Abstract
Staphylococcus aureus scavenges heme-iron from host hemoproteins using iron-regulated surface determinant (Isd) proteins. IsdC is the central conduit through which heme is passed across the cell wall and binds this molecule using a NEAr Transporter (NEAT) domain. NMR spectroscopy was used to determine the structure of IsdC in complex with a heme analog, zinc-substituted protoporphyrin IX (ZnPPIX). The backbone coordinates of the ensemble of conformers representing the structure exhibit a root mean square deviation to the mean structure of 0.53 +/- 0.11 angstroms. IsdC partially buries protoporphyrin within a large hydrophobic pocket that is located at the end of its beta-barrel structure. The central metal ion of the analog adopts a pentacoordinate geometry in which a highly conserved tyrosine residue serves as a proximal ligand. Consistent with the structure and its role in heme transfer across the cell wall, we show that IsdC weakly binds heme (K(D) = 0.34 +/- 0.12 microm) and that ZnPPIX rapidly dissociates from the protein at a rate of 126 +/- 30 s(-1). NMR studies of the apo-form of IsdC reveal that a 3(10) helix within the binding pocket undergoes a flexible to rigid transition as heme is captured. This structural plasticity may increase the efficiency of heme transfer across the cell wall by facilitating protein-protein interactions between apoIsdC and upstream hemoproteins.
Highlights
Iron), which contains ϳ80% of the total iron in the body (6)
We show that IsdCN recognizes zinc-substituted protoporphyrin IX (ZnPPIX) through a large cleft located at the end of its -barrel structure in which the zinc ion is bound in a pentacoordinate manner by a highly conserved tyrosine residue
IsdCN Binds Heme and Zinc-protoporphyrin IX—The IsdC protein is the central conduit through which heme is passed across the cell wall from surface-exposed heme receptors to the underlying transmembrane IsdDEF complex
Summary
Protein Purification and NMR Sample Preparation—The gene sequence encoding the IsdC NEAT (IsdCN) domain was cloned into pET15b (Novagen) using standard methods. The standard CANDID protocol of seven cycles of peak identification and assignment, followed by structure calculations, was executed This process assigned the vast majority of cross-peaks in the NOESY data and produced a set of conformers that converged to the structure of the IsdCN protein in the complex. As the relative moments vary significantly from a perfect sphere, the statistical significance of fitting the relaxation data to either axially symmetric or isotropic models of tumbling were explored using the R2R1 Diffusion program (54) These calculations were performed using a trimmed data set that included only those residues with R2/R1 ratios within 1 S.D. from the average R2/R1 ratio, and residues that had NOE ratios Ͼ0.65 (56). The reported error for the KD values is the standard deviation from three independent measurements
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