致病微生物铁代谢相关蛋白ViuP、FepB的结构与功能研究
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摘要
作为地球上分布最广的金属元素,铁在自然界中的含量可以说是相当丰富,但是大部分铁都是以难溶的氧化态存在,这种状态的铁元素难以被微生物利用。而且过量的铁对生物体还有潜在的危害性,在富氧条件下,细胞内的呼吸作用会产生一些过氧化物和H2O2,亚铁离子能够与过氧化氢通过Fenton反应产生对生物体有害的氢氧根自由基,氢氧根自由基能够通过氧化反应破坏诸如蛋白质、核酸、脂肪等生物大分子。因此,生物体需要精确控制体内游离铁的含量,在宿主体内,冗余的铁元素被转铁蛋白、乳铁蛋白等铁蛋白螯合掉,能供微生物利用的铁元素少之又少。而铁对于微生物的生长繁殖又是必需的,为了获得铁元素,一些细菌和真菌类能够合成一种能够高效螯合铁离子的小分子化合物-铁载体,来获得所需要的铁元素。从与铁配位的亚体的角度来看,铁载体可以分成四大类：儿茶酚型铁载体、羟基氧肟酸型铁载体、羧酸类铁载体,还有一类是前面三类的混合型铁载体。这些种类的铁载体中,儿茶酚型铁载体对铁的亲和力最高,这种铁载体大部分是通过2,3-二羟基苯甲酸亚体上的氧原子与铁进行六配位,其复合物表现出三个单位的负电荷。目前为止发现的对铁亲和力最高的儿茶酚型铁载体-肠菌素与铁作用,其解离常数可达10-52。儿茶酚型铁载体的另一个代表成员是由霍乱弧菌合成的弧菌素分子,它的分子骨架由反亚精胺、苏氨酸和2,3-二羟基苯甲酸三部分构成。
微生物进入宿主中之后,会通过合成铁载体的方式来获得铁元素,为了应对微生物的这种入侵,阻止微生物获得所需要的铁元素,哺乳动物能够合成一种叫siderocalin的免疫蛋白,这种免疫蛋白后来证实它能够高效地捕获儿茶酚型铁载体-肠菌素,Raymond等人通过用肠菌素对siderocalin荧光淬灭的实验得出siderocalin蛋白对肠菌素的亲和力相当高,解离常数可以达到纳摩尔的级别。因此,肠菌素分子虽然对铁的螯合性非常强,但在siderocalin免疫蛋白的作用下,螯合了铁离子的肠菌素分子并不能被大肠杆菌收回运送到细胞内供其利用,从而在这个过程中siderocalin蛋白起到了免疫杀菌的作用。为了逃避宿主siderocalin蛋白的免疫过程,一些致病肠道菌譬如沙门氏菌、克雷伯氏菌等能够对合成出来的肠菌素进行糖基化修饰,使得肠菌素的骨架变长,这样经过修饰的肠菌素由于骨架变大便不能被被siderocalin蛋白捕获。这样修饰肠菌素的代价是肠菌素不能保持像本体状态那样对铁离子的极高的亲和力,但是却能够逃避siderocalin免疫蛋白的捕获,为致病肠道菌提供所必要的铁元素。逃逸宿主siderocalin蛋白免疫过程的另个机制是炭疽杆菌的“偷袭机制”,它并不是在原有的铁载体基础上对其进行修饰,而是在铁载体合成过程中利用了一种在其他铁载体中很少见的3,4-二羟基苯甲酸亚体,通过这个亚体上的氧原子与铁进行配位,与铁配位后,Rebecca等人证实这种铁载体在与siderocalin蛋白作用时会产生空间位阻,使得siderocalin免疫蛋白无法捕获这种特殊的铁载体。
霍乱弧菌是霍乱的病原体,能够引起呕吐、腹泻等症状。弧菌素是霍乱弧菌在低铁环境下合成的一种儿茶酚型铁载体,关于弧菌素分子与铁离子的配位方式,Griffiths等人认为其第二个恶唑环上的氮原子也参与了配位,即五个氧原子和一个氮原子与铁配位,而Miethke等人认为是三个儿茶酚亚体上的六个氧原子与铁进行配位,那么弧菌素分子到底通过什么原子和铁离子配位,它是否也能逃逸siderocalin蛋白的免疫过程。并且,在体外环境中结合了铁离子之后,弧菌素分子是如何被识别并转运至霍乱弧菌细胞内部被利用也是人们比较关心的一个问题,这些都可以通过结构生物学的手段来解决。
在弧菌素分子由细胞外转运至细胞内的过程中,ViuP蛋白是其中的周质空间转运蛋白,它的作用是把带铁弧菌素由外膜孔蛋白ViuA转运到内膜上的ABC体系ViuDGC蛋白,在带铁弧菌素的运输中起到了承前启后的作用。我们的课题的第一部分就是试图通过解析Viup蛋白和ViuP与带铁弧菌素复合物的分子结构,来阐明ViuP蛋白与弧菌素分子的作用机制,以及弧菌素分子与铁离子真正的配位模式。通过这部分的实验,我们得到了以下结果：
1) ViuP蛋白分子整体结构是两个相对独立的球状结构域被一段α螺旋结构连接,属于第三类周质空间转运蛋白家族,在带铁弧菌素结合前和结合后ViuP蛋白的整体结构基本上不发生明显变化；
2)通过对比ViuP蛋白和其他儿茶酚型铁载体转运蛋白的三维结构,发现ViuP蛋白的弧菌素结合口袋的方向与其他的铁载体转运蛋白的底物结合口袋的方向相反,因此,ViuP蛋白从进化角度上来说可能来源于不同于其他的铁载体转运蛋白的新的家族；
3) ViuP与带铁弧菌素的复合物的高分辨率结构(1.45A)证实弧菌素分子是通过五个氧原子和一个氮原子与铁离子配位,这种独特的配位方式使带铁弧菌素分子表现出两个单位的负电荷；
4)通过对siderocalin蛋白的荧光淬灭实验,表明siderocalin不能像螯合肠菌素分子那样高效地捕获带铁弧菌素分子,分析判断可能与带铁弧菌素分子特殊的铁配位形式有关。
本课题的另一部分是关于肠菌素分子的周质空间转运蛋白的结构和功能分析。作为最高效的儿茶酚型铁载体,肠菌素结合蛋白目前已有一些研究,其外膜结合蛋白FepA的结构于1999年被发表。FepA蛋白识别并结合带铁肠菌素分子后,将肠菌素转运到周质空间,在周质空间内,再由FepB蛋白将带铁肠菌素分子转移给内膜上的ABC转运系统。FepB蛋白是如何识别并转运肠菌素分子,它与下游的ABC转运蛋白是如何相瓦作用的,我们通过解析FepB蛋白与带铁肠菌素复合物的结构对这些问题有了初步的回答。通过这部分的结构与功能实验,我们得到了以下实验结果：
1) FepB蛋白分子整体结构也是两个相对独立的球伏结构域被一段α螺旋结构连接,符合第三类周质空间转运蛋白家族的基本特征；
