该文以牦牛乳干酪苦味肽RPKHPIK(RK7)和VYPFPGPIPN(VN10)为研究对象,系统探究其抗血栓活性的分子间作用机制。使用ExPASy-ProtParam、Innovagen和PepDraw等工具计算RK7和VN10的理化性质,并结合体外试验测定肽段对凝血酶的抑制活性,通过分子对接技术和分子动力学揭示肽段抑制凝血酶活性的作用机制及抗凝血的分子作用。经研究表明,肽段RK7和VN10均能抑制凝血酶的活性,抗血栓活性显著。分子对接显示凝血酶中的Ser195、Glu192、Gly193、Gly216与RK7和VN10的结合起重要作用,并且Ser195、Glu192和Gly193是影响凝血酶活性的关键氨基酸;RK7和VN10通过氢键、疏水作用及静电相互作用与凝血酶(1A4W和2BVR)活性位点结合,结合能分别为-7.6和-8.6 kcal/mol以及-7.5和-7.7 kcal/mol,优于阳性对照比伐卢定(-7.0和-6.4 kcal/mol)。为明晰凝血酶与多肽RK7及VN10所形成复合物的动态特性,该文进行了分子动力学模拟,并对其关键结构参数进行了分析,包括均方根偏差(root-mean-square deviation,RMSD)、均方根波动(root mean square fluctuation,RMSF)、氢键数目、回转半径(radius of gyration,Rg)以及溶剂可及表面积(solvent accessible surface area,SASA),结果均表明肽与目标蛋白的复合体系稳定。经生物信息学预测与试验验证相结合印证,该试验从分子层面为牦牛乳酪蛋白降解肽的抗血栓活性提供重要理论参考,为功能性食品的开发设计提供理论依据与创新方向。
This study investigated the intermolecular mechanism underlying the antithrombotic activity of bitter peptides RPKHPIK (RK7) and VYPFPGPIPN (VN10) derived from yak milk cheese.The physicochemical properties of RK7 and VN10 were analyzed using ExPASy-ProtParam, Innovagen, and PepDraw tools.Through in vitro experiments, the inhibitory effects of these peptides on thrombin activity were determined.Furthermore, molecular docking and molecular dynamics simulations were employed to elucidate the mechanisms of thrombin inhibition and the molecular interactions involved in anticoagulation.The results showed that both RK7 and VN10 significantly inhibited thrombin activity, demonstrating notable antithrombotic effects.Molecular docking revealed that Ser195, Glu192, Gly193, and Gly216 in thrombin play critical roles in binding to RK7 and VN10, with Ser195, Glu192, and Gly193 being key residues influencing thrombin activity.RK7 and VN10 bind to the active sites of thrombin (PDB:1A4 W and 2BVR) via hydrogen bonding, hydrophobic interactions, and electrostatic interactions, with binding energies of -7.6 and -8.6 kcal/mol for RK7, and -7.5 and -7.7 kcal/mol for VN10, respectively.These values were lower than those of the positive control bivalirudin (-7.0 and -6.4 kcal/mol), indicating stronger binding.To further explore the dynamic behavior of the thrombin-peptide complexes, molecular dynamics simulations were conducted.Key structural parameters, including root mean square deviation (RMSD), root mean square fluctuation (RMSF), number of hydrogen bonds, radius of gyration (Rg), and solvent accessible surface area (SASA), were analyzed, collectively confirming the stability of the peptide–target protein complexes.By integrating bioinformatics predictions with experimental validation, this study provides important theoretical insights into the antithrombotic activity of yak milk casein-derived peptides at the molecular level, offering a foundation and innovative directions for developing functional foods.
[1] MENSAH G A, FUSTER V, MURRAY C J L, et al.Global burden of cardiovascular diseases and risks, 1990-2022[J].Journal of the American College of Cardiology, 2023, 82(25):2350-2473.
[2] LIU S D, LI S S, YUAN D, et al.Protease activated receptor 4 (PAR4) antagonists:Research progress on small molecules in the field of antiplatelet agents[J].European Journal of Medicinal Chemistry, 2021, 209:112893.
[3] RATHEE K, DHULL V, DHULL R, et al.Biosensors based on electrochemical lactate detection:A comprehensive review[J].Biochemistry and Biophysics Reports, 2016, 5:35-54.
[4] 杨保军, 梁琪, 宋雪梅.牦牛乳硬质干酪苦味肽RK7的抗氧化活性及其机制[J].中国食品学报, 2022, 22(8):40-50.
YANG B J, LIANG Q, SONG X M.Antioxidant activity and mechanism of yak milk hard cheese bitter peptide RK7[J].Journal of Chinese Institute of Food Science and Technology, 2022, 22(8):40-50.
[5] WANG W, LIANG Q, ZHAO B T, et al.Functional peptides from yak milk casein:Biological activities and structural characteristics[J].International Journal of Molecular Sciences, 2024, 25(16):9072.
[6] WANG P, SONG X M, LIANG Q.Study on the inhibitory effect of bioactive peptides derived from yak milk cheese on cholesterol esterase[J].Foods, 2024, 13(18):2970.
