Structural And Functional Researches On Siderophore Transporters Viup And FepB

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.