Abstract
Pathogens such as Staphylococcus aureus require iron to survive and have evolved specialized proteins to steal heme from their host. IsdC is the central conduit of the Isd (iron-regulated surface determinant) multicomponent heme uptake machinery; staphylococcal cell-surface proteins such as IsdA, IsdB, and IsdH are thought to funnel their molecular cargo to IsdC, which then mediates the transfer of the iron-containing nutrient to the membrane translocation system IsdDEF. The structure of the heme-IsdC complex reveals a novel heme site within an immunoglobulin-like domain and sheds light on its binding mechanism. The folding topology is reminiscent of the architecture of cytochrome f, cellobiose dehydrogenase, and ethylbenzene dehydrogenase; in these three proteins, the heme is bound in an equivalent position, but interestingly, IsdC features a distinct binding pocket with the ligand located next to the hydrophobic core of the beta-sandwich. The iron is coordinated with a tyrosine surrounded by several non-polar side chains that cluster into a tightly packed proximal side. On the other hand, the distal side is relatively exposed with a short helical peptide segment that acts as a lip clasping onto almost half of the porphyrin plane. This structural feature is argued to play a role in the mechanism of binding and release by switching to an open conformation and thus loosening the interactions holding the heme. The structure of the heme-IsdC complex provides a template for the understanding of other proteins, such as IsdA, IsdB, and IsdH, that contain the same heme-binding module as IsdC, known as the NEAT (near transporter) domain.
Highlights
Iron is essential to living organisms for it is used, for instance, by cytochromes in cellular energy transduction processes
In addition to data from various biochemical experiments, HasA, a heme-siderophore secreted by the pathogen Serratia marcescens, was the first bacterial heme carrier protein to have had its structure elucidated in complex with heme [2]
Since IsdC is the central conduit of the Isd heme uptake system, we have studied its x-ray crystal structure in complex with heme
Summary
Cloning and Overexpression—The gene encoding IsdC (National Center for Biotechnology Information number gi:13700931) was amplified from S. aureus genomic DNA (strain RN639OB) by PCR using the forward and reverse primers: IsdC, forward, 5Ј-gcgactttggatccgatagcggtactttgaattatg-3Ј, and IsdC reverse, 5Ј-cgcattttaagcttattctactttgtctttattcgcac-3Ј. The His-GST tag was removed by proteolytic cleavage with thrombin (10 units/mg of protein, Cambridge Biosciences) at room temperature overnight. Given the long molecular shape of IsdC and that changing conditions such as concentration of protein and salts made no difference to any peaks in terms of a possible monomer-dimer equilibrium, we believe that IsdC is not likely to be a dimer, exactly as observed in the case of gel filtration work on IsdA [21]. Crystallization—Crystals were obtained using the sitting drop vapor diffusion method with samples of IsdC at 95 mg/ml in 50 mM Tris-HCl, pH 8, 500 mM NaCl mixed in equal volumes (2 ϩ 2 l) with reservoir solution containing 26% polyethylene glycol 550 mono-methyl-ether, 18 mM zinc sulfate, and 0.1 M MES, pH 6.0.
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