Browsing by Author "Spirig, Thomas"

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Staphylococcus aureus Uses a Novel Multidomain Receptor to Break Apart Human Hemoglobin and Steal Its Heme
(2012-11) Spirig, Thomas; Malmirchegini, G. Reza; Zhang, Jiang; Robson, Scott A.; Sjodt, Megan; Liu, Mengyao; Krishna Kumar, Kaavya; Dickson, Claire; Gell, David A.; Lei, Benfang; Loo, Joseph A.; Clubb, Robert T.
Staphylococcus aureus is a leading cause of life-threatening infections in the United States. It requires iron to grow, which must be actively procured from its host to successfully mount an infection. Heme-iron within hemoglobin (Hb) is the most abundant source of iron in the human body and is captured by S. aureus using two closely related receptors, IsdH and IsdB. Here we demonstrate that each receptor captures heme using two conserved near iron transporter (NEAT) domains that function synergistically. NMR studies of the 39-kDa conserved unit from IsdH (IsdHN2N3, Ala326–Asp660) reveals that it adopts an elongated dumbbell-shaped structure in which its NEAT domains are properly positioned by a helical linker domain, whose three-dimensional structure is determined here in detail. Electrospray ionization mass spectrometry and heme transfer measurements indicate that IsdHN2N3 extracts heme from Hb via an ordered process in which the receptor promotes heme release by inducing steric strain that dissociates the Hb tetramer. Other clinically significant Gram-positive pathogens capture Hb using receptors that contain multiple NEAT domains, suggesting that they use a conserved mechanism.
Transient Weak Protein–Protein Complexes Transfer Heme Across the Cell Wall of Staphylococcus aureus
(2011-09) Villareal, Valerie A.; Spirig, Thomas; Robson, Scott A.; Liu, Mengyao; Lei, Benfang; Clubb, Robert T.
Iron is an essential nutrient for the bacterial pathogen Staphylococcus aureus. Heme in hemoglobin (Hb) is the most abundant source of iron in the human body and during infections is captured by S. aureus using iron-regulated surface determinant (Isd) proteins. A central step in this process is the transfer of heme between the cell wall associated IsdA and IsdC hemoproteins. Biochemical evidence indicates that heme is transferred via an activated IsdA:heme:IsdC heme complex. Transfer is rapid and occurs up to 70 000 times faster than indirect mechanisms in which heme is released into the solvent. To gain insight into the mechanism of transfer, we modeled the structure of the complex using NMR paramagnetic relaxation enhancement (PRE) methods. Our results indicate that IsdA and IsdC transfer heme via an ultraweak affinity “handclasp” complex that juxtaposes their respective 310 helices and β7/β8 loops. Interestingly, PRE also identified a set of transient complexes that could represent high-energy pre-equilibrium encounter species that form prior to the stereospecific handclasp complex. Targeted amino acid mutagenesis and stopped-flow measurements substantiate the functional relevance of a PRE-derived model, as mutation of interfacial side chains significantly slows the rate of transfer. IsdA and IsdC bind heme using NEAr Transporter (NEAT) domains that are conserved in many species of pathogenic Gram-positive bacteria. Heme transfer in these microbes may also occur through structurally similar transient stereospecific complexes.