Differential Function of Lip Residues in the Mechanism and Biology of an Anthrax Hemophore

Abstract

To replicate in mammalian hosts, bacterial pathogens must acquire iron. The majority of iron is coordinated to the protoporphyrin ring of heme, which is further bound to hemoglobin. Pathogenic bacteria utilize secreted hemophores to acquire heme from heme sources such as hemoglobin. Bacillus anthracis, the causative agent of anthrax disease, secretes two hemophores, IsdX1 and IsdX2, to acquire heme from host hemoglobin and enhance bacterial replication in iron-starved environments. Both proteins contain NEAr-iron Transporter (NEAT) domains, a conserved protein module that functions in heme acquisition in Gram-positive pathogens. Here, we report the structure of IsdX1, the first of a Gram-positive hemophore, with and without bound heme. Overall, IsdX1 forms an immunoglobin-like fold that contains, similar to other NEAT proteins, a 310-helix near the heme-binding site. Because the mechanistic function of this helix in NEAT proteins is not yet defined, we focused on the contribution of this region to hemophore and NEAT protein activity, both biochemically and biologically in cultured cells. Site-directed mutagenesis of amino acids in and adjacent to the helix identified residues important for heme and hemoglobin association, with some mutations affecting both properties and other mutations affecting only heme stabilization. IsdX1 with mutations that reduced the ability to associate with hemoglobin and bind heme failed to restore the growth of a hemophore-deficient strain of B. anthracis on hemoglobin as the sole iron source. These data indicate that not only is the 310-helix important for NEAT protein biology, but also that the processes of hemoglobin and heme binding can be both separate as well as coupled, the latter function being necessary for maximal heme-scavenging activity. These studies enhance our understanding of NEAT domain and hemophore function and set the stage for structure-based inhibitor design to block NEAT domain interaction with upstream ligands.

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

“…The eight ␤-strands of IsdX2 NEAT5 are arranged in two antiparallel ␤-sheets, which form a ␤-sandwich (Fig. 6A), consistent with the immunoglobulin-like fold, as observed in multiple NEAT domain crystal structures from B. anthracis and S. aureus (8,12,16,17,35,45,46).…”

“…The complete crystallographic statistics can be found in Table 1. The phases were calculated using AutoMR (31) utilizing holo-IsdX1 (Protein Data Bank code 3SIK) (12) as the search model for IsdX2 NEAT5 WT and IsdX2 NEAT5 WT as the search model for IsdX2 NEAT5 Q29T. The resulting electron density map was subjected to automated and manual model building procedures through a …”

“…To confirm that the mutations did not affect heme binding, recombinant NEAT domains expressed overnight were assayed for their ability to co-purify with endogenous heme during expression in E. coli. Each NEAT domain was purified as stated under "Experimental Procedures," and bound heme was detected by measuring the Soret absorbance at ϳ400 nm, a well documented spectroscopic assay used to detect the presence of bound heme iron (7,12,20,24,(35)(36)(37)(38)(39)(40). As indicated in Fig.…”

“…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).…”

“…NEAT domains fold into a ␤-sandwich and bind the heme iron atom through a conserved tyrosine located on the eighth ␤-strand (16 -18). The pocket is bordered by a 3 10 -helix that is thought to be critical for NEAT function, including heme and/or hemoprotein binding (9,12,19,20). NEAT-NEAT heme transfer can be facilitated by the conserved iron-coordinating tyrosine (21)(22)(23)(24).…”