Cysteine Mutants of the Major Facilitator Superfamily-Type Transporter CcoA Provide Insight into Copper Import

ABSTRACT CcoA belongs to the widely distributed bacterial copper (Cu) importer subfamily CalT (CcoA-like Transporters) of the Major Facilitator Superfamily (MFS) and provides cytoplasmic Cu needed for cbb3-type cytochrome c oxidase (cbb3-Cox) biogenesis. Earlier studies have supported a 12-transmemb...

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Autores principales: Bahia Khalfaoui-Hassani, Petru-Iulian Trasnea, Stefan Steimle, Hans-Georg Koch, Fevzi Daldal
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Publicado: American Society for Microbiology 2021
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spelling oai:doaj.org-article:012f265fa2074b8d8c69080b2abf52442021-11-10T18:37:51ZCysteine Mutants of the Major Facilitator Superfamily-Type Transporter CcoA Provide Insight into Copper Import10.1128/mBio.01567-212150-7511https://doaj.org/article/012f265fa2074b8d8c69080b2abf52442021-08-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01567-21https://doaj.org/toc/2150-7511ABSTRACT CcoA belongs to the widely distributed bacterial copper (Cu) importer subfamily CalT (CcoA-like Transporters) of the Major Facilitator Superfamily (MFS) and provides cytoplasmic Cu needed for cbb3-type cytochrome c oxidase (cbb3-Cox) biogenesis. Earlier studies have supported a 12-transmembrane helix (TMH) topology of CcoA with the well-conserved Met233xxxMet237 and His261xxxMet265 motifs in its TMH7 and TMH8, respectively. Of these residues, Met233 and His261 are essential for Cu uptake and cbb3-Cox production, whereas Met237 and Met265 contribute partly to these processes. CcoA also contains five Cys residues of unknown role and, remarkably, its structural models predict that three of these are exposed to the highly oxidizing periplasm. Here, we first demonstrate that elimination of both Met237 and Met265 completely abolishes Cu uptake and cbb3-Cox production, indicating that CcoA requires at least one of these two Met residues for activity. Second, using scanning mutagenesis to probe plausible metal-interacting Met, His, and Cys residues of CcoA, we found that the periplasm-exposed Cys49 located at the end of TMH2, the Cys247 on a surface loop between TMH7 and THM8, and the C367 located at the end of TMH11 are important for CcoA function. Analyses of the single and double Cys mutants revealed the occurrence of a disulfide bond in CcoA in vivo, possibly related to conformational changes it undergoes during Cu import as MFS-type transporter. Our overall findings suggest a model linking Cu import for cbb3-Cox biogenesis with a thiol:disulfide oxidoreduction step, advancing our understanding of the mechanisms of CcoA function. IMPORTANCE Copper (Cu) is a redox-active micronutrient that is both essential and toxic. Its cellular homeostasis is critical for supporting cuproprotein maturation while avoiding excessive oxidative stress. The Cu importer CcoA is the prototype of the widespread CalT subfamily of the MFS-type transporters. Hence, understanding its molecular mechanism of function is significant. Here, we show that CcoA undergoes a thiol:disulfide oxidoreduction cycle, which is important for its Cu import activity.Bahia Khalfaoui-HassaniPetru-Iulian TrasneaStefan SteimleHans-Georg KochFevzi DaldalAmerican Society for Microbiologyarticlecopper uptakecbb3-type cytochrome c oxidasecopper-binding residuesMFS-type transportersRhodobacter capsulatus CcoAbacterial copper importMicrobiologyQR1-502ENmBio, Vol 12, Iss 4 (2021)
institution DOAJ
collection DOAJ
language EN
topic copper uptake
cbb3-type cytochrome c oxidase
copper-binding residues
MFS-type transporters
Rhodobacter capsulatus CcoA
bacterial copper import
Microbiology
QR1-502
spellingShingle copper uptake
cbb3-type cytochrome c oxidase
copper-binding residues
MFS-type transporters
Rhodobacter capsulatus CcoA
bacterial copper import
Microbiology
QR1-502
Bahia Khalfaoui-Hassani
Petru-Iulian Trasnea
Stefan Steimle
Hans-Georg Koch
Fevzi Daldal
Cysteine Mutants of the Major Facilitator Superfamily-Type Transporter CcoA Provide Insight into Copper Import
description ABSTRACT CcoA belongs to the widely distributed bacterial copper (Cu) importer subfamily CalT (CcoA-like Transporters) of the Major Facilitator Superfamily (MFS) and provides cytoplasmic Cu needed for cbb3-type cytochrome c oxidase (cbb3-Cox) biogenesis. Earlier studies have supported a 12-transmembrane helix (TMH) topology of CcoA with the well-conserved Met233xxxMet237 and His261xxxMet265 motifs in its TMH7 and TMH8, respectively. Of these residues, Met233 and His261 are essential for Cu uptake and cbb3-Cox production, whereas Met237 and Met265 contribute partly to these processes. CcoA also contains five Cys residues of unknown role and, remarkably, its structural models predict that three of these are exposed to the highly oxidizing periplasm. Here, we first demonstrate that elimination of both Met237 and Met265 completely abolishes Cu uptake and cbb3-Cox production, indicating that CcoA requires at least one of these two Met residues for activity. Second, using scanning mutagenesis to probe plausible metal-interacting Met, His, and Cys residues of CcoA, we found that the periplasm-exposed Cys49 located at the end of TMH2, the Cys247 on a surface loop between TMH7 and THM8, and the C367 located at the end of TMH11 are important for CcoA function. Analyses of the single and double Cys mutants revealed the occurrence of a disulfide bond in CcoA in vivo, possibly related to conformational changes it undergoes during Cu import as MFS-type transporter. Our overall findings suggest a model linking Cu import for cbb3-Cox biogenesis with a thiol:disulfide oxidoreduction step, advancing our understanding of the mechanisms of CcoA function. IMPORTANCE Copper (Cu) is a redox-active micronutrient that is both essential and toxic. Its cellular homeostasis is critical for supporting cuproprotein maturation while avoiding excessive oxidative stress. The Cu importer CcoA is the prototype of the widespread CalT subfamily of the MFS-type transporters. Hence, understanding its molecular mechanism of function is significant. Here, we show that CcoA undergoes a thiol:disulfide oxidoreduction cycle, which is important for its Cu import activity.
format article
author Bahia Khalfaoui-Hassani
Petru-Iulian Trasnea
Stefan Steimle
Hans-Georg Koch
Fevzi Daldal
author_facet Bahia Khalfaoui-Hassani
Petru-Iulian Trasnea
Stefan Steimle
Hans-Georg Koch
Fevzi Daldal
author_sort Bahia Khalfaoui-Hassani
title Cysteine Mutants of the Major Facilitator Superfamily-Type Transporter CcoA Provide Insight into Copper Import
title_short Cysteine Mutants of the Major Facilitator Superfamily-Type Transporter CcoA Provide Insight into Copper Import
title_full Cysteine Mutants of the Major Facilitator Superfamily-Type Transporter CcoA Provide Insight into Copper Import
title_fullStr Cysteine Mutants of the Major Facilitator Superfamily-Type Transporter CcoA Provide Insight into Copper Import
title_full_unstemmed Cysteine Mutants of the Major Facilitator Superfamily-Type Transporter CcoA Provide Insight into Copper Import
title_sort cysteine mutants of the major facilitator superfamily-type transporter ccoa provide insight into copper import
publisher American Society for Microbiology
publishDate 2021
url https://doaj.org/article/012f265fa2074b8d8c69080b2abf5244
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