Determination of the Molecular Structures of Ferric Enterobactin and Ferric Enantioenterobactin Using Racemic Crystallography

Enterobactin is a secondary metabolite produced by Enterobacteriaceae for acquiring iron, an essential metal nutrient. The biosynthesis and utilization of enterobactin permits many Gram-negative bacteria to thrive in environments where low soluble iron concentrations would otherwise preclude survival. Despite extensive work carried out on this celebrated molecule since its discovery over 40 years ago, the ferric enterobactin complex has eluded crystallographic structural characterization. We report the successful growth of single crystals containing ferric enterobactin using racemic crystallization, a method that involves cocrystallization of a chiral molecule with its mirror image. The structures of ferric enterobactin and ferric enantioenterobactin obtained in this work provide a definitive assignment of the stereochemistry at the metal center and reveal secondary coordination sphere interactions. The structures were employed in computational investigations of the interactions of these complexes with two enterobactin-binding proteins, which illuminate the influence of metal-centered chirality on these interactions. This work highlights the utility of small-molecule racemic crystallography for obtaining elusive structures of coordination complexes

Siderophore-Mediated Cargo Delivery to the Cytoplasm of <i>Escherichia coli</i> and <i>Pseudomonas aeruginosa</i>: Syntheses of Monofunctionalized Enterobactin Scaffolds and Evaluation of Enterobactin–Cargo Conjugate Uptake

The design and syntheses of monofunctionalized entero­bactin (Ent, l- and d-isomers) scaffolds where one catecholate moiety of enterobactin houses an alkene, aldehyde, or carboxylic acid at the C5 position are described. These molecules are key precursors to a family of 10 enterobactin–cargo conjugates presented in this work, which were designed to probe the extent to which the Gram-negative ferric enterobactin uptake and processing machinery recognizes, transports, and utilizes derivatized enterobactin scaffolds. A series of growth recovery assays employing enterobactin-deficient <i>E. coli</i> ATCC 33475 (<i>ent</i>-) revealed that six conjugates based on l-Ent having relatively small cargos promoted <i>E. coli</i> growth under iron-limiting conditions whereas negligible-to-no growth recovery was observed for four conjugates with relatively large cargos. No growth recovery was observed for the enterobactin receptor-deficient strain of <i>E. coli</i> H1187 (<i>fepA</i>-) or the enterobactin esterase-deficient derivative of <i>E. coli</i> K-12 JW0576 (<i>fes</i>-), or when the d-isomer of enterobactin was employed. These results demonstrate that the <i>E. coli</i> ferric enterobactin transport machinery identifies and delivers select cargo-modified scaffolds to the <i>E. coli</i> cytoplasm. <i>Pseudomonas aeruginosa</i> PAO1 K648 (<i>pvd</i>-, <i>pch</i>-) exhibited greater promiscuity than that of <i>E. coli</i> for the uptake and utilization of the enterobactin–cargo conjugates, and growth promotion was observed for eight conjugates under iron-limiting conditions. Enterobactin may be utilized for delivering molecular cargos via its transport machinery to the cytoplasm of <i>E. coli</i> and <i>P. aeruginosa</i> thereby providing a means to overcome the Gram-negative outer membrane permeability barrier

Calcium Ion Gradients Modulate the Zinc Affinity and Antibacterial Activity of Human Calprotectin

Calprotectin (CP) is an antimicrobial protein produced and released by neutrophils that inhibits the growth of pathogenic microorganisms by sequestering essential metal nutrients in the extracellular space. In this work, spectroscopic and thermodynamic metal-binding studies are presented to delineate the zinc-binding properties of CP. Unique optical absorption and EPR spectroscopic signatures for the interfacial His₃Asp and His₄ sites of human calprotectin are identified by using Co­(II) as a spectroscopic probe. Zinc competition titrations employing chromophoric Zn­(II) indicators provide a 2:1 Zn­(II):CP stoichiometry, confirm that the His₃Asp and His₄ sites of CP coordinate Zn­(II), and reveal that the Zn­(II) affinity of both sites is calcium-dependent. The calcium-insensitive Zn­(II) competitor ZP4 affords dissociation constants of <i>K</i>_d1 = 133 ± 58 pM and <i>K</i>_d2 = 185 ± 219 nM for CP in the absence of Ca­(II). These values decrease to <i>K</i>_d1 ≤ 10 pM and <i>K</i>_d2 ≤ 240 pM in the presence of excess Ca­(II). The <i>K</i>_d1 and <i>K</i>_d2 values are assigned to the His₃Asp and His₄ sites, respectively. <i>In vitro</i> antibacterial activity assays indicate that the metal-binding sites and Ca­(II)-replete conditions are required for CP to inhibit the growth of both Gram-negative and -positive bacteria. Taken together, these data provide a working model whereby calprotectin responds to physiological Ca­(II) gradients to become a potent Zn­(II) chelator in the extracellular space

