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Thermostabilisation of the Serotonin Transporter in a Cocaine-Bound Conformation

Structure determination of mammalian integral membrane proteins is challenging due to their instability upon detergent solubilisation and purification. Recent successes in the structure determination of G-protein-coupled receptors (GPCRs) resulted from the development of GPCR-specific protein engine...

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Autores principales: Abdul-Hussein, Saba, Andréll, Juni, Tate, Christopher G.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3678023/
https://www.ncbi.nlm.nih.gov/pubmed/23706649
http://dx.doi.org/10.1016/j.jmb.2013.03.025
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author Abdul-Hussein, Saba
Andréll, Juni
Tate, Christopher G.
author_facet Abdul-Hussein, Saba
Andréll, Juni
Tate, Christopher G.
author_sort Abdul-Hussein, Saba
collection PubMed
description Structure determination of mammalian integral membrane proteins is challenging due to their instability upon detergent solubilisation and purification. Recent successes in the structure determination of G-protein-coupled receptors (GPCRs) resulted from the development of GPCR-specific protein engineering strategies. One of these, conformational thermostabilisation, could in theory facilitate structure determination of other membrane proteins by improving their tolerance to detergents and locking them in a specific conformation. We have therefore used this approach on the cocaine-sensitive rat serotonin transporter (SERT). Out of a panel of 554 point mutants throughout SERT, 10 were found to improve its thermostability. The most stabilising mutations were combined to make the thermostabilised mutants SAH6 (L99A + G278A + A505L) and SAH7 (L405A + P499A + A505L) that were more stable than SERT by 18 °C and 16 °C, respectively. Inhibitor binding assays showed that both of the thermostabilised SERT mutants bound [(125)I]RTI55 (β-CIT) with affinity similar to that of the wild-type transporter, although cocaine bound with increased affinity (17- to 56-fold) whilst ibogaine, imipramine and paroxetine all bound with lower affinity (up to 90-fold). Neither SAH6 nor SAH7 was capable of transporting [(3)H]serotonin into HEK293 cell lines stably expressing the mutants, although serotonin bound to them with an apparent K(i) of 155 μM or 82 μM, respectively. These data combined suggest that SAH6 and SAH7 are thermostabilised in a specific cocaine-bound conformation, making them promising candidates for crystallisation. Conformational thermostabilisation is thus equally applicable to membrane proteins that are transporters in addition to those that are GPCRs.
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spelling pubmed-36780232013-06-26 Thermostabilisation of the Serotonin Transporter in a Cocaine-Bound Conformation Abdul-Hussein, Saba Andréll, Juni Tate, Christopher G. J Mol Biol Article Structure determination of mammalian integral membrane proteins is challenging due to their instability upon detergent solubilisation and purification. Recent successes in the structure determination of G-protein-coupled receptors (GPCRs) resulted from the development of GPCR-specific protein engineering strategies. One of these, conformational thermostabilisation, could in theory facilitate structure determination of other membrane proteins by improving their tolerance to detergents and locking them in a specific conformation. We have therefore used this approach on the cocaine-sensitive rat serotonin transporter (SERT). Out of a panel of 554 point mutants throughout SERT, 10 were found to improve its thermostability. The most stabilising mutations were combined to make the thermostabilised mutants SAH6 (L99A + G278A + A505L) and SAH7 (L405A + P499A + A505L) that were more stable than SERT by 18 °C and 16 °C, respectively. Inhibitor binding assays showed that both of the thermostabilised SERT mutants bound [(125)I]RTI55 (β-CIT) with affinity similar to that of the wild-type transporter, although cocaine bound with increased affinity (17- to 56-fold) whilst ibogaine, imipramine and paroxetine all bound with lower affinity (up to 90-fold). Neither SAH6 nor SAH7 was capable of transporting [(3)H]serotonin into HEK293 cell lines stably expressing the mutants, although serotonin bound to them with an apparent K(i) of 155 μM or 82 μM, respectively. These data combined suggest that SAH6 and SAH7 are thermostabilised in a specific cocaine-bound conformation, making them promising candidates for crystallisation. Conformational thermostabilisation is thus equally applicable to membrane proteins that are transporters in addition to those that are GPCRs. Elsevier 2013-06-26 /pmc/articles/PMC3678023/ /pubmed/23706649 http://dx.doi.org/10.1016/j.jmb.2013.03.025 Text en © 2013 Elsevier Ltd. https://creativecommons.org/licenses/by/3.0/ Open Access under CC BY 3.0 (https://creativecommons.org/licenses/by/3.0/) license
spellingShingle Article
Abdul-Hussein, Saba
Andréll, Juni
Tate, Christopher G.
Thermostabilisation of the Serotonin Transporter in a Cocaine-Bound Conformation
title Thermostabilisation of the Serotonin Transporter in a Cocaine-Bound Conformation
title_full Thermostabilisation of the Serotonin Transporter in a Cocaine-Bound Conformation
title_fullStr Thermostabilisation of the Serotonin Transporter in a Cocaine-Bound Conformation
title_full_unstemmed Thermostabilisation of the Serotonin Transporter in a Cocaine-Bound Conformation
title_short Thermostabilisation of the Serotonin Transporter in a Cocaine-Bound Conformation
title_sort thermostabilisation of the serotonin transporter in a cocaine-bound conformation
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3678023/
https://www.ncbi.nlm.nih.gov/pubmed/23706649
http://dx.doi.org/10.1016/j.jmb.2013.03.025
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