title: Connectivity and orientation of the seven helical bundle in the tachykinin NK-1 receptor probed by zinc site engineering.
abstract: A high affinity, tridentate metal ion site has been constructed previously by His substitutions in an antagonist binding site located between transmembrane segment (TM)-V and TM-VI in the substance P NK-1 receptor. Here, an attempt is made to probe helix-helix interactions systematically in the NK-1 receptor by engineering of bis-His Zn(II) sites. His residues were introduced at selected positions individually and in combinations in the exterior segments of TM-II, III and V in both the wild-type background and after Ala substitution of naturally occurring His residues, and the increase in the affinity for Zn(II) was monitored in competition binding experiments with iodinated substance P or a tritiated non-peptide antagonist. In this way, two high affinity bis-His sites were constructed between position 193 in TM-V (Glu193, G1uV:01) and position 109 in TM-III (Asn1O9, AsnIII:05) as well as between the neighboring, naturally occurring His108 in TM-III (HisIII:04) and position 92 in TM-II (Tyr92, TyrII:24), respectively. Functionally, the coordination of zinc ions at these two sites blocked the receptor as it antagonized the substance P-induced increase in phosphatidylinositol turnover. It is concluded that the bis-His zinc sites from the central TM-III helix to TM-II and -V, respectively, together with the interconnected, previously constructed tridentate site between TM-V and -VI, constitute a basic network of distance constraints for the molecular models of receptors with seven transmembrane segments which, for example, strongly support an anti-clockwise orientation of the seven helical bundle as viewed from the extracellular space.
authors.affiliation: Laboratory for Molecular Pharmacology, The Laboratory Center, Rigshospitalet, University of Copenhagen, Denmark.
authors.forename: C E
authors.initials: CE
authors.lastname: Elling
authors.forename: T W
authors.initials: TW
authors.lastname: Schwartz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010716:Phosphatidylinositols / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D018040:Receptors, Neurokinin-1 / Q000737:chemistry* / Q000378:metabolism; D013373:Substance P; D015032:Zinc / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; D010716:Phosphatidylinositols; D018040:Receptors, Neurokinin-1; D013373:Substance P; D006639:Histidine; D015032:Zinc