2)通过对比FepB蛋白和ViuP蛋白以及其他的儿茶酚型铁载体转运蛋白的三维结构发现,FepB蛋白的肠菌素结合的口袋方向与ViuP蛋白一致,而与其他的铁载体转运蛋白都相反,这说明FepB蛋白可能与ViuP蛋白来源于同一个进化的家族,而不是目前已经报道的其他的铁载体转运蛋白家族；
3)复合物结构的一个重要特征是FepB蛋白与带铁肠菌素分子是以3：4的比例结合,每三个结合了肠菌素分子的FepB蛋白中间还结合了一个额外的带铁肠菌素分子,这种结合比例模式在其他铁载体转运蛋白中从未见报道。通过对加入不同浓度肠菌素分子的FepB蛋白溶液的动态光散射实验,我们发现高浓度的带铁肠菌素分子能够使FepB蛋白的聚合态由单体转变为三体,据此我们推测,FepB蛋白除了具有转运肠菌素的作用,可能还具有储存带铁肠菌素的功能,从而防止冗余的带铁肠菌素分子通过TolC系统溢出,提高了大肠杆菌对肠菌素分子的利用率；
4)通过序列比对和结构叠合我们找到了FepB蛋白上可能与下游FepDG蛋白作用的两个保守的酸性氨基酸残基Glu-109和Glu-251。在验证这两个氨基酸残基的重要性时我们发现这两个氨基酸的重要程度并不一致。Glu-251氨基酸突变掉之后,回补的fepB敲除的大肠杆菌在限铁LB平板上不能正常生长,而Glu-109氨基酸突变掉之后,回补的fepB敲除的大肠杆菌却依然能够正常生长。可能是由于Glu-251突变掉之后FepB蛋白不能识别下游的ABC转运蛋白,而FepB蛋白的Glu-109突变体依然能够识别下游ABC转运蛋白并将带铁肠菌素传递下去,这说明在识别下游ABC转运蛋白的过程中,FepB蛋白的C端结构域可能发挥了更重要的作用。
Despite being the most abundant metal element on the earth, iron is essentially inaccessible to biological systems in many conditions. Firstly, the earth's aerobic and aqueous environment stabilize iron's ferric state. Ferric irons are easy to combine with hydroxides forming insoluble ferric hydroxides, which makes the concentration of soluble ferric iron is reduced to levels below10-18M in the environment. Besides, iron also represents a potentially toxic element due to its propensity to react with oxygen and reactive oxygen species (ROS). Under aerobic conditions, cellular respiration produces considerable amounts of ROS such as superoxide and H2O2. Ferrous iron can react with those species through a serious of steps known as Fenton reactions to generate further ROS such as hydroxyl radicals. Hydroxyl radicals can damage biological macromolecules such as proteins, nucleic acids and lipids through oxidative mechanisms. For this reason, control of the concentration of ferrous iron is vitally important to biology system. So for bacteria that colonize host organisms, bioavailability of iron is reduced even further by sequestration strategies of the innate ferric-proteins such as transferrin and lactoferrin. In this niche, the concentration of bioavailable iron is as low as10-24M which is well below the micromolar amounts required for a single generation of a bacterium's life cycle. In order to obtain enough iron for growth, some bacteria and fungi have evolved the ability to synthesize low-molecular weight, high-affinity iron chelators known as siderophores to scavenge iron with high efficiency. Siderophores can be divided into four groups according to the moieties that coordinate with iron:catecholates, hydroxamates, carboxylates and 'mix type' that combining previous