Direct Heme Transfer Reactions in the Group A Streptococcus Heme Acquisition Pathway

The heme acquisition machinery in Group A Streptococcus (GAS) consists of the surface proteins Shr and Shp and ATP-binding cassette transporter HtsABC. Shp cannot directly acquire heme from methemoglobin (metHb) but directly transfers its heme to HtsA. It has not been previously determined whether Shr directly relays heme from metHb to Shp. Thus, the complete pathway for heme acquisition from metHb by the GAS heme acquisition machinery has remained unclear. In this study, the metHb-to-Shr and Shr-to-Shp heme transfer reactions were characterized by spectroscopy, kinetics and protein-protein interaction analyses. Heme is efficiently transferred from the β and α subunits of metHb to Shr with rates that are 7 and 60 times greater than those of the passive heme release from metHb, indicating that Shr directly acquires heme from metHb. The rapid heme transfer from Shr to Shp involves an initial heme donor/acceptor complex and a spectrally and kinetically detectable transfer intermediate, implying that heme is directly channeled from Shr to Shp. The present results show that Shr speeds up heme transfer from metHb to Shp, whereas Shp speeds up heme transfer from Shr to HtsA. Furthermore, the findings demonstrate that Shr can interact with metHb and Shp but not HtsA. Taken together with our published results on the Shp/HtsA reaction, these findings establish a model of the heme acquisition pathway in GAS in which Shr directly extracts heme from metHb and Shp relays it from Shr to HtsA

Differential Function of Lip Residues in the Mechanism and Biology of an Anthrax Hemophore

To replicate in mammalian hosts, bacterial pathogens must acquire iron. The majority of iron is coordinated to the protoporphyrin ring of heme, which is further bound to hemoglobin. Pathogenic bacteria utilize secreted hemophores to acquire heme from heme sources such as hemoglobin. Bacillus anthracis, the causative agent of anthrax disease, secretes two hemophores, IsdX1 and IsdX2, to acquire heme from host hemoglobin and enhance bacterial replication in iron-starved environments. Both proteins contain NEAr-iron Transporter (NEAT) domains, a conserved protein module that functions in heme acquisition in Gram-positive pathogens. Here, we report the structure of IsdX1, the first of a Gram-positive hemophore, with and without bound heme. Overall, IsdX1 forms an immunoglobin-like fold that contains, similar to other NEAT proteins, a 310-helix near the heme-binding site. Because the mechanistic function of this helix in NEAT proteins is not yet defined, we focused on the contribution of this region to hemophore and NEAT protein activity, both biochemically and biologically in cultured cells. Site-directed mutagenesis of amino acids in and adjacent to the helix identified residues important for heme and hemoglobin association, with some mutations affecting both properties and other mutations affecting only heme stabilization. IsdX1 with mutations that reduced the ability to associate with hemoglobin and bind heme failed to restore the growth of a hemophore-deficient strain of B. anthracis on hemoglobin as the sole iron source. These data indicate that not only is the 310-helix important for NEAT protein biology, but also that the processes of hemoglobin and heme binding can be both separate as well as coupled, the latter function being necessary for maximal heme-scavenging activity. These studies enhance our understanding of NEAT domain and hemophore function and set the stage for structure-based inhibitor design to block NEAT domain interaction with upstream ligands

QSOX1 has a protective role in the myocardium face to acute stress

International audienceno abstrac

QSOX1 has a protective role in the myocardium face to acute stress

Congress of the European-Society-of-Cardiology (ESC), Rome, ITALY, AUG 27-31, 2016International audienceno abstrac

Contribution of Specific Residues of the beta-Solenoid Fold to HET-s Prion Function, Amyloid Structure and Stability

The [Het-s] prion of the fungus Podospora anserina represents a good model system for studying the structure-function relationship in amyloid proteins because a high resolution solid-state NMR structure of the amyloid prion form of the HET-s prion forming domain (PFD) is available. The HET-s PFD adopts a specific β-solenoid fold with two rungs of β-strands delimiting a triangular hydrophobic core. A C-terminal loop folds back onto the rigid core region and forms a more dynamic semi-hydrophobic pocket extending the hydrophobic core. Herein, an alanine scanning mutagenesis of the HET-s PFD was conducted. Different structural elements identified in the prion fold such as the triangular hydrophobic core, the salt bridges, the asparagines ladders and the C-terminal loop were altered and the effect of these mutations on prion function, fibril structure and stability was assayed. Prion activity and structure were found to be very robust; only a few key mutations were able to corrupt structure and function. While some mutations strongly destabilize the fold, many substitutions in fact increase stability of the fold. This increase in structural stability did not influence prion formation propensity in vivo. However, if an Ala replacement did alter the structure of the core or did influence the shape of the denaturation curve, the corresponding variant showed a decreased prion efficacy. It is also the finding that in addition to the structural elements of the rigid core region, the aromatic residues in the C-terminal semi-hydrophobic pocket are critical for prion propagation. Mutations in the latter region either positively or negatively affected prion formation. We thus identify a region that modulates prion formation although it is not part of the rigid cross-β core, an observation that might be relevant to other amyloid models

Discovery and characterization of a unique mycobacterial heme acquisition system

Mycobacterium tuberculosis must import iron from its host for survival, and its siderophore-dependent iron acquisition pathways are well established. Here we demonstrate a newly characterized pathway, whereby M. tuberculosis can use free heme and heme from hemoglobin as an iron source. Significantly, we identified the genomic region, Rv0202c–Rv0207c, responsible for the passage of heme iron across the mycobacterial membrane. Key players of this heme uptake system were characterized including a secreted protein and two transmembrane proteins, all three specific to mycobacteria. Furthermore, the crystal structure of the key heme carrier protein Rv0203 was found to have a unique fold. The discovery of a unique mycobacterial heme acquisition pathway opens new avenues of exploration into mycobacterial therapeutics