The Interaction Mechanism Of Characteristic Structural Units Of Persimmon Tannin And Chinese Cobra Phospholipase A₂

Persimmon tannin（PT）exhibited significant inhibitory effects on Chinese cobra phospholipase A₂（PLA₂）,but detailed detoxifying mechanisms of PT were poorly understood due to the complex structure of persimmon tannin.Therefore,in this paper we studied the interaction between PLA₂ and four persimmon tannin characteristic structural units（EGCG,ECG,EGCG dimer and ECG dimer）and the binding mode and binding sites were also explored to obtain a better understanding of detoxifying mechanism of PT.It was confirmed that PT characteristic structural units showed both anti-enzymatic and anti-toxic activities.Multiple techniques,such as DLS,FT-IR and DSC et al.were used to explore the structural disturbing effects of PT structural units on PLA₂.And SPR,ITC and SDS-PAGE/NBT blotting methods were also used to determine the binding mode between PT structural units and PLA₂.Finally,we used molecular docking,key amino residue modification and LC-MS/MS methods to analysis the binding sites.The results were shown as follows:1.The detoxifying effects of PT characteristic structural units on PLA₂Enzymatic activity study showed that all the four PT structural units exhibited potent inhibitory effects on the catalytic activity of PLA₂,and the activity decreased with the increase of the concentrations of the PT structural units.The animal experiments showed that PT structural units could significantly inhibit the lethal toxicity,neurotoxicity,myotoxicity,anticoagulant activity,indirect hemolysis and edema inducing activity of PLA₂.Among the four PT characteristic structural units,EGCG dimer and ECG dimer showed more effective detoxifying ability than monomers.2.The structural disturbing effects of PT characteristic structural units on PLA₂PT characteristic structural units could bind to PLA₂,and induce aggregation of PLA₂,thus forming highly polymeric complexes.Binding with PT structural units led to a decrease in α-helix and β-turn contents of PLA₂,accompanying with an increase in β-sheet and random coil contents.The thermal stability of PLA₂ decreased after binding to PT characteristic elements,as reflected by the decrease in denaturation temperature and enthalpy of denaturation,suggesting the random coiled or unordered structures of PLA₂ increased.Besides,the surface hydrophobicity of PLA₂ declined after binding to PT units,indicating the conformation of PLA₂ was disturbed.It was also observed that the two dimers showed more potent influence on the structure of PLA₂ than monomers,manifesting EGCG dimer and ECG dimer might be the structural requirements for the binding of PT to PLA₂.We proposed that the structural disturbing effect of PT units on PLA₂ was probably one of the detoxifying mechanisms of PT.3.Both covalent and non-covalent binding existed in the interaction of PT characteristic structural units and PLA₂Non-covalent interaction was observed when the incubating time of PT structural units and PLA₂ was short,and in this case,hydrophobic interaction and electrostatic force were the predominant forces between them.The non-covalent binding was achieved fast but unstably.EGCG dimer and ECG dimer showed higher affinities to PLA₂ than PT monomers,further testifying that the two dimers were the structural requirements for the binding of PT to PLA₂.When prolonged the incubating time to 24 h,covalent bonds were formed between EGCG,ECG,EGCG dimer and PLA₂,and the B ring of polyphenol was involved in the covalent interaction.We proposed that only when the PT structural units were oxidized to quinone and PLA₂ existed in the natural conformation,did covalent interaction take place.4.PT characteristic structural units covalently bound to Lys,Trp and Tyr residues of PLA₂All of the four PT structural units bound to the catalytic site of PLA₂ and stabilized by hydrophobic forces and hydrogen bonds.His,Lys,Trp,Tyr and Gly were involved in the interaction between PT and PLA₂,and EGCG dimer and ECG dimer formed more non-covalent bonds with PLA₂.EGCG,ECG and EGCG dimer could covalently bind to PLA₂ through Lys6,Tyr27 and Trp18,19 respectively.PT units occupied the hydrophobic pocket of PLA₂,thus inhibiting the free access of substrate to the catalytic site,thus inhibiting the catalytic activity of PLA₂.Besides,PT units bound with the key active residues of PLA₂,such as lysine,histidine,tryptophan and tyrosine,restraining their toxicity.And the covalent binding with lysine,tryptophan and tyrosine further enhanced the detoxifying effects of persimmon tannin on snake venom.