The Five Near-iron Transporter (NEAT) Domain Anthrax Hemophore, IsdX2, Scavenges Heme from Hemoglobin and Transfers Heme to the Surface Protein IsdC

Honsa, Erin S.; Fabián, Marián; Cárdenas, Ana Marı́a; Olson, John S.; Maresso, Anthony W.

doi:10.1074/jbc.m111.241687

Cited by 44 publications

(69 citation statements)

References 62 publications

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“…Thrombin was removed from protein preparations using aminobenzamidine resin (Sigma). WT and mutant NEAT domains precipitated upon treatment with methyl ethyl ketone, a common procedure used to make apo forms of hemoproteins (13,26). The use of iron-depleted medium, which is a minimal medium, reduces the amount of endogenous heme from E. coli that co-purifies with the NEAT domains, providing essentially "apo" NEAT preparations (molar heme concentration calculations showed Ͻ1 M heme bound; data not shown).…”

Section: Methodsmentioning

confidence: 99%

“…2, C and D. The concentration of recombinant NEAT was determined using the Bradford assay (Bio-Rad) (27) or by using the extinction coefficients reported previously (13). Heme concentrations were measured as described previously (13).…”

Section: Methodsmentioning

confidence: 99%

“…Next, each GSTtagged NEAT n was eluted with 4 ml of 25 mM reduced glutathione, and relative heme content was determined by recording the intensity of the Soret band compared with an equivalent reaction of GST-NEAT n incubated with buffer only. SDS-PAGE was performed on each sample to verify that any differences in the Soret absorbance were not due to differences in NEAT protein amounts or carryover of metHb into NEAT elutions (data not shown) (13).…”

Section: Methodsmentioning

confidence: 99%

“…The creation of pGEX2TKgst-isdX2NEAT3 and -isdX2NEAT5 expression plasmids was described previously (13). For the creation of the NEAT point mutants, pGEX2TK plasmids containing wild type (WT) isdX2NEAT3 and isdX2NEAT5 inserts were subject to pfu-turbo (Agilent) PCR with primers containing specific nucleotide mismatches to produce the desired amino acid mutations: NEAT3 T29Q forward, 5Ј-CGA AAT TTC AAT GAT CAA CCA ATA TAC AAA AAG TCC AGG-3Ј; NEAT3 T29Q reverse, 5Ј-CCT GGA CTT TTT GTA TAT TGG TTC ATC ATT GAA ATT TCG-3Ј; NEAT3 T29N forward, 5Ј-CGA AAT TTC AAT GAT CAA CAA CTA TAC AAA AAG TCC AGG-3Ј; NEAT3 T29N reverse, 5Ј-CCT GGA CTT TTT GTA TAG TTG TTC ATC ATT GAA ATT TCG-3Ј; NEAT5 Q29T forward, 5Ј-GAA ATT TCA ATC ATG AAT ACA TAT GTT GTA AGT CCA GCA AGG-3Ј; NEAT5 Q29T reverse, 5Ј-CCT TGC TGG ACT TAC AAC ATA TGT ATT CAT CAT TGA AAT TTC-3Ј; NEAT5 Q29N forward, 5Ј-GAA ATT TCA ATC ATG AAT AAC TAT GTT GTA AGT CCA GCA AGG-3Ј; NEAT5 Q29N reverse, 5Ј-CCT TGC TGG ACT TAC AAC ATA GTT ATT CAT CAT TGA AAT TTC-3Ј.…”

Section: Methodsmentioning

confidence: 99%

“…Specifically, a conserved protein domain termed a near-iron transporter (NEAT) 2 is involved in heme uptake in pathogens, such as Bacillus anthracis, Bacillus cereus, Staphylococcus aureus, and Streptococcus pyogenes (7)(8)(9)(10)(11)(12)(13)(14)(15). NEAT domains fold into a ␤-sandwich and bind the heme iron atom through a conserved tyrosine located on the eighth ␤-strand (16 -18).…”

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confidence: 99%

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The Near-iron Transporter (NEAT) Domains of the Anthrax Hemophore IsdX2 Require a Critical Glutamine to Extract Heme from Methemoglobin

