Structural Basis for Multimeric Heme Complexation through a Specific Protein-Heme Interaction

Abstract

To elucidate the heme acquisition system in pathogenic bacteria, we investigated the heme-binding properties of the third NEAT domain of IsdH (IsdH-NEAT3), a receptor for heme located on the surfaces of pathogenic bacterial cells, by using x-ray crystallography, isothermal titration calorimetry, examination of absorbance spectra, mutation analysis, size-exclusion chromatography, and analytical ultracentrifugation. We found the following: 1) IsdH-NEAT3 can bind with multiple heme molecules by two modes; 2) heme was bound at the surface of IsdH-NEAT3; 3) candidate residues proposed from the crystal structure were not essential for binding with heme; and 4) IsdH-NEAT3 was associated into a multimeric heme complex by the addition of excess heme. From these observations, we propose a heme-binding mechanism for IsdH-NEAT3 that involves multimerization and discuss the biological importance of this mechanism.

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

“…All k d values were calculated as described previously (30 Tyr side chain in stabilization of the proximal iron ligand (first Tyr). A similar observation has been made for the first NEAT domain of IsdB and the first two NEAT domains of IsdH, both of which also lack the second tyrosine and instead act as hemoprotein receptors (8,12,17,40,42,50,64). However, unlike these NEAT domains, NEAT domain 2 of IsdX2 binds Hb poorly, and its rather rapid rate of Hb dissociation would seemingly preclude it from serving as a "receptor" for host Hb (Fig.…”

Iron-Regulated Surface Determinant (Isd) Proteins of Staphylococcus lugdunensis

“…IsdA (PDB ID 2ITE) was used to model the IsdJ NEAT1 and NEAT2 domains and the IsdK NEAT domain (27). S. aureus IsdH NEAT3 (PDB ID 2E7D) and IsdB NEAT2 (PDB ID: 3RTL) were used to model S. lugdunensis IsdB NEAT2 (24,72). The NEAT domain of IsdC was modeled on its orthologue from S. aureus (PDB ID 2O6P) (59,69).…”

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

“…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.…”