three types. Among those siderophores, catecholates are the most powerful iron chelators. Most of the catecholate siderophore donates six oxygen ligands from three moieties of2,3-DHBA to coordinate with ferric atom, which represents three units of negative charges. Enterobactin is a kind of catecholate siderophore, which represents the most efficient iron chelator recent to now, whose dissociate constant for ferric atom can reach10-52M. Vibriobactin is another kind of catecholate siderophore which is secreted by Vibrio cholerae. The schematic of vibriobactin include norspermidine, threonine and2,3-dihydroxybenzoyl moieties. As the microorganisms invades host such as mammalians, they will obtain the iron element through siderophore pathway. As a defense strategy, the innate immunity system of mammalian can synthesize a kind of protein named siderocalin. Through the fluorescence quenching analysis of siderocalin by enterobactin, Raymond et al discovered that the dissociate constant of siderocalin for enterobactin was so low that siderocalin could capture ferric-enterobactin in high efficiency. In this case, although enterobactin represents most efficient for ferric atom, bacteria are hard to obtain iron in mammalian niche through normal enterobactin because most of ferric-enterobactin are scavenged by innate immunity protein siderocalin. In the battle for iron, in order to evade the siderocalin immune system, some enteric pathogens such as Salmonella spp and K. pneumonia evolved the strategy to synthesized a special glucosyl-enterobactin. Comparing with normal enterobactin, the glucosyl-enterobactin represents weaker affinity for ferric atom, however, this kind of special enterobactin can evade the siderocalin system because of its huge molecular skeleton. Bacillus anthracis has evolved another stealth strategy to evade innate immune response. To capture iron from its environment, B. anthracis can synthesize two kinds of siderophores, bacillibactin (BB) and petrobactin (PB) by independent pathways. Despite the great efficiency of BB at chelating iron, BB can be captured by siderocalin protein. BB incorporates the common2,3-dihydroxylbenzoyl as iron chelating subunits, while PB comprises the very unusual3,4-dihydroxylbenzoyl chelating subunit. The structural variation of PB results in a large change in the shape of iron complex that precludes siderocalin binding. For this reason, B. anthracis could evade the siderocalin immune system by synthesizing the special siderophore petrobactin.
