FIGURE 10.

Cartoon model for the AzBCQ (CQ) binding site within PfCRT protein. On the basis of these and previous results, we propose that the AzBCQ 7-chloro-4-aminoquinoline pharmacophore (i.e., the CQ pharmacophore) binds within a cleft defined by helices 1 (purple), 9 (brown), and 10 (green) such that residues in helices 1 and 9 mutated in CQR vs CQS isoforms of PfCRT (near the asterisk) are proximal to the pharmacophore. This geometry easily permits the defined covalent attachment site within the loop between helices 9 and 10 (blue line) to contact the perfluoroazido side chain of the probe without disrupting predicted pharmacophore–helix interactions. Also, this geometry allows the biotin tag to be easily accessible outside the membrane in which the helices are imbedded. Notably, the model easily provides for a number of possible contacts between helix 1-mutated residues (near the asterisk, helix 1) and helix 9-mutated residues (near the asterisk, helix 9) as described in Discussion. Also, the model explains impressive isoform specific CQ, QN, and VPL competition, as flexible pfpa- and biotin-tagged side chains do not occupy the intrahelical CQ binding site but instead extend into the aqueous space. AzBCQ coordinates were computed with MM2, and the helices were built on the basis of the structure of the acetylcholine receptor m2 transmembrane segment (PDB entry 1EQ8). The AzBCQ molecule was manually docked onto the built helices using PyMOL (Delano Scientific LLC).