Coordination Chemistry of Biological Iron Transport Agents
Introduction
All multi-cellular and essentially all single-celled organisms require iron for growth, even though the biological availability of iron is extremely limited by the insolubility of iron hydroxide. To overcome the poor availability and potential toxicity of iron and achieve effective homeostasis in aqueous aerobic conditions, organisms must tightly regulate their iron balance.
While mammals use the serum protein transferrin to transport iron through the blood to cells and the protein shell ferritin for its storage, micro-organisms synthesize low-molecular-weight chelating agents called siderophores which bind and solubilize iron. Characterized by their high specificity and affinity towards ferric ion, siderophores are exported from the cell in their apo form and can remove iron from minerals and host iron-binding proteins to form stable ferric complexes that are transported into bacterial cells through specific receptor proteins.
Removal of iron from environmental or host sources by siderophores and incorporation of the iron-siderophore complex into the cell are often the rate limiting factors in bacterial growth and pathogenesis. Understanding siderophore chemistry (including the coordination chemistry of the ferric-siderophore complex, kinetics of iron acquisition from a host, and bacterial uptake mechanisms) is therefore of direct medical relevance. One specific aim of our iron project is to characterize the mechanisms of siderophore-mediated iron uptake and to understand the role of siderophores in bacterial diseases. We are also applying our understanding of siderophore chemistry to the design of therapeutic synthetic iron chelators for the treatment of iron-overload diseases.
Siderophore Structure and Coordination Chemistry
The recent isolation of bacillibactin (BB) from many Bacilli species has provided a Gram-positive counterpart to the siderophore archetype produced by enteric bacteria, enterobactin (Ent). In the molecular structures of Ent and BB, three 2,3-catecholate moieties are linked to a trilactone backbone through amide bonds. While Ent is formed with a triserine backbone, BB incorporates glycine spacers between the catecholate groups and a threonine-based lactone. The apo forms of the siderophores exported from the bacterial cells are predisposed for iron binding; coordinated to ferric ion, both Ent and BB form very stable complexes (Kf = 1049 and 1047.6 respectively) with the respective characteristic Δ and Λ configurations at the metal center.
Trilactone-based natural siderophores Modification of the seemingly perfect enterobactin structure invites many questions regarding the effect of the alterations on the uptake and stability of ferric bacillibactin as compared to ferric enterobactin. Several analogs of these trilactone-based siderophores have been synthesized to probe the effect of the spacer and trilactone structure on iron complex stability as well as on transport in Gram-negative and positive bacteria.
Current work focuses on structural characterization of the apo- and ferric-forms of these natural siderophores and their synthetic analogs. We are also investigating the effects of structure modifications on transport in Gram-negative and positive bacteria.
Raymond, K. N.; Dertz, E. A.; Kim, S. S. "Enterobactin -- an Archetype for Microbial Iron Transport." Proc. Nat. Acad. Sci. USA 2003, 100, 3584-3588.
Dertz, E. A. and Raymond, K. N. "Siderophores and Transferrins." in Que, L. (Ed.), Comprehensive Coordination Chemistry-II, Vol 8, (2003); pp. 141-168.
Dertz, E. A.; Xu, J.; Stintzi, A.; Raymond, K. N. "Bacillibactin-Mediated Iron Transport in Bacillus subtilis." J. Am. Chem. Soc. 2006, 128, 22-23.
Dertz, E. A.; Xu, J.; Raymond, K. N. "Tren Based Analogs of Bacillibactin: Structure and Stability." Inorg. Chem. 2006, 45, 5465-5478.
Abergel, R. J.; Warner, J. A.; Shuh, D. K.; Raymond, K. N. "Enterobactin Protonation and Iron Release: Structural Characterization of the Salicylate Coordination Shift in Ferric Enterobactin." J. Am. Chem. Soc. 2006, 128, 8920-8931.
Wilson, M. K.; Abergel, R. J.; Raymond, K. N.; Arceneaux, J. E. L.; Byers, B. R. "Siderophores of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis." Biochem. Biophys. Res. Commun. 2006, 348, 320-325.
Abergel, R. J.; Zawadzka, A. M.; Raymond, K. N. "Petrobactin-Mediated Iron Transport in Pathogenic Bacteria: Coordination Chemistry of an Unusual 3,4-Catecholate/Citrate Siderophore." J. Am. Chem. Soc. 2008, 130, 2124-2125.
Siderophore Mediated Iron Transport
Most of our studies with synthetic analogs of natural siderophores have established important correlations between the coordination chemistry of the ferric complex and recognition at the outer membrane of Gram-negative (double cell membrane) organisms such as E. coli and A. hydrophila. Siderophore receptor proteins on the outer membrane bind specifically iron siderophore complexes and energy from the cytoplasm is shuttled to the outer membrane through intermediate proteins. We have also shown that metal exchange between two siderophores was essential for iron transport. The ligand exchange step occurs at the cell surface and involves the transfer of iron from a ferric-siderophore to an iron-free siderophore bound to its receptor. This shuttle mechanism implies an increase in the iron uptake rate with increasing concentrations of iron-free siderophore, a characteristic of ferric-siderophore poor environments in vivo.