Some strains of V. cholerae cause the disease cholera. In the iron limited environment, V. cholerae can synthesize vibriobactin, a kind of catecholate siderophore. There are controversies about the coordination of vibriobactin and ferric atom. Griffiths et al. suggested that the nitrogen atom of the second oxazole may also participate in iron coordination, and the other oxygen atoms come from the2,3-DHBA moieties of vibriobactin, whereas Miethke et al. proposed that the six oxygen atoms from the three catechol moieties coordinate with the ferric atom. So what is the exact coordination type for vibriobactin and ferric atom? Besides, whether ferric vibriobactin has the ability to evade the scavenge of siderocalin. And many researchers also concerned that how vibriobactin is recognized and transported into the cytoplasm of V. cholerae. Those problems can be resolved by the method of structure biology. The secreted vibriobactin will chelate ferric iron in high efficiency and forms ferric-vibriobactin complex. The ferric-vibriobactin complex is then transported into the cytoplasm of V. cholerae by ViuAPDGC proteins. Of those transport proteins ViuA is a porin locating at the outer membrane of V. cholerae which can recognize and transport ferric vibriobactin to the periplasmic space of V. cholerae using the energy of TonB system. In the periplasmic space, ferric vibriobactin can be transferred to inner membrane ABC transport system ViuDGC by periplasm binding protein ViuP. So ViuP protein plays a vital feature in the transport pathway for ferric vibriobactin. First part of our research is to clarify the recognition mode for ViuP protein and ferric vibriobactin through the crystal structure of ViuP and holo-ViuP (ViuP and ferric vibriobactin complex), besides, we also hope to solve the problem about the exact coordination type for vibriobactin and ferric atom. Through the structural and functional research about ViuP and holo-ViuP, we get the following results.
(1) The overall structure of ViuP represents like a kidney, which contains two independently globular type domains, and those two domains are linked by a long a helix structure. The structure of ViuP belongs to type III periplasm binding protein and there is no apparent changeable after it binding ferric vibriobactin.
(2) The binding pocket of ferric vibriobactin is located at the opposite site of the ViuP protein comparing with other type III siderophore binding PBPs. This finding suggests that ViuP and other known catecholate siderophore binding PBPs may have evolved to the same fold via convergent evolution from different ancestral proteins.
(3) The high resolution structure (1.45A) of holo-ViuP demonstrates that five oxygen atoms and one nitrogen atom of vibriobactin participate in coordinating with ferric atom. Ferric vibriobactin represents two negative charges due to the unique coordination with iron atom.
(4) We found that the innate immune protein siderocalin can't capture ferric vibriobactin in high affinity like ferric enterobactin through fluorescence quenching analysis. That means V. cholerae may evade human siderocalin to get iron, although more in vivo data are needed to support this argument.
The other part of my thesis is about the structural and functional analysis of the enterobactin periplasm binding protein named FepB. FepA protein is a porin located at the outer membrane of E. coli which can recognize and transport ferric enterobactin into the periplasmic space. The crystal structure of FepA has been solved and published in the year1999. In periplasmic space, another PBP protein FepB will transfer ferric enterobactin into the inner membrane ABC transporter ViuDGC. We concerned that how FepB recognize and transport ferric enterobactin, and how FepB protein interacts with ViuDGC. Through the crystal structure of FepB combined with ferric enterobactin and some functional experiments we get the following results.
(1) The overall structure of FepB represents like a kidney with two independently globular domains linked by a long a helix structure. So FepB protein belongs to type Ⅲ periplasm binding protein family.
(2) The substrate binding pocket of FepB and ViuP are on the same position according to the superposing result of holo-FepB and holo-ViuP structures. The finding suggests that FepB and ViuP probably evolved to the fold from the same ancestral family protein while different from other known catecholate siderophore binding PBPs.
