Structural Probing of a Microdomain in the Dopamine Transporter by Engineering of Artificial Zn2+ Binding Sites

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• 作者：Lene Norregaard ; Irache Visiers ; Claus J. Loland ; Juan Ballesteros ; Harel Weinstein ; and Ulrik Gether
• 刊名：Biochemistry
• 出版年：2000
• 出版时间：December 26, 2000
• 年：2000
• 卷：39
• 期：51
• 页码：15836 - 15846
• 全文大小：470K
• 年卷期：v.39,no.51(December 26, 2000)
• ISSN：1520-4995

文摘

Previously, we have identified three Zn²⁺ binding residues in an endogenous Zn²⁺ bindingsite in the human dopamine transporter (hDAT): ¹⁹³His in extracellular loop 2 (ECL 2), ³⁷⁵His at theexternal end of transmembrane segment (TM) 7, and ³⁹⁶Glu at the external end of TM 8. Here we havegenerated a series of artificial Zn²⁺ binding sites in a domain situated around the external ends of TMs7 and 8 by taking advantage of the well-defined structural constraints for binding of the zinc(II) ion.Initially, we found that the Zn²⁺-coordinating ¹⁹³His in ECL 2 could be substituted with a histidine insertedat the i - 4 position relative to ³⁷⁵His in TM 7. In this mutant (H193K/M371H), Zn²⁺ potently inhibited[³H]dopamine uptake with an IC₅₀ value of 7 M as compared to a value of 300 M for the control(H193K). These data are consistent with the presence of an -helical configuration of TM 7. This inferencewas further corroborated by the observation that no increase in the apparent Zn²⁺ affinity was observedfollowing introduction of histidines at the i - 2, i - 3, and i - 5 positions. In contrast, introduction ofhistidines at positions i + 2, i + 3, and i + 4 all resulted in potent inhibition of [³H]dopamine uptake byZn²⁺ (IC₅₀ = 3-32 M). These observations are inconsistent with continuation of the helix beyond position375 and indicate an approximate boundary between the end of the helix and the succeeding loop. Insummary, the data presented here provide new insight into the structure of a functionally important domainin the hDAT and illustrate how engineering of Zn²⁺ binding sites can be a useful approach for probingboth secondary and tertiary structure relationships in membrane proteins of unknown structure.