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

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

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

A central role for PBP2 in the activation of peptidoglycan polymerization by the bacterial cell elongation machinery.

Cell elongation in rod-shaped bacteria is mediated by the Rod system, a conserved morphogenic complex that spatially controls cell wall assembly by the glycan polymerase RodA and crosslinking enzyme PBP2. Using Escherichia coli as a model system, we identified a PBP2 variant that promotes Rod system function when essential accessory components of the machinery are inactivated. This PBP2 variant hyperactivates cell wall synthesis in vivo and stimulates the activity of RodA-PBP2 complexes in vitro. Cells with the activated synthase also exhibited enhanced polymerization of the actin-like MreB component of the Rod system. Our results define an activation pathway governing Rod system function in which PBP2 conformation plays a central role in stimulating both glycan polymerization by its partner RodA and the formation of cytoskeletal filaments of MreB to orient cell wall assembly. In light of these results, previously isolated mutations that activate cytokinesis suggest that an analogous pathway may also control cell wall synthesis by the division machinery

Molecular mechanism of the wake-promoting agent TAK-925

Abstract The OX2 orexin receptor (OX2R) is a highly expressed G protein-coupled receptor (GPCR) in the brain that regulates wakefulness and circadian rhythms in humans. Antagonism of OX2R is a proven therapeutic strategy for insomnia drugs, and agonism of OX2R is a potentially powerful approach for narcolepsy type 1, which is characterized by the death of orexinergic neurons. Until recently, agonism of OX2R had been considered ‘undruggable.’ We harness cryo-electron microscopy of OX2R-G protein complexes to determine how the first clinically tested OX2R agonist TAK-925 can activate OX2R in a highly selective manner. Two structures of TAK-925-bound OX2R with either a Gq mimetic or Gi reveal that TAK-925 binds at the same site occupied by antagonists, yet interacts with the transmembrane helices to trigger activating microswitches. Our structural and mutagenesis data show that TAK-925’s selectivity is mediated by subtle differences between OX1 and OX2 receptor subtypes at the orthosteric pocket. Finally, differences in the polarity of interactions at the G protein binding interfaces help to rationalize OX2R’s coupling selectivity for Gq signaling. The mechanisms of TAK-925’s binding, activation, and selectivity presented herein will aid in understanding the efficacy of small molecule OX2R agonists for narcolepsy and other circadian disorders