Heme Coordination by Staphylococcus aureus IsdE

Jason C Grigg,Christie L Vermeiren,David E Heinrichs,Michael E.P Murphy

doi:10.1074/jbc.m704602200

Jason C Grigg, Christie L Vermeiren + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m704602200

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Abstract

Staphylococcus aureus is a Gram-positive bacterial pathogen and a leading cause of hospital acquired infections. Because the free iron concentration in the human body is too low to support growth, S. aureus must acquire iron from host sources. Heme iron is the most prevalent iron reservoir in the human body and a predominant source of iron for S. aureus. The iron-regulated surface determinant (Isd) system removes heme from host heme proteins and transfers it to IsdE, the cognate substrate-binding lipoprotein of an ATP-binding cassette transporter, for import and subsequent degradation. Herein, we report the crystal structure of the soluble portion of the IsdE lipoprotein in complex with heme. The structure reveals a bi-lobed topology formed by an N- and C-terminal domain bridged by a single alpha-helix. The structure places IsdE as a member of the helical backbone metal receptor superfamily. A six-coordinate heme molecule is bound in the groove established at the domain interface, and the heme iron is coordinated in a novel fashion for heme transporters by Met(78) and His(229). Both heme propionate groups are secured by H-bonds to IsdE main chain and side chain groups. Of these residues, His(229) is essential for IsdE-mediated heme uptake by S. aureus when growth on heme as a sole iron source is measured. Multiple sequence alignments of homologues from several other Gram-positive bacteria, including the human pathogens pyogenes, Bacillus anthracis, and Listeria monocytogenes, suggest that these other systems function equivalently to S. aureus IsdE with respect to heme binding and transport.

Highlights

Corroborating the structure, alanine substitutions in binding pocket residues showed that mutation of Met78 and His229 resulted in significant loss of heme binding and that IsdE H229A was incapable of supporting IsdE-mediated growth on heme as a sole source of iron in growth promotion assays
We demonstrate that S. aureus growth on heme as a sole iron source is impaired by inactivation of isdE
IsdE-mediated heme transport in S. aureus is reliant on the key heme iron axial ligand, His229 (Fig. 4)

Summary

The abbreviations used are

Iron-regulated surface determinant; Hts, heme transport system; NEAT, near transporter; ABC, ATP-binding cassette; MES, 4-morpholineethanesulfonic acid. We show that inactivation of S. aureus isdE impairs growth on heme as a sole source of iron. To gain insight into the function of IsdE in heme binding and transport, the crystal structure of the IsdE-heme complex was determined. The structure reveals His-Met heme iron coordination that is unique to heme transport proteins. Corroborating the structure, alanine substitutions in binding pocket residues showed that mutation of Met and His229 resulted in significant loss of heme binding and that IsdE H229A was incapable of supporting IsdE-mediated growth on heme as a sole source of iron in growth promotion assays. This work adds substantial mechanistic detail to the complex model of heme uptake by S. aureus and provides a framework for future studies into the mechanism of bacterial binding protein-dependent heme acquisition

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION