【作者】 王婧；

【导师】 牛立文； 滕脉坤；

【作者基本信息】 中国科学技术大学 ， 生物化学与分子生物学， 2021， 博士

【摘要】 裂殖酵母 Ofd2(2-Oxoglutarate-Fe(Ⅱ)dependent dioxygenase domain containing protein-2)蛋白属于Fe2+和α-酮戊二酸依赖的双加氧酶超家族,通过序列比对发现Ofd2属于双加氧酶家族中的AlkB亚家族。AlkB家族很多成员都参与核酸损伤修复,但研究表明Ofd2既不具有对DNA或者RNA的去甲基化酶活性,也不具有对DNA损伤试剂的敏感性,而是通过与组蛋白相互作用,在低氧条件下调控氧化磷酸化基因的表达。因此,裂殖酵母Ofd2是AlkB家族中与组蛋白相互作用的一种新的类型。本文解析了裂殖酵母Ofd2蛋白1.80 (?)的单体晶体结构。Ofd2蛋白由12条β链和3个α螺旋及5个310螺旋组成,整体结构具有双加氧酶家族保守的果冻卷样双股β折叠-螺旋(double-stranded β-helix,DSBH)结构。同时还具有保守的铁离子结合基序:His132,Asp134和His186。通过对比Ofd2与AlkB家族其它蛋白质的结构,发现Ofd2缺失了以核酸为底物的双加氧酶保守的核苷酸识别盖子 2(Nucleotide Recognition Lid 2,NRL2),并在 βⅣ-βⅤ loop 部位的构象有很大不同。另外,在βⅦ-βⅧ loop之间多出了同源家族少见的由2股β折叠组成的β折叠片。通过GST pull-down实验,研究了 Ofd2蛋白和组蛋白的相互作用,结果显示Ofd2可以在低盐条件下与组蛋白相互作用,而在高盐情况下与组蛋白无相互作用。若将βⅦ-βⅧ之间的β折叠片突变为AlkB家族对应的loop时,发现其仍然可以与组蛋白相互作用,说明该β折叠片部位并不参与Ofd2与组蛋白的相互作用。以上是论文第一篇内容的摘要。根据实验室的课题、生物技术学习、科学研究策略学习安排,本论文第一篇内容是以原核表达系统来研究蛋白质的结构与功能,第二篇内容以真核表达系统来研究蛋白质的结构与功能,对于不同的课题选择合适的技术和策略进行研究。下面是论文第二篇研究内容的摘要。缺血性脑卒中和心血管疾病是严重威胁人类健康的疾病,引起这些疾病的一个重要原因是血栓。GPIbα(glycoprotein Ibα)是血小板膜糖蛋白,在血小板凝集中有着重要的作用,可作为抗血栓药物的靶标。Agkisacucetin(以下简称Agk)是皖南尖吻蝮蛇蛇毒中分离得到的C型凝集素类似蛋白,因其可以抑制GPIb与vWF(von Willebrand Factor)相互作用而具有抗血栓作用,同时在体内不易造成凝血障碍,因此是一种潜在抗血栓药物。本论文通过真核系统重组表达了血小板膜糖蛋白GPIbαN端结构域(GPIbαN端1-305氨基酸),并通过酶联免疫吸附法检测了其与Agk的结合活性。通过凝胶过滤层析纯化获得GPIbα与Agk的复合物(以下简称为GAGK)蛋白,通过晶体筛选获得可供衍射的GAGK晶体,其衍射分辨率可达到3.30 (?)。通过分子置换法解析了 GAGK的结构。结合定点突变实验和晶体结构分析确认了 GPIbα与Agk结合的区域:Agk的β链通过GPIbα C端的保守的富亮氨酸结构域和β-switch区域结合,Agk的α链通过GPIbα C端多肽结合。该复合物结构的解析,解释了 Agk何以竞争性抑制GPIb与vWF结合。通过生化实验,验证了蛇毒Agk可以竞争性抑制GPIb与凝血酶的结合。由于蛇毒蛋白间的结构相似性和功能多样性,GAGK复合物结构的解析为Agk在临床上的应用提供结构基础。更多还原

【Abstract】 Schizosaccharomyces pombe 2-Oxoglutarate-Fe(Ⅱ)dependent Dioxygenase domain containing protein-2(Ofd2),member of the AlkB which belongs to 2Fe(II)/Oxoglutarate dependent dioxygenase superfamily.To date,dozens of AlkB family members have been characterized,which recognize methylated nucleic acid substrates,while Ofd2(histone H2A dioxygenase)play an important role in regulating chromatin structure.In this study,the crystal structure of spOfd2 was resolved at 1.80 (?) resolution.Ofd2 is composed of 12 β-strands,3 α-helices and 5 310-helices.eight β strands form a central jelly-roll fold consisting of double-stranded β-helix(DSBH)conserved in 20G/Fe(Ⅱ)-dependent dioxygenase family.Several characteristics including nucleotide recognition lid(NRL),βIV-βV loop and additional two β-strands make Ofd2 significantly different from other nucleic acid oxygenases in AlkB family.We also used GST pull-down to analyze the interaction of Ofd2 with histones.The above is an abstract of the first content of this thesis.According to the laboratory task,biotechnology and scientific research strategy,we use prokaryotic expression system to study the structure and function of protein in the first part of this thesis and use eukaryotic expression system to study the structure and functions of protein in second part.The following is a summary of the second part of this thesis.Platelet receptor glycoprotein Ibα(GPIbα)has important role in aggregation of platelets,it could be a good target for anti-thrombonic drugs.Agkisacucetin is a snake C-type lectin-like protein(snaclec)isolated from the venom of Deinagkistrodon acutus(formerly Agkistrodon acutus),which is a novel anti-thrombotic drug candidate currently in phase 2 clinical trials.Agkisacucetin specifically recognizes GPIba to block GPIb and von Willebrand Factor(VWF)interaction.In this study,we expressed GPIba in 293T cells and measured the affinity between GPIba and Agk by ELISA,solved the crystal structure of GPIba N-terminal domain(residue 1-305)in complex with Agkisacucetin and refined to 3.30 (?).Crystal structure showed that Agkisacucetin mainly contacts GPIbα at the C-terminal part of the conserved leucine rich repeat(LRR)domain(LRR-6 to LRR-8)and the previously described "β-switch" region through βchain.In addition,we found that Agkisacucetin a chain also contacts part of the GPIba C-terminal peptide after the LRR domain through complementary charge interactions.This C-terminal peptide plays a key role in GPIba and thrombin recognition.Therefore,our structure revealed that Agkisacucetin can sterically block the interaction of GPIb receptor with both VWF and thrombin proteins to inhibit platelet function.Our structural work provides key molecular insights into how an anti-thrombonic drug candidate recognizes GPIb receptor to modulate platelet function to inhibit thrombosis.更多还原