(3) The crystal structure of holo-FepB reveals the3:4stoichiometry for FepB protein and ferric enterobactin, in which every three FepB molecules contain one additional ferric enterobactin. The dynamic light scattering experiment for FepB demonstrated that FepB would form trimer state as the concentration of ferric enterobactin increased. According to those results, we can speculate that FepB also has the ability to store ferric enterobactin as the concentration of enterobactin increased. This mechanism could enhance the efficiency for E. coli to utilize enterobactin because the redundant ferric enterobactin in periplasmic space would escape through TolC system and rebound to FepA, which is a futile leakage cycle for iron metabolism.
(4) We found two conserved residues of FepB, Glu-109and Glu-251that may interact with FepDG through sequence alignment and structure superposing. However, the in vivo data revealed that residue Glu-251was more important than the residue Glu-109, which demonstrate that the C terminal domain of FepB plays vital feature in the recognition of FepDG proteins. The characteristic of FepB was not reported for other siderophore binding proteins.
微生物进入宿主中之后,会通过合成铁载体的方式来获得铁元素,为了应对微生物的这种入侵,阻止微生物获得所需要的铁元素,哺乳动物能够合成一种叫siderocalin的免疫蛋白,这种免疫蛋白后来证实它能够高效地捕获儿茶酚型铁载体-肠菌素,Raymond等人通过用肠菌素对siderocalin荧光淬灭的实验得出siderocalin蛋白对肠菌素的亲和力相当高,解离常数可以达到纳摩尔的级别。因此,肠菌素分子虽然对铁的螯合性非常强,但在siderocalin免疫蛋白的作用下,螯合了铁离子的肠菌素分子并不能被大肠杆菌收回运送到细胞内供其利用,从而在这个过程中siderocalin蛋白起到了免疫杀菌的作用。为了逃避宿主siderocalin蛋白的免疫过程,一些致病肠道菌譬如沙门氏菌、克雷伯氏菌等能够对合成出来的肠菌素进行糖基化修饰,使得肠菌素的骨架变长,这样经过修饰的肠菌素由于骨架变大便不能被被siderocalin蛋白捕获。这样修饰肠菌素的代价是肠菌素不能保持像本体状态那样对铁离子的极高的亲和力,但是却能够逃避siderocalin免疫蛋白的捕获,为致病肠道菌提供所必要的铁元素。逃逸宿主siderocalin蛋白免疫过程的另个机制是炭疽杆菌的“偷袭机制”,它并不是在原有的铁载体基础上对其进行修饰,而是在铁载体合成过程中利用了一种在其他铁载体中很少见的3,4-二羟基苯甲酸亚体,通过这个亚体上的氧原子与铁进行配位,与铁配位后,Rebecca等人证实这种铁载体在与siderocalin蛋白作用时会产生空间位阻,使得siderocalin免疫蛋白无法捕获这种特殊的铁载体。
霍乱弧菌是霍乱的病原体,能够引起呕吐、腹泻等症状。弧菌素是霍乱弧菌在低铁环境下合成的一种儿茶酚型铁载体,关于弧菌素分子与铁离子的配位方式,Griffiths等人认为其第二个恶唑环上的氮原子也参与了配位,即五个氧原子和一个氮原子与铁配位,而Miethke等人认为是三个儿茶酚亚体上的六个氧原子与铁进行配位,那么弧菌素分子到底通过什么原子和铁离子配位,它是否也能逃逸siderocalin蛋白的免疫过程。并且,在体外环境中结合了铁离子之后,弧菌素分子是如何被识别并转运至霍乱弧菌细胞内部被利用也是人们比较关心的一个问题,这些都可以通过结构生物学的手段来解决。
在弧菌素分子由细胞外转运至细胞内的过程中,ViuP蛋白是其中的周质空间转运蛋白,它的作用是把带铁弧菌素由外膜孔蛋白ViuA转运到内膜上的ABC体系ViuDGC蛋白,在带铁弧菌素的运输中起到了承前启后的作用。我们的课题的第一部分就是试图通过解析Viup蛋白和ViuP与带铁弧菌素复合物的分子结构,来阐明ViuP蛋白与弧菌素分子的作用机制,以及弧菌素分子与铁离子真正的配位模式。通过这部分的实验,我们得到了以下结果：
1) ViuP蛋白分子整体结构是两个相对独立的球状结构域被一段α螺旋结构连接,属于第三类周质空间转运蛋白家族,在带铁弧菌素结合前和结合后ViuP蛋白的整体结构基本上不发生明显变化；
2)通过对比ViuP蛋白和其他儿茶酚型铁载体转运蛋白的三维结构,发现ViuP蛋白的弧菌素结合口袋的方向与其他的铁载体转运蛋白的底物结合口袋的方向相反,因此,ViuP蛋白从进化角度上来说可能来源于不同于其他的铁载体转运蛋白的新的家族；
3) ViuP与带铁弧菌素的复合物的高分辨率结构(1.45A)证实弧菌素分子是通过五个氧原子和一个氮原子与铁离子配位,这种独特的配位方式使带铁弧菌素分子表现出两个单位的负电荷；
4)通过对siderocalin蛋白的荧光淬灭实验,表明siderocalin不能像螯合肠菌素分子那样高效地捕获带铁弧菌素分子,分析判断可能与带铁弧菌素分子特殊的铁配位形式有关。
