Abstract
Abstract
The superior larvicidal activity of crude neem-based formulations over isolated azadirachtin has long been attributed to uncharacterized synergies among co-extracted volatiles. Using all-atom molecular dynamics (5.6 µs total ensemble sampling, comprising N = 3 independent replicates for APO, AZA, and DOUBLE systems, and an extended N = 5 replicates for the EUG-alone system to statistically validate surface contact-loss events), we characterised co-binding of azadirachtin (AZA) and eugenol (EUG) to the ecdysone receptor–ultraspiracle (EcR–USP) ligand-binding domain heterodimer. When bound alone, EUG samples a broad, diffuse configurational workspace. Orthosteric occupation by AZA induces a conformational response consistent with allosteric basin squeezing: the Boltzmann-inverted free-energy profile W (ξ) transitions from a shallow, broad well to a narrow, focused basin. This geometric confinement is accompanied by a 4.96% reduction in EUG-site backbone flexibility (∆RMSF = −0.066 Å), a switch in dominant polar contact from ARG383 (EUG-alone, 46.8% occupancy) to ALA475:N (DOUBLE, 75.6%), and state-selective recruitment of al-losteric relay residues ARG131/LEU494/PRO450 exclusively under dual occupancy. The cooperativity is directional—AZA stabilises EUG (retained in 3/3 DOUBLE replicates vs. 1/3 alone)—and is best described as apparent residence stabilization. These findings provide a structural rationale for the entourage-like synergy of neem-derived formulations and suggest a general design principle for combination biopesticides exploiting orthosteric-to-distal-site communication.