Mutational analysis of the high-affinity zinc binding site validates a refined human dopamine transporter homology model.

The high-resolution crystal structure of the leucine transporter (LeuT) is frequently used as a template for homology models of the dopamine transporter (DAT). Although similar in structure, DAT differs considerably from LeuT in a number of ways: (i) when compared to LeuT, DAT has very long intracel...

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Autores principales:	Thomas Stockner, Therese R Montgomery, Oliver Kudlacek, Rene Weissensteiner, Gerhard F Ecker, Michael Freissmuth, Harald H Sitte
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/036fd3d1414a46e38bcca194053a7f18
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spelling	oai:doaj.org-article:036fd3d1414a46e38bcca194053a7f182021-11-18T05:52:26ZMutational analysis of the high-affinity zinc binding site validates a refined human dopamine transporter homology model.1553-734X1553-735810.1371/journal.pcbi.1002909https://doaj.org/article/036fd3d1414a46e38bcca194053a7f182013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23436987/pdf/?tool=EBIhttps://doaj.org/toc/1553-734Xhttps://doaj.org/toc/1553-7358The high-resolution crystal structure of the leucine transporter (LeuT) is frequently used as a template for homology models of the dopamine transporter (DAT). Although similar in structure, DAT differs considerably from LeuT in a number of ways: (i) when compared to LeuT, DAT has very long intracellular amino and carboxyl termini; (ii) LeuT and DAT share a rather low overall sequence identity (22%) and (iii) the extracellular loop 2 (EL2) of DAT is substantially longer than that of LeuT. Extracellular zinc binds to DAT and restricts the transporter's movement through the conformational cycle, thereby resulting in a decrease in substrate uptake. Residue H293 in EL2 praticipates in zinc binding and must be modelled correctly to allow for a full understanding of its effects. We exploited the high-affinity zinc binding site endogenously present in DAT to create a model of the complete transmemberane domain of DAT. The zinc binding site provided a DAT-specific molecular ruler for calibration of the model. Our DAT model places EL2 at the transporter lipid interface in the vicinity of the zinc binding site. Based on the model, D206 was predicted to represent a fourth co-ordinating residue, in addition to the three previously described zinc binding residues H193, H375 and E396. This prediction was confirmed by mutagenesis: substitution of D206 by lysine and cysteine affected the inhibitory potency of zinc and the maximum inhibition exerted by zinc, respectively. Conversely, the structural changes observed in the model allowed for rationalizing the zinc-dependent regulation of DAT: upon binding, zinc stabilizes the outward-facing state, because its first coordination shell can only be completed in this conformation. Thus, the model provides a validated solution to the long extracellular loop and may be useful to address other aspects of the transport cycle.Thomas StocknerTherese R MontgomeryOliver KudlacekRene WeissensteinerGerhard F EckerMichael FreissmuthHarald H SittePublic Library of Science (PLoS)articleBiology (General)QH301-705.5ENPLoS Computational Biology, Vol 9, Iss 2, p e1002909 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Biology (General) QH301-705.5
spellingShingle	Biology (General) QH301-705.5 Thomas Stockner Therese R Montgomery Oliver Kudlacek Rene Weissensteiner Gerhard F Ecker Michael Freissmuth Harald H Sitte Mutational analysis of the high-affinity zinc binding site validates a refined human dopamine transporter homology model.
description	The high-resolution crystal structure of the leucine transporter (LeuT) is frequently used as a template for homology models of the dopamine transporter (DAT). Although similar in structure, DAT differs considerably from LeuT in a number of ways: (i) when compared to LeuT, DAT has very long intracellular amino and carboxyl termini; (ii) LeuT and DAT share a rather low overall sequence identity (22%) and (iii) the extracellular loop 2 (EL2) of DAT is substantially longer than that of LeuT. Extracellular zinc binds to DAT and restricts the transporter's movement through the conformational cycle, thereby resulting in a decrease in substrate uptake. Residue H293 in EL2 praticipates in zinc binding and must be modelled correctly to allow for a full understanding of its effects. We exploited the high-affinity zinc binding site endogenously present in DAT to create a model of the complete transmemberane domain of DAT. The zinc binding site provided a DAT-specific molecular ruler for calibration of the model. Our DAT model places EL2 at the transporter lipid interface in the vicinity of the zinc binding site. Based on the model, D206 was predicted to represent a fourth co-ordinating residue, in addition to the three previously described zinc binding residues H193, H375 and E396. This prediction was confirmed by mutagenesis: substitution of D206 by lysine and cysteine affected the inhibitory potency of zinc and the maximum inhibition exerted by zinc, respectively. Conversely, the structural changes observed in the model allowed for rationalizing the zinc-dependent regulation of DAT: upon binding, zinc stabilizes the outward-facing state, because its first coordination shell can only be completed in this conformation. Thus, the model provides a validated solution to the long extracellular loop and may be useful to address other aspects of the transport cycle.
format	article
author	Thomas Stockner Therese R Montgomery Oliver Kudlacek Rene Weissensteiner Gerhard F Ecker Michael Freissmuth Harald H Sitte
author_facet	Thomas Stockner Therese R Montgomery Oliver Kudlacek Rene Weissensteiner Gerhard F Ecker Michael Freissmuth Harald H Sitte
author_sort	Thomas Stockner
title	Mutational analysis of the high-affinity zinc binding site validates a refined human dopamine transporter homology model.
title_short	Mutational analysis of the high-affinity zinc binding site validates a refined human dopamine transporter homology model.
title_full	Mutational analysis of the high-affinity zinc binding site validates a refined human dopamine transporter homology model.
title_fullStr	Mutational analysis of the high-affinity zinc binding site validates a refined human dopamine transporter homology model.
title_full_unstemmed	Mutational analysis of the high-affinity zinc binding site validates a refined human dopamine transporter homology model.
title_sort	mutational analysis of the high-affinity zinc binding site validates a refined human dopamine transporter homology model.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/036fd3d1414a46e38bcca194053a7f18
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Mutational analysis of the high-affinity zinc binding site validates a refined human dopamine transporter homology model.

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