新型抗菌化合物HL-J6与金黄色葡萄球菌PBP1的相互作用研究  

Interaction between a novel antimicrobial compound HL-J6 and Staphylococcus aureus PBP1

作　　者：徐铭琦 史祥睿 刘威[4] 段浩 魏静 邓炎 江悦 高英英[1,5] 李海波 XU Mingqi;SHI Xiangrui;LIU Wei;DUAN Hao;WEI Jing;DENG Yan;JIANG Yue;GAO Yingying;LI Haibo(Clinical Medical College,Jiamusi University,Jiamusi,Heilongjiang;Department of Microbiology and Biochemical Pharmacy,National Engineering Research Center of Immunological Products,Faculty of Pharmacy and Laboratory Medicine,Chongqing;School of Pharmacy,Chongqing University,Chongqing;Institute of Immunology,Army Medical University(Third Military Medical University),Chongqing;Department of Clinical Laboratory,Banan Hospital Affiliated to Chongqing Medical University,Chongqing,China)

机构地区：[1]佳木斯大学临床医学院,黑龙江佳木斯 [2]陆军军医大学(第三军医大学)药学与检验医学系微生物与生化药学教研室,国家免疫生物制品工程技术研究中心,重庆 [3]重庆大学药学院,重庆 [4]陆军军医大学(第三军医大学)全军免疫学研究所,重庆 [5]重庆医科大学附属巴南医院检验科,重庆

出　　处：《陆军军医大学学报》2025年第9期912-921,共10页Journal of Army Medical University

基　　金：国家自然科学基金面上项目(32270988);重庆市自然科学基金面上项目(CSTB2024NSCQ-MSX0981)。

摘　　要：目的 探究新型抗菌化合物HL-J6与金黄色葡萄球菌青霉素结合蛋白1(penicillinbinding protein 1, PBP1)的相互作用。方法 以MRSA252基因组DNA为模板,PBP1F/R为上下游引物,以Nde I/Xho I为酶切位点构建表达质粒pET30a-pbp1-39-608。将质粒转入大肠杆菌中进行表达并通过亲和层析纯化获得PBP1-39-608蛋白;利用肽聚糖侧链主干肽的硫酯类似物S2d作为底物,测定HL-J6对PBP1-39-608转肽酶活性的抑制作用;用微量热泳动技术(microscale thermophoresis,MST)检测HL-J6与PBP1-39-608的亲和力,并通过细胞热转移实验(cell thermal shift assay,CETSA)确证结合作用;分别利用AutoDock Vina和Desmond软件进行分子对接和动力学模拟,明确HL-J6与PBP1-39-608蛋白的结合模式和关键氨基酸残基。结果 成功构建pET30a-pbp1-39-608重组质粒,诱导表达后,经纯化获得相对分子质量约为65×103的PBP1-39-608蛋白;HL-J6可呈时间依赖性抑制PBP1-39-608的转肽酶活性(P<0.001);HL-J6与PBP1-39-608的结合解离常数Kd约为64.92μmol/L;分子对接结果显示HL-J6通过与ILE-348、ASN-370、THR-516、PHE-423等主要氨基酸残基相互作用结合在PBP1-39-608的活性口袋,结合评分为-8.38 kcal/mol(<-5.00 kcal/mol);动力学模拟结果显示二者结合后50 ns趋于稳定。结论 HL-J6可显著抑制金黄色葡萄球菌PBP1的转肽酶活性,并与其存在稳定的相互作用。Objective To investigate the interaction between a novel antimicrobial compound, HL-J6,and penicillin-binding protein 1(PBP1) of Staphylococcus aureus. Methods With MRSA252 genomic DNA as the template and PBP1F and PBP1R as primers, the expression plasmid pET30a-pbp1-39-608 was constructed by amplifying the target gene fragment followed by cloning into the Nde I/Xho I restriction sites of the pET30a vector. Then the obtained plasmids were transformed into Escherichia coli for the expression of PBP1-39-608 protein, and the product was purified by affinity chromatography. The inhibitory effect of HL-J6on the transpeptidase activity of PBP1-39-608 was measured using peptidoglycan side chain backbone peptide, with thiol ester analog S2d as the substrate. The affinity between HL-J6 and PBP1-39-608 was detected using microscale thermophoresis(MST), and the binding interaction was confirmed by cellular thermal shift assay(CETSA). Molecular docking and dynamics simulation were performed using AutoDock Vina and Desmond software, respectively, to elucidate the binding mode of HL-J6 with the PBP1-39-608protein and the key amino acid residues involved. Results The recombinant plasmid pET30a-pbp1-39-608was successfully constructed, and PBP1-39-608 protein was produced after induction and purified, yielding a protein with an approximate molecular mass of 65×103. HL-J6 inhibited the transpeptidase activity of PBP1-39-608 in a time-dependent manner(P<0. 001). The dissociation constant Kd of the binding between HL-J6and PBP1-39-608 was 64. 92 μmol/L. Molecular docking results showed that HL-J6 bound to the active pocket of PBP1-39-608 by interacting with key residues such as ILE-348, ASN-370, THR-516 and PHE-423,with a binding score of-8. 38 kcal/mol(<-5. 00 kcal/mol). Dynamics simulation results indicated that the complex became stable after 50 ns. Conclusion HL-J6 effectively inhibits the transpeptidase activity of Staphylococcus aureus PBP1, and shows stable interaction with the protein.

关 键 词：金黄色葡萄球菌 青霉素结合蛋白 HL-J6 重组蛋白:相互作用 

分 类 号：R378.11[医药卫生—病原生物学] R966[医药卫生—基础医学] R978.19