Investigation of Siderophore–Platinum(IV) Conjugates Reveals Differing Antibacterial Activity and DNA Damage Depending on the Platinum Cargo

The growing threat of bacterial infections coupled with the dwindling arsenal of effective antibiotics has heightened the urgency for innovative strategies to combat bacterial pathogens, particularly Gram-negative strains, which pose a significant challenge due to their outer membrane permeability barrier. In this study, we repurpose clinically approved anticancer agents as targeted antibacterials. We report two new siderophore–platinum(IV) conjugates, both of which consist of an oxaliplatin-based Pt(IV) prodrug (oxPt(IV)) conjugated to enterobactin (Ent), a triscatecholate siderophore employed by Enterobacteriaceae for iron acquisition. We demonstrate that l/d-Ent-oxPt(IV) (l/d-EOP) are selectively delivered into the Escherichia coli cytoplasm, achieving targeted antibacterial activity, causing filamentous morphology, and leading to enhanced Pt uptake by bacterial cells but reduced Pt uptake by human cells. d-EOP exhibits enhanced potency compared to oxaliplatin and l-EOP, primarily attributed to the intrinsic antibacterial activity of its non-native siderophore moiety. To further elucidate the antibacterial activity of Ent–Pt(IV) conjugates, we probed DNA damage caused by l/d-EOP and the previously reported cisplatin-based conjugates l/d-Ent-Pt(IV) (l/d-EP). A comparative analysis of these four conjugates reveals a correlation between antibacterial activity and the ability to induce DNA damage. This work expands the scope of Pt cargos targeted to the cytoplasm of Gram-negative bacteria via Ent conjugation, provides insight into the cellular consequences of Ent–Pt(IV) conjugates in E. coli, and furthers our understanding of the potential of Pt-based therapeutics for antibacterial applications

Esterase-Catalyzed Siderophore Hydrolysis Activates an Enterobactin–Ciprofloxacin Conjugate and Confers Targeted Antibacterial Activity

Enteric Gram-negative bacteria, including <i>Escherichia coli</i>, biosynthesize and deploy the triscatecholate siderophore enterobactin (Ent) in the vertebrate host to acquire iron, an essential nutrient. We report that Ent–Cipro, a synthetic siderophore–antibiotic conjugate based on the native Ent platform that harbors an alkyl linker at one of the catechols with a ciprofloxacin cargo attached, affords targeted antibacterial activity against <i>E. coli</i> strains that express the pathogen-associated <i>iroA</i> gene cluster. Attachment of the siderophore to ciprofloxacin, a DNA gyrase inhibitor and broad-spectrum antibiotic that is used to treat infections caused by <i>E. coli</i>, generates an inactive prodrug and guides the antibiotic into the cytoplasm of bacteria that express the Ent uptake machinery (FepABCDG). Intracellular hydrolysis of the siderophore restores the activity of the antibiotic. Remarkably, Fes, the cytoplasmic Ent hydrolase expressed by all <i>E. coli</i>, does not contribute to Ent–Cipro activation. Instead, this processing step requires IroD, a cytoplasmic hydrolase that is expressed only by <i>E. coli</i> that harbor the <i>iroA</i> gene cluster and are predominantly pathogenic. In the uropathogenic <i>E. coli</i> UTI89 and CFT073, Ent–Cipro provides antibacterial activity comparable to unmodified ciprofloxacin. This work highlights the potential of leveraging and targeting pathogen-associated microbial enzymes in narrow-spectrum antibacterial approaches. Moreover, because <i>E. coli</i> include harmless gut commensals as well as resident microbes that can contribute to disease, Ent–Cipro may provide a valuable chemical tool for strain-selective modulation of the microbiota