本课题的另一部分是关于肠菌素分子的周质空间转运蛋白的结构和功能分析。作为最高效的儿茶酚型铁载体,肠菌素结合蛋白目前已有一些研究,其外膜结合蛋白FepA的结构于1999年被发表。FepA蛋白识别并结合带铁肠菌素分子后,将肠菌素转运到周质空间,在周质空间内,再由FepB蛋白将带铁肠菌素分子转移给内膜上的ABC转运系统。FepB蛋白是如何识别并转运肠菌素分子,它与下游的ABC转运蛋白是如何相瓦作用的,我们通过解析FepB蛋白与带铁肠菌素复合物的结构对这些问题有了初步的回答。通过这部分的结构与功能实验,我们得到了以下实验结果：
1) FepB蛋白分子整体结构也是两个相对独立的球伏结构域被一段α螺旋结构连接,符合第三类周质空间转运蛋白家族的基本特征；
2)通过对比FepB蛋白和ViuP蛋白以及其他的儿茶酚型铁载体转运蛋白的三维结构发现,FepB蛋白的肠菌素结合的口袋方向与ViuP蛋白一致,而与其他的铁载体转运蛋白都相反,这说明FepB蛋白可能与ViuP蛋白来源于同一个进化的家族,而不是目前已经报道的其他的铁载体转运蛋白家族；
3)复合物结构的一个重要特征是FepB蛋白与带铁肠菌素分子是以3：4的比例结合,每三个结合了肠菌素分子的FepB蛋白中间还结合了一个额外的带铁肠菌素分子,这种结合比例模式在其他铁载体转运蛋白中从未见报道。通过对加入不同浓度肠菌素分子的FepB蛋白溶液的动态光散射实验,我们发现高浓度的带铁肠菌素分子能够使FepB蛋白的聚合态由单体转变为三体,据此我们推测,FepB蛋白除了具有转运肠菌素的作用,可能还具有储存带铁肠菌素的功能,从而防止冗余的带铁肠菌素分子通过TolC系统溢出,提高了大肠杆菌对肠菌素分子的利用率；
4)通过序列比对和结构叠合我们找到了FepB蛋白上可能与下游FepDG蛋白作用的两个保守的酸性氨基酸残基Glu-109和Glu-251。在验证这两个氨基酸残基的重要性时我们发现这两个氨基酸的重要程度并不一致。Glu-251氨基酸突变掉之后,回补的fepB敲除的大肠杆菌在限铁LB平板上不能正常生长,而Glu-109氨基酸突变掉之后,回补的fepB敲除的大肠杆菌却依然能够正常生长。可能是由于Glu-251突变掉之后FepB蛋白不能识别下游的ABC转运蛋白,而FepB蛋白的Glu-109突变体依然能够识别下游ABC转运蛋白并将带铁肠菌素传递下去,这说明在识别下游ABC转运蛋白的过程中,FepB蛋白的C端结构域可能发挥了更重要的作用。
Despite being the most abundant metal element on the earth, iron is essentially inaccessible to biological systems in many conditions. Firstly, the earth's aerobic and aqueous environment stabilize iron's ferric state. Ferric irons are easy to combine with hydroxides forming insoluble ferric hydroxides, which makes the concentration of soluble ferric iron is reduced to levels below10-18M in the environment. Besides, iron also represents a potentially toxic element due to its propensity to react with oxygen and reactive oxygen species (ROS). Under aerobic conditions, cellular respiration produces considerable amounts of ROS such as superoxide and H2O2. Ferrous iron can react with those species through a serious of steps known as Fenton reactions to generate further ROS such as hydroxyl radicals. Hydroxyl radicals can damage biological macromolecules such as proteins, nucleic acids and lipids through oxidative mechanisms. For this reason, control of the concentration of ferrous iron is vitally important to biology system. So for bacteria that colonize host organisms, bioavailability of iron is reduced even further by sequestration strategies of the innate ferric-proteins such as transferrin and lactoferrin. In this niche, the concentration of bioavailable iron is as low as10-24M which is well below the micromolar amounts required for a single generation of a bacterium's life cycle. In order to obtain enough iron for growth, some bacteria and fungi have evolved the ability to synthesize low-molecular weight, high-affinity iron chelators known as siderophores to scavenge iron with high efficiency. Siderophores