【作者】 张燕；

【导师】 林志；

【作者基本信息】 天津大学 ， 生物学， 2023， 博士

【摘要】 铜绿假单胞菌（Pseudomonas aeruginosa,PAO1）是医院内感染常见的条件致病菌,由于其具有很强的环境适应性和耐药性,使得抗感染治疗变得十分困难。在细菌中,与感染性、致病性相关的生物膜、运动性、毒力等表型均受第二信使环鸟苷二磷酸（c-di-GMP）的调控。C-di-GMP由含GGDEF（Gly-Gly-Asp-Glu-Phe）结构域的鸟苷酸环化酶催化合成,由含EAL（Glu-Ala-Leu）或HD-GYP结构域的磷酸二酯酶分解,这些蛋白被称为c-di-GMP代谢蛋白。对这些c-di-GMP代谢相关基因的深入研究不仅有利于了解病原菌的致病和耐药机理,而且也可为开发新型药物提供靶标和理论基础。细菌中通常存在多个c-di-GMP代谢蛋白,控制着胞内c-di-GMP水平稳态。C-di-GMP代谢蛋白的活性是受到精细调控的,近年来的研究表明,涉及环鸟苷二磷酸代谢的蛋白往往可以感应环境信号,从而影响胞内c-di-GMP的水平及表型改变。其中,c-di-GMP代谢蛋白的N-端结构域可能发挥了信号识别作用,但对各种信号的识别特异性及信号输入方式还很不清楚。CHASE4是存在于含GGDEF结构域的PA0847和含GGDEF-EAL结构域的PA2072蛋白N端假定的感应结构域,其在c-di-GMP信号网络中的作用尚未得到研究。本项研究主要以铜绿假单胞菌中PA0847和PA2072为研究对象,对其结构和功能进行了探索,主要包括以下几个部分:1.对PA0847和PA2072的c-di-GMP代谢相关功能进行了验证。根据已有研究,PA0847胞内结构域具有二鸟苷酸环化酶活性。通过刚果红染色实验验证了PA2072的体内磷酸二酯酶活性,通过噻唑橙荧光测定实验验证了PA2072含EAL的胞内结构域在Mg²⁺存在下具有体外磷酸二酯酶活性。2.PA2072 CHASE4结构域在溶液中以单体和二聚体状态之间的平衡存在。通过NMR解析了PA2072 CHASE4的单体结构。PA2072 CHASE4结构域属于dCache构象,由两个PAS样亚结构域通过长N-末端α-螺旋（α1）连接组成。通过晶体X射线晶体衍射解析了PA2072 CHASE4二聚体的结构。主要的二聚界面位于两个单体的螺旋α1之间,其包埋面积约为2284（?）²。PA2072 CHASE4结构域在单体和二聚体构象中的整体结构相似,其RMSD为1.73（?）。3.对PA2072 CHASE4的直接相互作用物质进行了体外筛选。经NMR滴定发现Mg²⁺存在下,外源ATP与PA2072 CHASE4单体具有弱相互作用,在相同的条件下,AMP,ADP,GTP,CTP,UTP,d ATP,Adenine,Adenosine与PA2072 CHASE4的互作不明显。4.通过HPLC和³¹P NMR谱检测证实,ATP与PA2072 CHASE4单体的直接结合导致ATP水解为ADP和AMP,而磷酸基团向CHASE4蛋白的转移未被检测到。因此,PA2072 CHASE4结构域具有ATPase活性。与单体相比,PA2072CHASE4结构域的二聚体形式显示出大约低10倍的ATPase活性。5.基于NMR滴定和突变实验的结果,通过HADDOCK获得了ATP结合PA2072 CHASE4单体的对接模型,产生了10个收敛良好的最低能量复合结构,进一步表明了ATP和CHASE4结构域之间的特异性相互作用。在复合模型中,ATP分子与PA2072 CHASE4的近膜端PAS样域结合。ATP的O’和N6与S230-O和PA2072 CHASE4的H53-N^（?）2之间形成的关键分子间氢键,以及Mg²⁺和D227侧链之间形成的两个配位共价键,决定了ATP底物的结合特异性。PA2072CHASE4的单体和二聚体之间的结构比较揭示了二聚化触发螺旋α1和链β7的构象变化,导致更开放的口袋,这种变化可能显著削弱ATP结合并阻碍ATP的有效水解。6.最后,细胞外ATP被认为是“危险分子”。我们提出了一个细胞外CHASE4结构域感受ATP的模型,该结构域可能变构调节细胞内PDE的激活。这项研究的发现为理解PA2072调节细胞内c-di-GMP水平以响应危险的细胞外ATP信号提供了结构基础。更多还原

