Abstract
Staphylococcus aureus is a leading cause of life-threatening infections in the United States. It actively acquires the essential nutrient iron from human hemoglobin (Hb) using the iron-regulated surface-determinant (Isd) system. This process is initiated when the closely related bacterial IsdB and IsdH receptors bind to Hb and extract its hemin through a conserved tri-domain unit that contains two NEAr iron Transporter (NEAT) domains that are connected by a helical linker domain. Previously, we demonstrated that the tri-domain unit within IsdH (IsdHN2N3) triggers hemin release by distorting Hb's F-helix. Here, we report that IsdHN2N3 promotes hemin release from both the α- and β-subunits. Using a receptor mutant that only binds to the α-subunit of Hb and a stopped-flow transfer assay, we determined the energetics and micro-rate constants of hemin extraction from tetrameric Hb. We found that at 37 °C, the receptor accelerates hemin release from Hb up to 13,400-fold, with an activation enthalpy of 19.5 ± 1.1 kcal/mol. We propose that hemin removal requires the rate-limiting hydrolytic cleavage of the axial HisF8 Nϵ-Fe3+ bond, which, based on molecular dynamics simulations, may be facilitated by receptor-induced bond hydration. Isothermal titration calorimetry experiments revealed that two distinct IsdHN2N3·Hb protein·protein interfaces promote hemin release. A high-affinity receptor·Hb(A-helix) interface contributed ∼95% of the total binding standard free energy, enabling much weaker receptor interactions with Hb's F-helix that distort its hemin pocket and cause unfavorable changes in the binding enthalpy. We present a model indicating that receptor-introduced structural distortions and increased solvation underlie the IsdH-mediated hemin extraction mechanism.
Highlights
Staphylococcus aureus is a leading cause of life-threatening infections in the United States
We developed an assay to quantitatively measure the kinetics of IsdH-mediated hemin removal from the ␣-globin chain within tetrameric Hb
The receptors extract Hb’s hemin using a conserved tridomain unit that is formed by two NEAr iron Transporter (NEAT) domains that are separated by a helix linker domain (Fig. 1a) [15]
Summary
Harvard Medical School Dept. of Biological Chemistry and Molecular Pharmacology Boston, MA 02115. An understanding of the molecular mechanism of heme scavenging will provide insight into how this pathogen survives and persists within its human host, and it could lead to new anti-infective agents that work by limiting microbial access to iron. S. aureus uses the iron-regulated surface determinant (Isd) system to extract the oxidized form of heme from Hb (hereafter called hemin). The Isd system is composed of nine proteins that form a hemin relay system that captures Hb and rapidly extracts its hemin, transfers hemin across the cell wall, and pumps the hemin into the cytoplasm where it is degraded to release free iron (8 –10). Four Isd proteins (IsdA, IsdB, IsdC, and IsdH/ HarA) are covalently linked to the bacterial cell wall via sortase transpeptidases [11, 12]. The Isd system is important for S. aureus virulence, as strains lacking genes that encode for components of the system display attenuated infec-
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