Investigating Hemoglobin Capture and Heme Acquisition by the Pathogen Staphylococcus aureus

Staphylococcus aureus is a medically important Gram-positive bacterial pathogen that actively procures heme from human hemoglobin (Hb) using the iron-regulated surface determinant (Isd) system. Research described in this dissertation investigated how the Isd system uses the IsdH receptor protein to capture Hb and extract its hemin (the oxidized form of heme). To rapidly extract Hb’s hemin, IsdH employs a conserved tri-domain unit that contains two NEAr iron Transporter (NEAT) domains that are connected by a helical linker domain. The work described in chapter 2 used UV-Vis spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, and electrospray ionization mass spectrometry (ESI-MS) methods to define the importance of the conserved linker domain in hemin extraction and revealed that this domain enables the NEAT domains within the tri-domain unit to work together to synergistically extract hemin. Chapter 3 of this thesis describes the structure and dynamics of the tri-domain unit (IsdHN2N3). The solution structure of the apo-receptor was defined using small angle X-ray scattering, and advanced NMR methods such as paramagnetic relaxation enhancement (PRE), residual dipolar coupling (RDC), and selective methyl labeling approaches. The structure and inter-domain dynamics of IsdHN2N3 in the absence of Hb were further defined using ensemble modeling calculations. The results of these studies illustrated that the receptor adaptively recognizes Hb using a combination of conformational selection and induced fit mechanisms, and suggests that the linker domain may facilitate hemin transfer by destabilizing the iron-coordinating F-helix in Hb. Chapter 4 describes studies that investigated the kinetic and thermodynamic basis of hemin transfer using stopped-flow UV-Vis spectroscopy, analytical ultracentrifugation sedimentation equilibrium, and isothermal titration methods. The results of this work provide insight into the kinetic and thermodynamic determinants that facilitate receptor-mediated hemin release from Hb. Lastly, Chapter 5 describes the methods that were used to site-specifically label proteins with nitroxide spin-label probes and the subsequent derivation of paramagnetic NMR distance restraints. Altogether, the results of the work described in this dissertation have advanced our knowledge of Hb recognition and hemin acquisition by the pathogen S. aureus

The PRE-Derived NMR Model of the 38.8-kDa Tri-Domain IsdH Protein from Staphylococcus aureus Suggests That It Adaptively Recognizes Human Hemoglobin

Staphylococcus aureus is a medically important bacterial pathogen that, during infections, acquires iron from human hemoglobin (Hb). It uses two closely related iron-regulated surface determinant (Isd) proteins to capture and extract the oxidized form of heme (hemin) from Hb, IsdH and IsdB. Both receptors rapidly extract hemin using a conserved tri-domain unit consisting of two NEAT (near iron transporter) domains connected by a helical linker domain. To gain insight into the mechanism of extraction, we used NMR to investigate the structure and dynamics of the 38.8-kDa tri-domain IsdH protein (IsdHN2N3, A326–D660 with a Y642A mutation that prevents hemin binding). The structure was modeled using long-range paramagnetic relaxation enhancement (PRE) distance restraints, dihedral angle, small-angle X-ray scattering, residual dipolar coupling and inter-domain NOE nuclear Overhauser effect data. The receptor adopts an extended conformation wherein the linker and N3 domains pack against each other via a hydrophobic interface. In contrast, the N2 domain contacts the linker domain via a hydrophilic interface and, based on NMR relaxation data, undergoes inter-domain motions enabling it to reorient with respect to the body of the protein. Ensemble calculations were used to estimate the range of N2 domain positions compatible with the PRE data. A comparison of the Hb-free and Hb-bound forms reveals that Hb binding alters the positioning of the N2 domain. We propose that binding occurs through a combination of conformational selection and induced-fit mechanisms that may promote hemin release from Hb by altering the position of its F helix

A switch in surface polymer biogenesis triggers growth-phase-dependent and antibiotic-induced bacteriolysis