Proposed model of the siderophore shuttle iron exchange mechanism for iron transport in Gram-negative bacteria. While most studies about bacterial iron transport have been performed with Gram-negative organisms, very little is known about siderophore-mediated iron transport in Gram-positive organisms, even though this family includes many important human pathogens. We are now interested in characterizing the mechanisms of siderophore-mediated iron uptake in some representative Gram-positive bacteria such as B. subtilis and B. cereus.
Stintzi, A.; Barnes, C.; Xu, J.; Raymond, K. N. "Microbial Iron Transport via a Siderophore Shuttle: A Membrane Ion Transport Paradigm." Proc. Natl. Acad. Sci. USA 2000, 97, 10691-10696.
Dertz, E. A.; Stintzi, A.; Raymond, K. N. "Siderophore-mediated Iron Transport in B. subtilis and C. glutamicum." J. Biol. Inorg. Chem. 2006, 11, 1087-1097.
Bacterial Siderophore-Human Protein Interaction
The binding of enterobactin by siderocalin (formally NGAL) is the first indication that human proteins may be produced to bind siderophores as an immunoresponse. Just as some siderophore receptors can bind a variety of siderophores, the generality of siderocalin-siderophore interaction is a major aspect of this project.
Determining which siderophores siderocalin binds can help elucidate the relevant molecular interactions and the function of the protein as an antagonist for siderophore-mediated iron transport. The specificity of siderocalin for a variety of siderophores and analogs is being probed in this collaboration with the research group of Dr. Roland Strong.
The first human protein-
siderophore interactionSiderocalin binds ferric enterobactin with an affinity close to that of the bacterial outer membrane receptor protein, FepA, however, the binding specificity of siderocalin differs notably from that of FepA. We have determined that the binding of ferric enterobactin by siderocalin is not significantly altered by changing the metal center, the siderophore scaffold or the chirality of the metal complex, but substitution of the 5' position on the catecholate rings can introduce enough steric hindrance to preclude protein binding.
Siderocalin can then act as an innate immune response to siderophore-mediated iron acquisition by sequestering siderophores such as enterobactin and bacillibactin, found in enteric bacteria and Bacilli species, respectively. Therefore, despite the great efficiency of these catecholate ligands at chelating iron, independent synthesis of additional stealth siderophores like aerobactin, salmochelin or petrobactin, which can evade siderocalin binding, is necessary to insure full virulence of the lethal pathogens.
The arms race between the mammalian immune system and bacteria in the search for iron: enterobactin removes iron from transferrin (a), siderocalin intercepts the ferric complex of enterobactin (b), bacteria produce alternative siderophores such as salmochelin S4 (c). Current work in our lab focuses on synthesizing siderophore analogs to further probe the specificity of the protein and to characterize spectroscopically the interactions between the protein and its substrates (mainly hybrid electrostatic/cation-π interactions). We are also investigating the effect of siderocalin and similar proteins on the growth of different human pathogens.
Goetz, D. H.; Holms, M. A.; Borregaard, N.; Bluhm, M. E.; Raymond, K. N.; Strong, R. K. "The Neutrophil Lipocalin NGAL is a Bacteriostatic Agent that Interferes with Siderophore-Mediated Iron Acquisition." Mol. Cell 2002, 10, 1033-1043. (Cover article.)
Flo, T. H.; Smith, K. D.; Sato, S.; Rodriguez, D. J.; Holmes, M. A.; Strong, R. K.; Akira, S.; Aderem, A. Nature 2004, 432, 917-921.
Abergel, R. J.; Moore, E. G.; Strong, R. K.; Raymond, K. N. "Microbial Evasion of the Immune System: Structural Modifications of Enterobactin Impair Siderocalin Recognition." J. Am. Chem. Soc. 2006, 128, 10998-10999.
Fischbach, M. A.; Lin, H.; Zhou, L.; Yu, Y.; Abergel, R. J.; Liu, D. R.; Raymond, K. N.; Wanner, B. L.; Strong, R. K.; Walsh, C. T.; Aderem, A.; Smith, K. D. "The Pathogen-associated IroA Gene Cluster Mediates Bacterial Evasion of Lipocalin 2." Proc. Nat. Acad. Sci. USA 2006, 103, 16502-16507.
Abergel, R. J.; Wilson, M. L.; Arceneaux, J. E. L.; Hoette, T. M.; Strong, R. K.; Byers, B. R.; Raymond, K. N. "The Anthrax Pathogen Evades the Mammalian Immune System Through Stealth Siderophore Production." Proc. Natl. Acad. Sci. USA 2006, 103, 18499-18503.
Therapeutic Iron Chelators
Treatments of blood-transfusion requiring diseases such as β-thalassemia, hemochromatosis or sickle cell anemia generate an iron overload condition in humans. Such serious clinical condition can be treated by iron chelation therapy effectively, but the goal of developing readily available, orally-active, effective and non-toxic sequestering agents has proven to be remarkably difficult to achieve. Desferrioxamine (Desferal©), a trihydroxamate ligand, remains the iron chelator of clinical use in the United States. Desferal© is expensive, has a short half-life in vivo, does not efficiently remove iron from transferrin, must be given on a regular, frequent basis by a subcutaneous or intravenous route, and its use can result in significant, irreversible toxicity. While other agents such as the tridentate chelator Deferasirox©, developed by Novartis, are being investigated as potential successors to Desferal©, orally active multidentate chelators that can bind iron more efficiently at lower doses are still desperately needed. The goal of this project is to develop new sequestering agents that would meet that need.