can be divided into four groups according to the moieties that coordinate with iron:catecholates, hydroxamates, carboxylates and 'mix type' that combining previous three types. Among those siderophores, catecholates are the most powerful iron chelators. Most of the catecholate siderophore donates six oxygen ligands from three moieties of2,3-DHBA to coordinate with ferric atom, which represents three units of negative charges. Enterobactin is a kind of catecholate siderophore, which represents the most efficient iron chelator recent to now, whose dissociate constant for ferric atom can reach10-52M. Vibriobactin is another kind of catecholate siderophore which is secreted by Vibrio cholerae. The schematic of vibriobactin include norspermidine, threonine and2,3-dihydroxybenzoyl moieties. As the microorganisms invades host such as mammalians, they will obtain the iron element through siderophore pathway. As a defense strategy, the innate immunity system of mammalian can synthesize a kind of protein named siderocalin. Through the fluorescence quenching analysis of siderocalin by enterobactin, Raymond et al discovered that the dissociate constant of siderocalin for enterobactin was so low that siderocalin could capture ferric-enterobactin in high efficiency. In this case, although enterobactin represents most efficient for ferric atom, bacteria are hard to obtain iron in mammalian niche through normal enterobactin because most of ferric-enterobactin are scavenged by innate immunity protein siderocalin. In the battle for iron, in order to evade the siderocalin immune system, some enteric pathogens such as Salmonella spp and K. pneumonia evolved the strategy to synthesized a special glucosyl-enterobactin. Comparing with normal enterobactin, the glucosyl-enterobactin represents weaker affinity for ferric atom, however, this kind of special enterobactin can evade the siderocalin system because of its huge molecular skeleton. Bacillus anthracis has evolved another stealth strategy to evade innate immune response. To capture iron from its environment, B. anthracis can synthesize two kinds of siderophores, bacillibactin (BB) and petrobactin (PB) by independent pathways. Despite the great efficiency of BB at chelating iron, BB can be captured by siderocalin protein. BB incorporates the common2,3-dihydroxylbenzoyl as iron chelating subunits, while PB comprises the very unusual3,4-dihydroxylbenzoyl chelating subunit. The structural variation of PB results in a large change in the shape of iron complex that precludes siderocalin binding. For this reason, B. anthracis could evade the siderocalin immune system by synthesizing the special siderophore petrobactin.