Honsa

Owens

Goulding

et al. 2013

Journal of Biological Chemistry

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

The Near-iron Transporter (NEAT) Domains of the Anthrax Hemophore IsdX2 Require a Critical Glutamine to Extract Heme from Methemoglobin

Honsa

Owens

Goulding

et al. 2013

Journal of Biological Chemistry

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Selective binding of antimicrobial porphyrins to the heme‐receptor IsdH‐NEAT3 of Staphylococcus aureus

Moriwaki

Caaveiro

et al. 2013

Protein Science

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The Isd (iron-regulated surface determinant) system of the human pathogen Staphylococcus aureus is responsible for the acquisition of heme from the host organism. We recently reported that the extracellular heme receptor IsdH-NEAT3 captures and transfers noniron antimicrobial porphyrins containing metals in oxidation state (III). However, it is unclear if geometric factors such as the size of the metal (ionic radius) affect binding and transfer of metalloporphyrins. We carried out an ample structural, functional, and thermodynamic analysis of the binding properties of antimicrobial indium(III)-porphyrin, which bears a much larger metal ion than the iron(III) of the natural ligand heme. The results demonstrate that the NEAT3 receptor recognizes the In(III)-containing PPIX in a manner very similar to that of heme. Site-directed mutagenesis identifies Tyr642 as the central element in the recognition mechanism as suggested from the crystal structures. Importantly, the NEAT3 receptor possesses the remarkable ability to capture dimers of metalloporphyrin. Molecular dynamics simulations reveal that IsdH-NEAT3 does not require conformational changes, or large rearrangements of the residues within its binding site, to accommodate the much larger (heme) 2 ligand. We discuss the implications of these findings for the design of potent inhibitors against this family of key receptors of S. aureus.

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NMR experiments redefine the hemoglobin binding properties of bacterial NEAr‐iron Transporter domains

et al. 2019

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Iron is a versatile metal cofactor that is used in a wide range of essential cellular processes. During infections, many bacterial pathogens acquire iron from human hemoglobin (Hb), which contains the majority of the body's total iron content in the form of heme (iron protoporphyrin IX). Clinically important Gram-positive bacterial pathogens scavenge heme using an array of secreted and cell-wall-associated receptors that contain NEAr-iron Transporter (NEAT) domains. Experimentally defining the Hb binding properties of NEAT domains has been challenging, limiting our understanding of their function in heme uptake. Here we show that solution-state NMR spectroscopy is a powerful tool to define the Hb binding properties of NEAT domains. The utility of this method is demonstrated using the NEAT domains from Bacillus anthracis and Listeria monocytogenes. Our results are compatible with the existence of at least two types of NEAT domains that are capable of interacting with either Hb or heme. These binding properties can be predicted from their primary sequences, with Hb-and heme-binding NEAT domains being distinguished by the presence of (F/Y)YH(Y/F) and S/YXXXY motifs, respectively. The results of this work should enable the functions of a wide range of NEAT domain containing proteins in pathogenic bacteria to be reliably predicted.Additional Supporting Information may be found in the online version of this article.Significance statement: During infections, many bacterial pathogens use NEAr-iron Transporter (NEAT) domains to acquire iron, an essential nutrient that is required for microbial growth. Here we show that NMR is a powerful tool to assess the hemoglobin binding properties of these domains. Our results resolve prior discrepancies in the literature and help to define the functions of a wide range of NEAT domains based on their amino acid primary sequences.

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The Five Near-iron Transporter (NEAT) Domain Anthrax Hemophore, IsdX2, Scavenges Heme from Hemoglobin and Transfers Heme to the Surface Protein IsdC

Cited by 44 publications

References 62 publications

The Near-iron Transporter (NEAT) Domains of the Anthrax Hemophore IsdX2 Require a Critical Glutamine to Extract Heme from Methemoglobin

The Near-iron Transporter (NEAT) Domains of the Anthrax Hemophore IsdX2 Require a Critical Glutamine to Extract Heme from Methemoglobin

Selective binding of antimicrobial porphyrins to the heme‐receptor IsdH‐NEAT3 of Staphylococcus aureus

NMR experiments redefine the hemoglobin binding properties of bacterial NEAr‐iron Transporter domains

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