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receptor, it has not yet been obtained in crystalline form, nor is it likely to be in the near future.
Page 131
The bradykinin receptor is a member of a family of receptors for which an intracellular interaction with
a G-protein is a critical part of the signal transduction pathway following agonist binding. Structurally,
these G-protein-coupled receptors extend from beyond the extracellular boundary of the cell membrane
into the cytoplasm. The tertiary structure is such that the protein crosses the bilayer of the cell membrane
seven times, thus forming three intracellular loops, three extracellular loops, and giving rise to
cytoplasmic C-terminal and extra-cellular N-terminal strands. It is generally presumed that the
transmembrane domains of these receptors exist as a bundle of helical strands. This assumption is
derived primarily from the known structure of the trans-membrane portions of a structurally related
protein, bacteriorhodopsin [40].
G-Protein-coupled receptors do not lend themselves to analysis by either NMR or x-ray crystallography
due to their structural dependence on an intact cell membrane. In our laboratories we pursued this
valuable structural information by utilizing a combination of structural homology modeling, molecular
dynamics, systematic conformational searching methods, and mutagenesis experiments. The
combination of these techniques led to a proposed model of bradykinin bound to the agonist site on its
receptor [41].
A hydrophobicity (Kyte-Doolittle) calculation [42] on the amino acid sequence of the rat bradykinin
receptor yielded seven segments, each of which were 21 to 25 contiguous residues with predominantly
hydrophobic side chains. These were presumed to be the seven transmembrane portions of the receptor.
Cartesian coordinates of the backbone atoms within each of these seven segments were built by
structural homology from the cryomicroscopic structure of the analogous segments of
bacteriorhodopsin. Subsequently, side chains were added to these seven segments as appropriate for the
rat bradykinin receptor, and the resulting geometry was optimized via constrained energy minimization
to alleviate bad contacts. Extracellular and intracellular loops were extracted from the Protein Data Bank
library, following a geometric search based upon a vector defined by terminal alpha carbons in adjacent
helices. The model was subsequently subjected to a series of constrained and unconstrained energy
minimizations as well as molecular dynamics simulations. The resulting structure of the receptor was
used in a novel two-step docking procedure.
Following our hypothesis that bradykinin adopts a C-terminal β turn upon complexation with the
receptor, the φ, ψ backbone dihedral angles in the tetrapeptide corresponding to the C-terminus of
bradykinin (Ser-Pro-Phe-Agr) were constrained in a harmonic fashion (force constant = 15 Kcal Å -1 mol -
1) to values that define a type II' β-turn [43]. This tetrapeptide probe was then systematically translated
about the interior of a theoretical box inscribing the rat
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