Nickel Sequestration by the Host-Defense Protein Human Calprotectin

The human innate immune protein calprotectin (CP, S100A8/S100A9 oligomer, calgranulin A/calgranulin B oligomer, MRP-8/MRP-14 oligomer) chelates a number of first-row transition metals, including Mn­(II), Fe­(II), and Zn­(II), and can withhold these essential nutrients from microbes. Here we elucidate the Ni­(II) coordination chemistry of human CP. We present a 2.6-Å crystal structure of Ni­(II)- and Ca­(II)-bound CP, which reveals that CP binds Ni­(II) ions at both its transition-metal-binding sites: the His₃Asp motif (site 1) and the His₆ motif (site 2). Further biochemical studies establish that coordination of Ni­(II) at the hexahistidine site is thermodynamically preferred over Zn­(II). We also demonstrate that CP can sequester Ni­(II) from two human pathogens, Staphylococcus aureus and Klebsiella pneumoniae, that utilize this metal nutrient during infection, and inhibit the activity of the Ni­(II)-dependent enzyme urease in bacterial cultures. In total, our findings expand the biological coordination chemistry of Ni­(II)-chelating proteins in nature and provide a foundation for evaluating putative roles of CP in Ni­(II) homeostasis at the host–microbe interface and beyond

Contributions of the S100A9 C‑Terminal Tail to High-Affinity Mn(II) Chelation by the Host-Defense Protein Human Calprotectin

Human calprotectin (CP) is an antimicrobial protein that coordinates Mn­(II) with high affinity in a Ca­(II)-dependent manner at an unusual histidine-rich site (site 2) formed at the S100A8/S100A9 dimer interface. We present a 16-member CP mutant family where mutations in the S100A9 C-terminal tail (residues 96–114) are employed to evaluate the contributions of this region, which houses three histidines and four acidic residues, to Mn­(II) coordination at site 2. The results from analytical size-exclusion chromatography, Mn­(II) competition titrations, and electron paramagnetic resonance spectroscopy establish that the C-terminal tail is essential for high-affinity Mn­(II) coordination by CP in solution. The studies indicate that His103 and His105 (HXH motif) of the tail complete the Mn­(II) coordination sphere in solution, affording an unprecedented biological His₆ site. These solution studies are in agreement with a Mn­(II)-CP crystal structure reported recently (Damo, S. M.; et al. <i>Proc. Natl. Acad. Sci. U.S.A. </i> <b>2013</b>, <i>110</i>, 3841). Remarkably high-affinity Mn­(II) binding is retained when either H103 or H105 are mutated to Ala, when the HXH motif is shifted from positions 103–105 to 104–106, and when the human tail is substituted by the C-terminal tail of murine S100A9. Nevertheless, antibacterial activity assays employing human CP mutants reveal that the native disposition of His residues is important for conferring growth inhibition against <i>Escherichia coli</i> and <i>Staphylococcus aureus</i>. Within the S100 family, the S100A8/S100A9 heterooligomer is essential for providing high-affinity Mn­(II) binding; the S100A7, S100A9­(C3S), S100A12, and S100B homodimers do not exhibit such Mn­(II)-binding capacity

High-Affinity Manganese Coordination by Human Calprotectin Is Calcium-Dependent and Requires the Histidine-Rich Site Formed at the Dimer Interface

Calprotectin (CP) is a transition metal-chelating antimicrobial protein of the calcium-binding S100 family that is produced and released by neutrophils. It inhibits the growth of various pathogenic microorganisms by sequestering the transition metal ions manganese and zinc. In this work, we investigate the manganese-binding properties of CP. We demonstrate that the unusual His₄ motif (site 2) formed at the S100A8/S100A9 dimer interface is the site of high-affinity Mn­(II) coordination. We identify a low-temperature Mn­(II) spectroscopic signal for this site consistent with an octahedral Mn­(II) coordination sphere with simulated zero-field splitting parameters <i>D</i> = 270 MHz and <i>E</i>/<i>D</i> = 0.30 (<i>E</i> = 81 MHz). This analysis, combined with studies of mutant proteins, suggests that four histidine residues (H17 and H27 of S100A8; H91 and H95 of S100A9) coordinate Mn­(II) in addition to two as-yet unidentified ligands. The His₃Asp motif (site 1), which is also formed at the S100A8/S100A9 dimer interface, does not provide a high-affinity Mn­(II) binding site. Calcium binding to the EF-hand domains of CP increases the Mn­(II) affinity of the His₄ site from the low-micromolar to the mid-nanomolar range. Metal-ion selectivity studies demonstrate that CP prefers to coordinate Zn­(II) over Mn­(II). Nevertheless, the specificity of Mn­(II) for the His₄ site provides CP with the propensity to form mixed Zn:Mn:CP complexes where one Zn­(II) ion occupies site 1 and one Mn­(II) ion occupies site 2. These studies support the notion that CP responds to physiological calcium ion gradients to become a high-affinity transition metal ion chelator in the extracellular space where it inhibits microbial growth

