ENGINEERING OF METAL-ION SITES AS DISTANCE CONSTRAINTS IN STRUCTURAL AND FUNCTIONAL-ANALYSIS OF 7TM RECEPTORS

G-protein-coupled receptors with their seven transmembrane (7TM) segme nts constitute the largest superfamily of proteins known. Unfortunatel y, still only relatively low resolution structures derived from electr on cryo-microscopy analysis of 2D crystals are available for these pro teins. We have used artificially designed Zn(II) metal-ion binding sit es to probe 7TM receptors structurally and functionally and to define some basic distance constraints for molecular modeling. In this way, t he relative helical rotation and vertical translocation of transmembra ne helices TM-II, TM-III, TM-V, and TM-VI of the tachykinin NK-1 recep tor have been restricted. Collectively, these zinc sites constitute a basic network of distance constraints that limit the degrees of freedo m of the interhelical contact faces in molecular models of 7TM recepto rs. The construction of artificially designed metal-ion sites is discu ssed also in the context of probes for conformational changes occurrin g during receptor activation.