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might be jointly interacting either with the guanidino group in the side chain of Arg1 or the N-terminal
amino group in bradykinin. Therefore a receptor containing a double mutation (Asp268,286 rarrow.gif
Ala268,286) would be expected to show a much more dramatic loss in affinity for bradykinin than would
receptors containing the individual point mutations. The appropriate double mutation experiment
confirmed this by causing a 500-fold loss in affinity for bradykinin, as predicted (Table 2). The
mutagenized residues of this double mutant B2 receptor are colored dark gray in Figure 5. This type of
an ionic interaction is also precedented by the body of literature that exists supporting the requirement of
an N-terminal arginine residue and a free N-terminal amino group in both bradykinin peptide agonists
and antagonists for high affinity binding. All of these mutant receptors were demonstrated to be
functional receptors on the basis of bradykinin-induced membrane depolarization in a Xenopus oocyte
expression system [44,45].
The selected agonist site model is characterized by an overall twisted S-shape ligand, similar to the
conformation of bradykinin determined previously in a hydrophobic environment by NMR [19,46].
Overall, the model suggested that the N-terminal amino and guanidine groups of Arg 1 interact directly
with negatively charged amino acids in extracellular loop three, and the C-terminal end is in a β-turn
conformation buried just below the extracellular boundary of the trans-membrane domain of the
receptor. Noteworthy is the presence of a hydrophobic cavity in our receptor model located adjacent to
Pro 7 of the bradykinin ligand. This cavity is made up, in part, by the residues Phe 261, Leu 104, Val 108, and
Ile 112. Given the historical significance of position seven in peptide bradykinin-like ligands, these
residues represent interesting targets for further mutagenesis experiments. One such result, the mutation
of Phe 261 to Ala 261, has already been described, and the results were supportive of this proposed model
[47]. Antibodies to the extracellular loops two and three have also been shown to compete with
bradykinin binding, lending further experimental support for an extracellular domain on this agonist
binding site.
More recently, chemical crosslinking combined with site-directed mutagenesis was used to analyze the
bradykinin binding site in the human B2 bradykinin receptor [48]. Previous studies using the bovine B2
receptor showed that heterobifiunctional reagents reactive to amines and free sulfhydryls crosslink the
bound bradykinin N-terminus to a sulfhydryl(s) in the receptor [49]. To identify this sulfhydryl(s), two
conserved candidate residues in the human B2 receptor—Cys 20 in the N-terminal domain and Cys 277 in
extracellular loop 3—were mutated to serine residues. Single and double mutants were expressed in Cos
7 cells. All mutants bound [ 3H]bradykinin with typical B2 receptor specificity. The heterobifunctional
reagent m-maleimidobenzoyl-N-hydroxysuccinimide ester crosslinked bradykinin to wild-type and
mutants with maximum efficiencies of 35% (wild type), 40% (Ser 20), 20% (Ser 277), and 0%
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