Biophysical Examination of the Calcium-Modulated Nickel-Binding Properties of Human Calprotectin Reveals Conformational Change in the EF-Hand Domains and His₃Asp Site

Calprotectin (CP, S100A8/S100A9 oligomer, MRP-8/MRP-14 oligomer) is a host-defense protein that sequesters nutrient transition metals from microbes. Each S100A8/S100A9 heterodimer contains four EF-hand domains and two transition-metal-binding sites. We investigate the effect of Ca­(II) ions on the structure and Ni­(II)-binding properties of human CP. By employing energy dispersive X-ray (EDX) spectroscopy, we evaluate the metal content of Ni­(II)-bound CP-Ser [oligomer of S100A8­(C42S) and S100A9­(C3S)] crystals obtained in the absence and presence of Ca­(II). We present a 2.1 Å resolution crystal structure of Ni­(II)-bound CP-Ser and compare this structure to a reported Ni­(II)- and Ca­(II)-bound CP-Ser structure [Nakashige, T. G., et al. (2017) <i>J. Am. Chem. Soc.</i> <i>139</i>, 8828–8836]. This analysis reveals conformational changes associated with coordination of Ca­(II) to the EF-hands of S100A9 and that Ca­(II) binding affects the coordination number and geometry of the Ni­(II) ion bound to the His₃Asp site. In contrast, negligible differences are observed for the Ni­(II)-His₆ site in the absence and presence of Ca­(II). Biochemical studies show that, whereas the His₆ site has a thermodynamic preference for Ni­(II) over Zn­(II), the His₃Asp site selects for Zn­(II) over Ni­(II), and relatively rapid metal exchange occurs at this site. These observations inform the working model for how CP withholds nutrient metals in the extracellular space

Biophysical Examination of the Calcium-Modulated Nickel-Binding Properties of Human Calprotectin Reveals Conformational Change in the EF-Hand Domains and His₃Asp Site

Calprotectin (CP, S100A8/S100A9 oligomer, MRP-8/MRP-14 oligomer) is a host-defense protein that sequesters nutrient transition metals from microbes. Each S100A8/S100A9 heterodimer contains four EF-hand domains and two transition-metal-binding sites. We investigate the effect of Ca­(II) ions on the structure and Ni­(II)-binding properties of human CP. By employing energy dispersive X-ray (EDX) spectroscopy, we evaluate the metal content of Ni­(II)-bound CP-Ser [oligomer of S100A8­(C42S) and S100A9­(C3S)] crystals obtained in the absence and presence of Ca­(II). We present a 2.1 Å resolution crystal structure of Ni­(II)-bound CP-Ser and compare this structure to a reported Ni­(II)- and Ca­(II)-bound CP-Ser structure [Nakashige, T. G., et al. (2017) <i>J. Am. Chem. Soc.</i> <i>139</i>, 8828–8836]. This analysis reveals conformational changes associated with coordination of Ca­(II) to the EF-hands of S100A9 and that Ca­(II) binding affects the coordination number and geometry of the Ni­(II) ion bound to the His₃Asp site. In contrast, negligible differences are observed for the Ni­(II)-His₆ site in the absence and presence of Ca­(II). Biochemical studies show that, whereas the His₆ site has a thermodynamic preference for Ni­(II) over Zn­(II), the His₃Asp site selects for Zn­(II) over Ni­(II), and relatively rapid metal exchange occurs at this site. These observations inform the working model for how CP withholds nutrient metals in the extracellular space