[7] 杨保军, 梁琪, 宋雪梅.牦牛乳干酪苦味肽ACE抑制活性表征的分子机制[J].中国食品学报, 2022, 22(5):8-17.
YANG B J, LIANG Q, SONG X M.Molecular mechanism of ACE inhibitory activity characterization of yak milk cheese bitter peptide[J].Journal of Chinese Institute of Food Science and Technology, 2022, 22(5):8-17.
[8] 王鹏, 梁琪, 赵保堂, 等.结合分子对接技术研究牦牛乳苦味肽RK7和KQ7的HMG-CoA还原酶抑制活性[J].食品与发酵工业, 2025, 51(11):208-215.
WANG P, LIANG Q, ZHAO B T, et al.Application of molecular docking technique to study the inhibitory activity of yak milk hard cheese bittering peptides RK7 and KQ7 and positive control on HMG-CoA reductase[J].Food and Fermentation Industries, 2025, 51(11):208-215.
[9] 李梦瑶, 梁琪, 宋雪梅.结合分子对接技术研究牦牛乳干酪苦味肽RK7和KQ7的α-淀粉酶抑制活性[J].食品科学, 2023, 44(2):132-138.
LI M Y, LIANG Q, SONG X M.Using molecular docking to investigate the alpha-amylase inhibitory activity of bitter peptides RK7 and KQ7 derived from yak cheese[J].Food Science, 2023, 44(2):132-138.
[10] MAZRAEDOOST S, SEDIGH MALEKROODI H, ŽUVELA P, et al.Prediction of chromatographic retention time of a small molecule from SMILES representation using a hybrid transformer-LSTM model[J].Journal of Chemical Information and Modeling, 2025, 65(7):3343-3356.
[11] LIU H X, TU M L, CHENG S Z, et al.An anticoagulant peptide from beta-casein:Identification, structure and molecular mechanism[J].Food & Function, 2019, 10(2):886-892.
[12] AGYEI D, ONGKUDON C M, WEI C Y, et al.Bioprocess challenges to the isolation and purification of bioactive peptides[J].Food and Bioproducts Processing, 2016, 98:244-256.
[13] TONDAR A, SÁNCHEZ-HERRERO S, BEPARI A K, et al.Virtual screening of small molecules targeting BCL2 with machine learning, molecular docking, and MD simulation[J].Biomolecules, 2024, 14(5):544.
[14] ROJAS-RONQUILLO R, CRUZ-GUERRERO A, FLORES-NÁJERA A, et al.Antithrombotic and angiotensin-converting enzyme inhibitory properties of peptides released from bovine casein by Lactobacillus casei Shirota[J].International Dairy Journal, 2012, 26(2):147-154.
[15] SINGH S, BANI BAKER Q, SINGH D B.Chapter 18 - Molecular Docking and Molecular Dynamics Simulation[M]//SINGH D B, PATHAK R K.Bioinformatics.Academic Press.2022:291-304.
[16] NASRI R, BEN AMOR I, BOUGATEF A, et al.Anticoagulant activities of goby muscle protein hydrolysates[J].Food Chemistry, 2012, 133(3):835-841.
[17] NAR H.The role of structural information in the discovery of direct thrombin and factor Xa inhibitors[J].Trends in Pharmacological Sciences, 2012, 33(5):279-288.
[18] HAMPOELZ B, ANDRES-PONS A, KASTRITIS P, et al.Structure and assembly of the nuclear pore complex[J].Annual Review of Biophysics, 2019, 48:515-536.
[19] MORSTEIN J, TRAUNER D.Photopharmacological control of lipid function[J].Methods in Enzymology, 2020, 638:219-232.
[20] TALAGAYEV V, CHEN Y, DOERING N P, et al.OpenMMDL-simplifying the complex:Building, simulating, and analyzing protein-ligand systems in OpenMM[J].Journal of Chemical Information and Modeling, 2025, 65(4):1967-1978.
[21] NELSON L, BARIAMI S, RINGROSE C, et al.Implementation of the QUBE force field in SOMD for high-throughput alchemical free-energy calculations[J].Journal of Chemical Information and Modeling, 2021, 61(5):2124-2130.
[22] WANG Y K, WAN H Y, XU J, et al.Bifunctional electron-transporting agent for red colloidal quantum dot light-emitting diodes[J].Journal of the American Chemical Society, 2023, 145(11):6428-6433.
[23] LI X L, LI C M, ZHU J Y, et al.A scheme for rapid evaluation of the intermolecular three-body polarization effect in water clusters[J].Journal of Computational Chemistry, 2023, 44(5):677-686.
[24] QIAO M L, TU M L, WANG Z Y, et al.Identification and antithrombotic activity of peptides from blue mussel (Mytilus edulis) protein[J].International Journal of Molecular Sciences, 2018, 19(1):138.
[25] LI Y R, HUANG Y N, ZHAO B, et al.RGD-hirudin-based low molecular weight peptide prevents blood coagulation via subcutaneous injection[J].Acta Pharmacologica Sinica, 2020, 41(6):753-762.
