins / chemistry
Bacterial Proteins / genetics
Bacterial Proteins / metabolism
Binding Sites
Cation Transport Proteins / chemistry
Cation Transport Proteins / genetics
Cation Transport Proteins / metabolism
Cations, Monovalent
Cloning, Molecular
Crystallography, X-Ray
Dinucleoside Phosphates / chemistry
Dinucleoside Phosphates / metabolism
Escherichia coli / genetics
Escherichia coli / metabolism
Gene Expression
Genetic Complementation Test
Genetic Vectors / chemistry
Genetic Vectors / metabolism
Ion Transport
Models, Molecular
Potassium / chemistry
Potassium / metabolism
Protein Binding
Protein Conformation, alpha-Helical
Protein Conformation, beta-Strand
Protein Interaction Domains and Motifs
Protein Isoforms / chemistry
Protein Isoforms / genetics
Protein Isoforms / metabolism
Protein Multimerization
Recombinant Proteins / chemistry
Recombinant Proteins / genetics
Recombinant Proteins / metabolism
topic Adenosine Diphosphate / chemistry
Adenosine Diphosphate / metabolism
Adenosine Triphosphate / chemistry
Adenosine Triphosphate / metabolism
Amino Acid Motifs
Bacillus subtilis / chemistry
Bacillus subtilis / metabolism
Bacterial Proteins / chemistry
Bacterial Proteins / genetics
Bacterial Proteins / metabolism
Binding Sites
Cation Transport Proteins / chemistry
Cation Transport Proteins / genetics
Cation Transport Proteins / metabolism
Cations, Monovalent
Cloning, Molecular
Crystallography, X-Ray
Dinucleoside Phosphates / chemistry
Dinucleoside Phosphates / metabolism
Escherichia coli / genetics
Escherichia coli / metabolism
Gene Expression
Genetic Complementation Test
Genetic Vectors / chemistry
Genetic Vectors / metabolism
Ion Transport
Models, Molecular
Potassium / chemistry
Potassium / metabolism
Protein Binding
Protein Conformation, alpha-Helical
Protein Conformation, beta-Strand
Protein Interaction Domains and Motifs
Protein Isoforms / chemistry
Protein Isoforms / genetics
Protein Isoforms / metabolism
Protein Multimerization
Recombinant Proteins / chemistry
Recombinant Proteins / genetics
Recombinant Proteins / metabolism
description RCK (regulating conductance of K+) domains are common regulatory domains that control the activity of eukaryotic and prokaryotic K+ channels and transporters. In bacteria these domains play roles in osmoregulation, regulation of turgor and membrane potential and in pH homeostasis. Whole-genome sequencing unveiled RCK gene redundancy, however the biological role of this redundancy is not well understood. In Bacillus subtilis, there are two closely related RCK domain proteins (KtrA and KtrC) that regulate the activity of the Ktr cation channels. KtrA has been well characterized but little is known about KtrC. We have characterized the structural and biochemical proprieties of KtrC and conclude that KtrC binds ATP and ADP, just like KtrA. However, in solution KtrC exist in a dynamic equilibrium between octamers and non-octameric species that is dependent on the bound ligand, with ATP destabilizing the octameric ring relative to ADP. Accordingly, KtrC-ADP crystal structures reveal closed octameric rings similar to those in KtrA, while KtrC-ATP adopts an open assembly with RCK domains forming a super-helix. In addition, both KtrC-ATP and -ADP octamers are stabilized by the signaling molecule cyclic-di-AMP, which binds to KtrC with high affinity. In contrast, c-di-AMP binds with 100-fold lower affinity to KtrA. Despite these differences we show with an E. coli complementation assay that KtrC and KtrA are interchangeable and able to form functional transporters with both KtrB and KtrD. The distinctive properties of KtrC, in particular ligand-dependent assembly/disassembly, suggest that this protein has a specific physiological role that is distinct from KtrA.
publishDate 2019
dc.date.none.fl_str_mv 2019
2019-01-01T00:00:00Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv https://hdl.handle.net/10216/127799
url https://hdl.handle.net/10216/127799
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv 1047-8477
10.1016/j.jsb.2019.02.002
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dc.publisher.none.fl_str_mv Academic Press
publisher.none.fl_str_mv Academic Press
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