Structure of the G119S Mutant Acetylcholinesterase of the Malaria Vector Anopheles gambiae Reveals Basis of Insecticide Resistance

Abstract

Malaria is a devastating disease in sub-Saharan Africa and is transmitted by the mosquito Anopheles gambiae. While indoor residual spraying of anticholinesterase insecticides has been useful in controlling the spread of malaria, widespread application of these compounds has led to the rise of an insecticide-resistant mosquito strain that harbors a G119S mutation in the nervous system target enzyme acetylcholinesterase. We demonstrate the atomic basis of insecticide resistance through structure determination of the G119S mutant acetylcholinesterase of An. gambiae in the ligand-free state and bound to a potent difluoromethyl ketone inhibitor. These structures reveal specific features within the active-site gorge distinct from human acetylcholinesterase, including an open channel at the base of the gorge, and provide a means for improving species selectivity in the rational design of improved insecticides for malaria vector control.

Keywords: acetylcholinesterase; channel; difluoromethyl ketone; insecticide; malaria; resistance; selectivity.

Figure 1. Structural analysis of ^apoAgAChE^G280S

(A) The structure of the ^apoAgAChE^G280S biological dimer is drawn as yellow and red ribbons. (B) As ribbons, the catalytic domain of ^apoAgAChE^G280S (yellow) is shown superimposed with hAChE (turquoise). Ordered N-linked carbohydrates are shown as sticks. (C) Active site gorge residues in superimposed ^apoAgAChE^G280S (yellow carbons) and hAChE (turquoise carbons) structures are drawn as sticks with hAChE residue numbering in parentheses. (D) Active site gorge residues of superimposed ^apoAgAChE^G280 (yellow carbons) and WT AgAChE (gray carbons) structures are shown as sticks (WT AgAChE residue type in parenthesis). Panels B, C, and D show the same protein orientation and O, N, and S atoms are colored red, blue, and orange respectively. See also Figure S1.

Figure 2. Surface analysis of ^apoAgAChE^G280S

(A), (B) In a cut-away view through the active site gorge, the molecular surface is drawn with gray interiors. Solvent-exposed surfaces of ^apoAgAChE^G280 (left) and hAChE (right) are colored semi-transparently by electrostatic potential from red to blue (−10 to +10 k_BT/e_c). Select residues are drawn as sticks and colored in the same scheme as Figure 1. See also Figure S2.

Figure 3. Structural analysis of PRC1214:AgAChE^G280S

(A) The chemical structures of PRC1214 and An. gambiae-selective carbamates 1a–c are shown with non-hydrogen atoms of PRC1214 numbered in red. (B) The active site of PRC1214:AgAChE^G280S is shown with residues drawn as sticks and colored by atom type (C, O, N, S, and F colored green, red, blue, orange, and light purple, respectively). Water is drawn as a turquoise sphere and hydrogen bonds are drawn as magenta dashes with distances (Å units) labeled. (C) In the color scheme of B, a cut-away view through the active site gorge shows the molecular surface with gray interiors colored semi-transparent green in solvent exposed areas. (D) Similar to panel C, the structure of DmAChE (PDB ID: 1DX4) is shown in a superimposed orientation with carbons and solvent exposed areas of the molecular surface colored purple. See also Figure S3.