Some strains of V. cholerae cause the disease cholera. In the iron limited environment, V. cholerae can synthesize vibriobactin, a kind of catecholate siderophore. There are controversies about the coordination of vibriobactin and ferric atom. Griffiths et al. suggested that the nitrogen atom of the second oxazole may also participate in iron coordination, and the other oxygen atoms come from the2,3-DHBA moieties of vibriobactin, whereas Miethke et al. proposed that the six oxygen atoms from the three catechol moieties coordinate with the ferric atom. So what is the exact coordination type for vibriobactin and ferric atom? Besides, whether ferric vibriobactin has the ability to evade the scavenge of siderocalin. And many researchers also concerned that how vibriobactin is recognized and transported into the cytoplasm of V. cholerae. Those problems can be resolved by the method of structure biology. The secreted vibriobactin will chelate ferric iron in high efficiency and forms ferric-vibriobactin complex. The ferric-vibriobactin complex is then transported into the cytoplasm of V. cholerae by ViuAPDGC proteins. Of those transport proteins ViuA is a porin locating at the outer membrane of V. cholerae which can recognize and transport ferric vibriobactin to the periplasmic space of V. cholerae using the energy of TonB system. In the periplasmic space, ferric vibriobactin can be transferred to inner membrane ABC transport system ViuDGC by periplasm binding protein ViuP. So ViuP protein plays a vital feature in the transport pathway for ferric vibriobactin. First part of our research is to clarify the recognition mode for ViuP protein and ferric vibriobactin through the crystal structure of ViuP and holo-ViuP (ViuP and ferric vibriobactin complex), besides, we also hope to solve the problem about the exact coordination type for vibriobactin and ferric atom. Through the structural and functional research about ViuP and holo-ViuP, we get the following results.
(1) The overall structure of ViuP represents like a kidney, which contains two independently globular type domains, and those two domains are linked by a long a helix structure. The structure of ViuP belongs to type III periplasm binding protein and there is no apparent changeable after it binding ferric vibriobactin.
(2) The binding pocket of ferric vibriobactin is located at the opposite site of the ViuP protein comparing with other type III siderophore binding PBPs. This finding suggests that ViuP and other known catecholate siderophore binding PBPs may have evolved to the same fold via convergent evolution from different ancestral proteins.
(3) The high resolution structure (1.45A) of holo-ViuP demonstrates that five oxygen atoms and one nitrogen atom of vibriobactin participate in coordinating with ferric atom. Ferric vibriobactin represents two negative charges due to the unique coordination with iron atom.
(4) We found that the innate immune protein siderocalin can't capture ferric vibriobactin in high affinity like ferric enterobactin through fluorescence quenching analysis. That means V. cholerae may evade human siderocalin to get iron, although more in vivo data are needed to support this argument.
The other part of my thesis is about the structural and functional analysis of the enterobactin periplasm binding protein named FepB. FepA protein is a porin located at the outer membrane of E. coli which can recognize and transport ferric enterobactin into the periplasmic space. The crystal structure of FepA has been solved and published in the year1999. In periplasmic space, another PBP protein FepB will transfer ferric enterobactin into the inner membrane ABC transporter ViuDGC. We concerned that how FepB recognize and transport ferric enterobactin, and how FepB protein interacts with ViuDGC. Through the crystal structure of FepB combined with ferric enterobactin and some functional experiments we get the following results.
(1) The overall structure of FepB represents like a kidney with two independently globular domains linked by a long a helix structure. So FepB protein belongs to type Ⅲ periplasm binding protein family.
(2) The substrate binding pocket of FepB and ViuP are on the same position according to the superposing result of holo-FepB and holo-ViuP structures. The finding suggests that FepB and ViuP probably evolved to the fold from the same ancestral family protein while different from other known catecholate siderophore binding PBPs.
(3) The crystal structure of holo-FepB reveals the3:4stoichiometry for FepB protein and ferric enterobactin, in which every three FepB molecules contain one additional ferric enterobactin. The dynamic light scattering experiment for FepB demonstrated that FepB would form trimer state as the concentration of ferric enterobactin increased. According to those results, we can speculate that FepB also has the ability to store ferric enterobactin as the concentration of enterobactin increased. This mechanism could enhance the efficiency for E. coli to utilize enterobactin because the redundant ferric enterobactin in periplasmic space would escape through TolC system and rebound to FepA, which is a futile leakage cycle for iron metabolism.
(4) We found two conserved residues of FepB, Glu-109and Glu-251that may interact with FepDG through sequence alignment and structure superposing. However, the in vivo data revealed that residue Glu-251was more important than the residue Glu-109, which demonstrate that the C terminal domain of FepB plays vital feature in the recognition of FepDG proteins. The characteristic of FepB was not reported for other siderophore binding proteins.
引文
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