Christie Vermeiren

Haem recognition by a Staphylococcus aureus NEAT domain

Successful pathogenic organisms have developed mechanisms to thrive under extreme levels of iron restriction. Haem‐iron represents the largest iron reservoir in the human body and is a significant source of iron for some bacterial pathogens. NEAT (NEAr Transporter) domains are found exclusively in a family of cell surface proteins in Gram‐positive bacteria. Many NEAT domain‐containing proteins, including IsdA in Staphylococcus aureus, are implicated in haem binding. Here, we show that overexpression of IsdA in S. aureus enhances growth and an inactivation mutant of IsdA has a growth defect, compared with wild type, when grown in media containing haem as the sole iron source. Furthermore, the haem‐binding property of IsdA is contained within the NEAT domain. Crystal structures of the apo‐IsdA NEAT domain and in complex with haem were solved and reveal a clathrin adapter‐like β‐sandwich fold with a large hydrophobic haem‐binding pocket. Haem is bound with the propionate groups directed at the molecular surface and the iron is co‐ordinated solely by Tyr166. The phenol groups of Tyr166 and Tyr170 form an H‐bond that may function in regulating haem binding and release. An analysis of IsdA structure‐sequence alignments indicate that conservation of Tyr166 is a predictor of haem binding by NEAT domains.

Heme Coordination by Staphylococcus aureus IsdE.

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 α-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 Met78 and His229. Both heme propionate groups are secured by H-bonds to IsdE main chain and side chain groups. Of these residues, His229 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.

Protoporphyrin IX and heme binding properties of Staphylococcus aureus IsdC

Staphylococcus aureus is a human pathogen that results in numerous infections in hospital settings and recently also in the wider community. Its antibiotic resistant forms are causing considerable alarm. A series of surface-anchored proteins that have heme uptake and transport properties have been reported. Through the use of absorption and magnetic circular dichroism spectroscopies and mass spectrometry, the iron-free, protoporphyrin IX and the iron-containing, heme-binding characteristics of bacterial rIsdC have been obtained. Mass spectrometry showed that following isolation and purification, the rIsdC is bound predominantly to protoporphyrin IX and to a lesser extent heme, unlike the case of rIsdA, which binds predominantly heme. Magnetic circular dichroism analysis provided further information regarding porphyrin binding because the characteristic magnetic circular dichroism band envelopes for the iron-free protoporphyrin IX and the iron-containing heme can be clearly distinguished in the spectrum of the rIsdC. Analysis of these spectral data showed that the minor heme component exists as a high-intermediate spin state ferric heme when bound to rIsdC, similar to the high-spin ferric heme reported for the rIsdA protein.