Penicillin and related antibiotics disrupt cell wall synthesis to induce bacteriolysis. Lysis in response to these drugs requires the activity of cell wall hydrolases called autolysins, but how penicillins misactivate these deadly enzymes has long remained unclear. Here, we show that alterations in surface polymers called teichoic acids (TAs) play a key role in penicillin-induced lysis of the Gram-positive pathogen Streptococcus pneumoniae (Sp). We find that during exponential growth, Sp cells primarily produce lipid-anchored TAs called lipoteichoic acids (LTAs) that bind and sequester the major autolysin LytA. However, penicillin-treatment or prolonged stationary phase growth triggers the degradation of a key LTA synthase, causing a switch to the production of wall-anchored TAs (WTAs). This change allows LytA to associate with and degrade its cell wall substrate, thus promoting osmotic lysis. Similar changes in surface polymer assembly may underlie the mechanism of antibiotic- and/or growth phase-induced lysis for other important Gram-positive pathogens

Investigating Hemoglobin Capture and Heme Acquisition by the Pathogen Staphylococcus aureus

Staphylococcus aureus is a medically important Gram-positive bacterial pathogen that actively procures heme from human hemoglobin (Hb) using the iron-regulated surface determinant (Isd) system. Research described in this dissertation investigated how the Isd system uses the IsdH receptor protein to capture Hb and extract its hemin (the oxidized form of heme). To rapidly extract Hb’s hemin, IsdH employs a conserved tri-domain unit that contains two NEAr iron Transporter (NEAT) domains that are connected by a helical linker domain. The work described in chapter 2 used UV-Vis spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, and electrospray ionization mass spectrometry (ESI-MS) methods to define the importance of the conserved linker domain in hemin extraction and revealed that this domain enables the NEAT domains within the tri-domain unit to work together to synergistically extract hemin. Chapter 3 of this thesis describes the structure and dynamics of the tri-domain unit (IsdHN2N3). The solution structure of the apo-receptor was defined using small angle X-ray scattering, and advanced NMR methods such as paramagnetic relaxation enhancement (PRE), residual dipolar coupling (RDC), and selective methyl labeling approaches. The structure and inter-domain dynamics of IsdHN2N3 in the absence of Hb were further defined using ensemble modeling calculations. The results of these studies illustrated that the receptor adaptively recognizes Hb using a combination of conformational selection and induced fit mechanisms, and suggests that the linker domain may facilitate hemin transfer by destabilizing the iron-coordinating F-helix in Hb. Chapter 4 describes studies that investigated the kinetic and thermodynamic basis of hemin transfer using stopped-flow UV-Vis spectroscopy, analytical ultracentrifugation sedimentation equilibrium, and isothermal titration methods. The results of this work provide insight into the kinetic and thermodynamic determinants that facilitate receptor-mediated hemin release from Hb. Lastly, Chapter 5 describes the methods that were used to site-specifically label proteins with nitroxide spin-label probes and the subsequent derivation of paramagnetic NMR distance restraints. Altogether, the results of the work described in this dissertation have advanced our knowledge of Hb recognition and hemin acquisition by the pathogen S. aureus

Novel Mechanism of Hemin Capture by Hbp2, the Hemoglobin-binding Hemophore from Listeria monocytogenes *

Iron is an essential nutrient that is required for the growth of the bacterial pathogen Listeria monocytogenes. In cell cultures, this microbe secretes hemin/hemoglobin-binding protein 2 (Hbp2; Lmo2185) protein, which has been proposed to function as a hemophore that scavenges heme from the environment. Based on its primary sequence, Hbp2 contains three NEAr transporter (NEAT) domains of unknown function. Here we show that each of these domains mediates high affinity binding to ferric heme (hemin) and that its N- and C-terminal domains interact with hemoglobin (Hb). The results of hemin transfer experiments are consistent with Hbp2 functioning as an Hb-binding hemophore that delivers hemin to other Hbp2 proteins that are attached to the cell wall. Surprisingly, our work reveals that the central NEAT domain in Hbp2 binds hemin even though its primary sequence lacks a highly conserved YXXXY motif that is used by all other previously characterized NEAT domains to coordinate iron in the hemin molecule. To elucidate the mechanism of hemin binding by Hbp2, we determined crystal structures of its central NEAT domain (Hbp2(N2); residues 183-303) in its free and hemin-bound states. The structures reveal an unprecedented mechanism of hemin binding in which Hbp2(N2) undergoes a major conformational rearrangement that facilitates metal coordination by a non-canonical tyrosine residue. These studies highlight previously unrecognized plasticity in the hemin binding mechanism of NEAT domains and provide insight into how L. monocytogenes captures heme iron

Energetics underlying hemin extraction from human hemoglobin by Staphylococcus aureus

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