Pyrethroids in an AlphaFold2 Model of the Insect Sodium Channel

Abstract

Pyrethroid insecticides stabilize the open state of insect sodium channels. Previous mutational, electrophysiological, and computational analyses led to the development of homology models predicting two pyrethroid receptor sites, PyR1 and PyR2. Many of the naturally occurring sodium channel mutations, which confer knockdown resistance (kdr) to pyrethroids, are located within or close to these receptor sites, indicating that these mutations impair pyrethroid binding. However, the mechanism of the state-dependent action of pyrethroids and the mechanisms by which kdr mutations beyond the receptor sites confer resistance remain unclear. Recent advances in protein structure prediction using the AlphaFold2 (AF2) neural network allowed us to generate a new model of the mosquito sodium channel AaNav1-1, with the activated voltage-sensing domains (VSMs) and the presumably inactivated pore domain (PM). We further employed Monte Carlo energy minimizations to open PM and deactivate VSM-I and VSM-II to generate additional models. The docking of a Type II pyrethroid deltamethrin in the models predicted its interactions with many known pyrethroid-sensing residues in the PyR1 and PyR2 sites and revealed ligand-channel interactions that stabilized the open PM and activated VSMs. Our study confirms the predicted two pyrethroid receptor sites, explains the state-dependent action of pyrethroids, and proposes the mechanisms of the allosteric effects of various kdr mutations on pyrethroid action. The AF2-based models may assist in the structure-based design of new insecticides.

Keywords: channel gating; insecticides; kdr mutations; pyrethroids; sodium channel.

Figure 1

(A) Transmembrane topology of voltage-gated insect sodium channels indicating residues within PyR1 (circles) or PyR2 (triangles), or beyond PyR1 and PyR2 (squares), for which kdr mutations have been functionally confirmed in Xenopus oocytes. Residues are labeled according to the scheme, which is universal for P-loop channels [11,23]. A residue label includes the amino acid one-letter code, the domain number (I–IV), segment type (k, the linker-helix S4–S5; i, the inner helix S6; p, the P-loop; and o, the outer helix S5), and relative number of the residue in the segment. (B) Ensemble of PMT starting poses in the PyR1 site of the mosquito sodium channel model with inactivated pore domain, ⁱAaNav1-1. (C) A low-energy binding pose of PMT in PyR1. Side chains of residues within 4 Å from PMT are shown as sticks. Labels of residues for which kdr mutations are reported (Table 1) are bold-underlined. Labels of residues for which kdr mutations are unknown, but engineered mutations are shown to affect the action of pyrethroids (Table 2), are bold-typed.

Figure 2

Alternative binding modes of deltamethrin in the PyR1 (A,B) and PyR2 (C,D) sites of the mosquito sodium channel with an in silico opened pore domain, ^oAaNav1-1. Side chains of residues within 4 Å from DMT are shown. Labels of residues for which kdr mutations are reported (Table 1) are bold-underlined. Labels of residues for which kdr mutations are unknown, but the engineered mutations that are shown to affect the action of pyrethroids (Table 2) are bold-typed.

Figure 3

In silico inactivating DMT-bound model ^oAaNav1-1. (A) Superposition of models ^oAaNav1-1 (green) and ^o-iAaNav1-1 (pink) with DMT in PyR1. (B,C) Surface representation of models ^oAaNav1-1 (B) and ^o-iAaNav1-1 (C) with DMT in site PyR1. Note a significant shift of the CBr₂ group that lost halogen bonds with T^2o10 and C^2o14. (D) Superposition of models ^oAaNav1-1 and ^o-iAaNav1-1 with DMT in site PyR2. (E,F) Surface representation of models ^oAaNav1-1. (E) and ^o-iAaNav1-1 (F) with DMT in PyR2. Note a significant shift of L²ⁱ¹⁶ that lost favorable contacts with the DMT aromatic ring.

Figure 4

In-silico deactivating VSMs in DMT-bound model ^oAaNav1-1. (A) In site PyR1, the side chain of M^2k11 moves significantly from DMT. (B) In site PyR2, the side chains of I²ⁱ¹³ and L²ⁱ¹⁶ move from DMT.

Figure 5

Residues beyond the PyR1 and PyR2 sites form intersegment contacts whose destruction would allosterically affect the action of pyrethroids. Labels of residues that are subject to kdr mutations are bold-underlined (see Section 3.6 for more detail). Panels (A–F) show, respectively, contacts of residues M⁸²⁷, G^2o24, L^2p47/S^2.p55, I^1k7, S¹ⁱ²⁹ and G^1k3.

Figure 6

Residues beyond the PyR1 and PyR2 sites form intersegment contacts whose destruction would allosterically affect the action of pyrethroids. Labels of residues that are subject to kdr mutations are bold-underlined (see Section 3.6 for more detail). Labels of residues, which are not subject of kdr mutations, but to their engineered substitutions, are explored in functional studies and are bold-typed. (see Section 3.8 for more detail). Panels (A–F) show, respectively, contacts of residues A^3k10, A^3p4, M³ⁱ³, F³ⁱ⁷, D³ⁱ²⁸/E³ⁱ³² and L^2k7.