Structure of the peptidoglycan polymerase RodA resolved by evolutionary coupling analysis.

The shape, elongation, division and sporulation (SEDS) proteins are a large family of ubiquitous and essential transmembrane enzymes with critical roles in bacterial cell wall biology. The exact function of SEDS proteins was for a long time poorly understood, but recent work has revealed that the prototypical SEDS family member RodA is a peptidoglycan polymerase-a role previously attributed exclusively to members of the penicillin-binding protein family. This discovery has made RodA and other SEDS proteins promising targets for the development of next-generation antibiotics. However, little is known regarding the molecular basis of SEDS activity, and no structural data are available for RodA or any homologue thereof. Here we report the crystal structure of Thermus thermophilus RodA at a resolution of 2.9 Å, determined using evolutionary covariance-based fold prediction to enable molecular replacement. The structure reveals a ten-pass transmembrane fold with large extracellular loops, one of which is partially disordered. The protein contains a highly conserved cavity in the transmembrane domain, reminiscent of ligand-binding sites in transmembrane receptors. Mutagenesis experiments in Bacillus subtilis and Escherichia coli show that perturbation of this cavity abolishes RodA function both in vitro and in vivo, indicating that this cavity is catalytically essential. These results provide a framework for understanding bacterial cell wall synthesis and SEDS protein function.

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.

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 ß-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.

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.

Staphylococcus aureus uses a novel multidomain receptor to break apart human hemoglobin and steal its heme.

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 (IsdH(N2N3), Ala(326)-Asp(660)) 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 IsdH(N2N3) 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.

Molecular mechanism of the wake-promoting agent TAK-925.

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.

Structural coordination of polymerization and crosslinking by a SEDS-bPBP peptidoglycan synthase complex.

The shape, elongation, division and sporulation (SEDS) proteins are a highly conserved family of transmembrane glycosyltransferases that work in concert with class B penicillin-binding proteins (bPBPs) to build the bacterial peptidoglycan cell wall1-6. How these proteins coordinate polymerization of new glycan strands with their crosslinking to the existing peptidoglycan meshwork is unclear. Here, we report the crystal structure of the prototypical SEDS protein RodA from Thermus thermophilus in complex with its cognate bPBP at 3.3 Å resolution. The structure reveals a 1:1 stoichiometric complex with two extensive interaction interfaces between the proteins: one in the membrane plane and the other at the extracytoplasmic surface. When in complex with a bPBP, RodA shows an approximately 10 Å shift of transmembrane helix 7 that exposes a large membrane-accessible cavity. Negative-stain electron microscopy reveals that the complex can adopt a variety of different conformations. These data define the bPBP pedestal domain as the key allosteric activator of RodA both in vitro and in vivo, explaining how a SEDS-bPBP complex can coordinate its dual enzymatic activities of peptidoglycan polymerization and crosslinking to build the cell wall.

FtsW is a peptidoglycan polymerase that is functional only in complex with its cognate penicillin-binding protein.

The peptidoglycan cell wall is essential for the survival and morphogenesis of bacteria1. For decades, it was thought that only class A penicillin-binding proteins (PBPs) and related enzymes effected peptidoglycan synthesis. Recently, it was shown that RodA-a member of the unrelated SEDS protein family-also acts as a peptidoglycan polymerase2-4. Not all bacteria require RodA for growth; however, its homologue, FtsW, is a core member of the divisome complex that appears to be universally essential for septal cell wall assembly5,6. FtsW was previously proposed to translocate the peptidoglycan precursor lipid II across the cytoplasmic membrane7,8. Here, we report that purified FtsW polymerizes lipid II into peptidoglycan, but show that its polymerase activity requires complex formation with its partner class B PBP. We further demonstrate that the polymerase activity of FtsW is required for its function in vivo. Thus, our findings establish FtsW as a peptidoglycan polymerase that works with its cognate class B PBP to produce septal peptidoglycan during cell division.

Nitroxide Labeling of Proteins and the Determination of Paramagnetic Relaxation Derived Distance Restraints for NMR Studies.

Site-specific attachment of paramagnetic spin labels to biomolecules causes distance-dependent line-broadening effects, which can be exploited to study the structure and dynamics of these molecules in solution. This protocol describes how to attach nitroxide spin labels to proteins and how to collect and analyze NMR data using these labeled samples. We also explain how to derive distance restraints for paramagnetic relaxation enhancement nuclear magnetic resonance (PRE-NMR) studies.

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 (IsdH(N2N3), 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.

Identification of agents effective against multiple toxins and viruses by host-oriented cell targeting.

A longstanding and still-increasing threat to the effective treatment of infectious diseases is resistance to antimicrobial countermeasures. Potentially, the targeting of host proteins and pathways essential for the detrimental effects of pathogens offers an approach that may discover broad-spectrum anti-pathogen countermeasures and circumvent the effects of pathogen mutations leading to resistance. Here we report implementation of a strategy for discovering broad-spectrum host-oriented therapies against multiple pathogenic agents by multiplex screening of drugs for protection against the detrimental effects of multiple pathogens, identification of host cell pathways inhibited by the drug, and screening for effects of the agent on other pathogens exploiting the same pathway. We show that a clinically used antimalarial drug, Amodiaquine, discovered by this strategy, protects host cells against infection by multiple toxins and viruses by inhibiting host cathepsin B. Our results reveal the practicality of discovering broadly acting anti-pathogen countermeasures that target host proteins exploited by pathogens.