| Phenamacril(experimental code JS399-19),developed independently in China,is a novel cyanoacrylate fungicide.With the advantages of low toxicity,high activity and environmental friendliness,it has special effects to Fusarium head blight and rice bakanae disease.Previous studies have shown that the target of phenamacril is Myosin-5(type I),a specific motor protein of F.graminearum,which is the third target discovered in the history of fungicides following enzyme protein and skeleton protein.This study intends to study interaction mechanism between phenamacril and Myosin-5 combining with structural biology and fungicide biology.The expression and purification system of Myosin-5 were optimized.Firstly,the prokaryotic expression system was used and it was found that the target protein formed inclusion bodies with a large amount of expression.Then,the Sf9 insect expression system was used to construct plasmids with different purification tags and different truncated fragments of Myosin-5,however,Myosin-5 formed aggregation.Calmodulin was coexpressed with Myosin-5 and it successfully stabilized Myosin-5.Myosin-5 dimer protein with high ATPase activity was obtained by this method.Phenamacril at the concentration of 5 μM had a significant inhibitory effect on the ATPase activity of Myosin-5.The plasmids for expression were optimized with Fluorescence-detection Size-Exclusion Chromatography(FSEC)and a sufficient amount of Myosin-5 monomer protein for crystal screening was successfully obtained.The ATPase activity of Myosin-5 was increased by adding 0.1 μM CaM.It was found that phenamacril at the concentration of 2.5 μM almost completely inhibited the ATPase activity of Myosin-5 protein,and the IC50 of phenamacril to Myosin-5 protein was 0.36 μM.The crystals of Myosin-5-CaM-phenamacril were obtained by the sitting drop method.X-ray diffraction data was collected and analysed.Finally,the structure of the phenamacrilbound Myosin-5 and CaM of F.graminearum with a resolution of 2.65 (?) was obtained(PDB ID:6UI4).The structure showed that the binding cavity is 238 (?)3,including 17 phenamacrilinteracting residues:R189,L213,L214,E215,K216,S217,M375,Y409,F419,E420,C423,1424,D536,K537,N538,D540 and A577.The pocket is adjacent to the binding site of Blebbistatin,a mammalian Myosin Ⅱ inhibitor,but is different from each other.We mutated the important residues interacting with phenamacril to amino acids(K216E,S217L,M375K,Y409A,S418R,F419A,E420G,C423D,and A577F),which can break the original interaction,further proving the correctness of the structure.The mutant protein was purified and inhibition of 5 μM phenamacril to the ATPase activity of the mutant protein was tested in vitro,and it showed that the inhibition was decreased.F.graminearum mutants were obtained with homologous recombination.With the mycelial growth-inhibitory method,the sensitivity of mutants to phenamacril was tested for in vivo,showing that F.graminearum mutants developed varying resistance level to phenamacril.The correctness of the resolved crystal structure was proved both in vitro and in vivo.There are 17 amino acids directly interacting with phenamacril,including the 5 amino acid sites(K216E/R,S217P/L,E420K/G/D,I424R,and A577G),which had been reported to show high or medium resistance to F.graminearum or F.asiaticum.These mutations directly break down the interaction between the binding cavity and phenamacril,leading to resistance.The reported resistance site S418 doesn’t directly interact with phenamacril,but stabilize the interaction between phenamacril and other important amino acid residues.Other reported mutations that showed mild resistance to phenamacril also directly or indirectly weaken the interaction between the binding cavity and the phenamacril,resulting in low resistance to phenamacril.There is an actin-binding cleft in the motor domain of Myosin-5.In the ATPase cycle of Myosin-5,the acting-binding cleft opens and closes during the contractile,mediating the binding of actin,releasing Pi and ADP,and completing the ATPase cycle.The small molecule phenamacril occupies the space where the actin-binding cleft is closed.Meanwhile,both the amino acid L213,C423 and 1424 in the pocket and phenamacril interact with the Y409 site of the Switch 2 loop,which prevent transition of the Switch 2 loop structure from open conformation(pre-powerstroke)to closed conformation(powerstroke),arresting Myosin-5 in the pre-powerstroke state,blocking the release of Pi,interfering with the cycling process of Myosin-5 ATPase,and finally inhibiting the motor function of the protein.The important amino acid site 375 interacting with phenamacril was found.The sequence alignment showed that the 375 site of the pathogens sensitive to phenamacril is M,whereas it is K in the pathogens insensitive to phenamacril.The wild-type and K375M mutant Myosin-5 protein of Magnaporthe grisea were purified,and the inhibitions of phenamacril to their ATPase activity were tested.The results showed that the ATPase activity of wildtype was in-sensitive to phenamacril,while the activity of K375M mutant protein was sensitive to phenamacril,indicating that the genetic differentiation of the amino acid site 375 in different fungi might determine their sensitivities to phenamacril.Furthermore,the structure of Myosin-5-CaM in M grisea with a resolution of 1.82 A was obtained.And it was found that the binding pocket of phenamacril in F.graminearum is nearly consistent with that in M.grisea,except that the important amino acid 378(corresponding to amino acid 375 in F.graminearum)is K in M grisea Myosin-5 structure,which resulted in the insensitivity of M.grisea to phenamacril.The three-dimensional structure of the complex solved in this study clarified the specific interaction mechanism between F.graminearum Myosin-5 and phenamacril,intuitively revealed the resistance mechanism to phenamacril and determined the reason for the high specificity of phenamacril,which has important significance for the development of a series of derivative pesticides and medicines with Myosin as the target. |
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