【Abstract】 Pseudomonas aeruginosa（PAO1）is an important opportunistic pathogen in hospi-tal infection.Biofilm,motility,and virulence,which are significant phenotypes related to infection and pathogenesis,are regulated by c-di-GMP,a small secondary messenger.C-di-GMP is synthesized by diguanylate cyclases（DGCs）containing GGDEF（Gly-Gly-Asp-Glu-Phe）domain and degraded by phosphodiesterases（PDEs）containing EAL（Glu-Ala-Leu）or HD-GYP domains.The in-depth structural and functional studies of these c-di-GMP metabolizing proteins will not only help understand the pathogenic and drug resistance mechanisms of PAO1 but also provide targets and a theoretical basis for the development of novel drugs.Multiple c-di-GMP metabolizing proteins are usually present in bacteria and con-trol the homeostasis of intracellular c-di-GMP levels.The activities of c-di-GMP me-tabolizing proteins are fine-tuned by different signals.In recent years,it has been found that membrane-bound c-di-GMP-metabolizing proteins are involved in sensing environ-mental signals,leading to variations in intracellular c-di-GMP levels and phenotypical changes.However,sensing specificity and signal transduction mechanisms are unclear.CHASE4 domain is a putative extracellular sensing domain found in the N termini of GGDEF-containing PA0847 and GGDEF-EAL-containing PA2072 transmembrane pro-teins from P.aeruginosa.This study aimed to investigate the structures and functions of PA0847 and PA2072.The focus was on the following aspects:1.The c-di-GMP metabolism-related functions of PA0847 and PA2072 were val-idated.The intracellular domain of PA0847 possesses DGC activity.The in vivo PDE activity of PA2072 was verified by Congo red staining assay,and the in vitro PDE ac-tivity of the EAL-containing intracellular domain of PA2072 in the presence of Mg²⁺was confirmed by thiazole orange fluorescence assay.2.PA2072 CHASE4 domain exists in equilibrium between monomeric and dimeric states in solution.The monomeric structure of PA2072 CHASE4 was resolved by NMR spectroscopy.PA2072 CHASE4 domain belongs to double Cache（dCache）fold con-sisting of two PAS-like subdomains connected by a long N-terminalα-helix（α1）.The dimeric structure of the PA2072 CHASE4 was determined by X-ray crystallography.The main dimerization interface,with a buried area of～2284（?）²,is located between the helicesα1 of the two monomers.The overall structures of PA2072 CHASE4 domain in the monomeric and dimeric conformations are similar,with a pairwise RMSD of 1.73（?）.3.NMR titration showed that ATP can interact with PA2072 CHASE4 monomer in the presence of Mg²⁺,with a weak binding affinity in the sub-m M range.However,other nucleotides and ATP derivatives,including AMP,ADP,GTP,CTP,UTP,d ATP,adenine,and adenosine,showed no observable specific interactions with the CHASE4domain.4.Direct binding of ATP to PA2072 CHASE4 monomer leads to ATP hydrolysis to ADP and AMP,as confirmed by HPLC and³¹P NMR spectroscopy,while the trans-fer of phosphate groups to the CHASE4 protein was not detected.Therefore,PA2072CHASE4 domain has ATPase activity.Compared to the monomer,the dimeric form of PA2072 CHASE4 domain shows approximately 10 times lower ATPase activity.5.HADDOCK structure of ATP-bound PA2072 CHASE4 domain was calculated based on experimental data obtained from NMR titration and mutagenesis studies,yield-ing ten well-converged lowest-energy conformations and further indicating specific in-teraction between ATP and the CHASE4 domain.In the complex model,theATP molecule binds to the membrane-proximal PAS-like subdomain of PA2072 CHASE4.Two critical intermolecular hydrogen bonding between O’and N⁶of ATP and S230-O and H53-N^（?）2of PA2072 CHASE4,respectively,together with two coordinated covalent bonds between Mg²⁺and the side chain of D227,dictate the binding specificity for ATP substrate.Structural comparison between the monomer and dimer of PA2072 CHASE4reveals that dimerization triggers conformational changes in helixα1 and strandβ7,lead-ing to a more open pocket which may significantly weaken ATP binding and impedes efficient ATP hydrolysis.6.Lastly,extracellular ATP（eATP）is considered a“dangerous molecule”.We proposed a model for ATP-sensing by the extracellular CHASE4 domain,which may allosterically modulate intracellular PDE activation.The findings from this study pro-vide a structural basis for understanding PA2072-regulated intracellular c-di-GMP level in response to the dangerous extracellular ATP signal.更多还原