Structural probing of a microdomain in the dopamine transporter by engineering of artificial Zn2+ binding sites.
Previously, we have identified three Zn(2+) binding residues in an endogenous Zn(2+) binding site in the human dopamine transporter (hDAT): (193)His in extracellular loop 2 (ECL 2), (375)His at the external end of transmembrane segment (TM) 7, and (396)Glu at the external end of TM 8. Here we have generated a series of artificial Zn(2+) binding sites in a domain situated around the external ends of TMs 7 and 8 by taking advantage of the well-defined structural constraints for binding of the zinc(II) ion. Initially, we found that the Zn(2+)-coordinating (193)His in ECL 2 could be substituted with a histidine inserted at the i - 4 position relative to (375)His in TM 7. In this mutant (H193K/M371H), Zn(2+) potently inhibited [(3)H]dopamine uptake with an IC(50) value of 7 microM as compared to a value of 300 microM for the control (H193K). These data are consistent with the presence of an alpha-helical configuration of TM 7. This inference was further corroborated by the observation that no increase in the apparent Zn(2+) affinity was observed following introduction of histidines at the i - 2, i - 3, and i - 5 positions. In contrast, introduction of histidines at positions i + 2, i + 3, and i + 4 all resulted in potent inhibition of [(3)H]dopamine uptake by Zn(2+) (IC(50) = 3-32 microM). These observations are inconsistent with continuation of the helix beyond position 375 and indicate an approximate boundary between the end of the helix and the succeeding loop. In summary, the data presented here provide new insight into the structure of a functionally important domain in the hDAT and illustrate how engineering of Zn(2+) binding sites can be a useful approach for probing both secondary and tertiary structure relationships in membrane proteins of unknown structure.
Norregaard
L
L
Division of Cellular and Molecular Physiology, Department of Medical Physiology, Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark.
Visiers
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I
Loland
C J
CJ
Ballesteros
J
J
Weinstein
H
H
Gether
U
U
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive / Q000235:genetics; D019556:COS Cells; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003198:Computer Simulation; D050483:Dopamine Plasma Membrane Transport Proteins; D006639:Histidine / Q000235:genetics; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism
D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D006639:Histidine; D015032:Zinc