All bactericidal antibiotics were recently proposed to kill by inducing reactive oxygen species (ROS) production, causing destabilization of iron-sulfur (Fe-S) clusters and generating Fenton chemistry. We find that the ROS response is dispensable upon treatment with bactericidal antibiotics. Furthermore, we demonstrate that Fe-S clusters are required for killing only by aminoglycosides. In contrast to cells, using the major Fe-S cluster biosynthesis machinery, ISC, cells using the alternative machinery, SUF, cannot efficiently mature respiratory complexes I and II, resulting in impendence of the proton motive force (PMF), which is required for bactericidal aminoglycoside uptake. Similarly, during iron limitation, cells become intrinsically resistant to aminoglycosides by switching from ISC to SUF and down-regulating both respiratory complexes. We conclude that Fe-S proteins promote aminoglycoside killing by enabling their uptake.

The reactive species of oxygen (ROS) and chlorine (RCS) damage cellular components, potentially leading to cell death. In proteins, the sulfur-containing amino acid methionine (Met) is converted to methionine sulfoxide (Met-O), which can cause a loss of biological activity. To rescue proteins with Met-O residues, living cells express methionine sulfoxide reductases (Msrs) in most subcellular compartments, including the cytosol, mitochondria and chloroplasts [1][2][3] . Here, we report the identification of an enzymatic system, MsrPQ, repairing Met-O containing proteins in the bacterial cell envelope, a compartment particularly exposed to the ROS and RCS generated by the host defense mechanisms. MsrP, a molybdo-enzyme, and MsrQ, a heme-binding membrane protein, are widely conserved throughout Gram-negative bacteria, including major human pathogens. MsrPQ synthesis is induced by hypochlorous acid (HOCl), a powerful antimicrobial released by neutrophils. Consistently, MsrPQ is essential for the maintenance of envelope integrity under bleach stress, rescuing a wide series of structurally unrelated periplasmic proteins from Met

DNA double-strand breaks pose a significant threat to cell survival and must be repaired. In higher eukaryotes, such damage is repaired efficiently by non-homologous end joining (NHEJ). Within this pathway, XRCC4 and XLF fulfill key roles required for end joining. Using DNA-binding and -bridging assays, combined with direct visualization, we present evidence for how XRCC4–XLF complexes robustly bridge DNA molecules. This unanticipated, DNA Ligase IV-independent bridging activity by XRCC4–XLF suggests an early role for this complex during end joining, in addition to its more well-established later functions. Mutational analysis of the XRCC4–XLF C-terminal tail regions further identifies specialized functions in complex formation and interaction with DNA and DNA Ligase IV. Based on these data and the crystal structure of an extended protein filament of XRCC4–XLF at 3.94 Å, a model for XRCC4–XLF complex function in NHEJ is presented.

XRCC4 and XLF are structurally related proteins important for DNA Ligase IV function. XRCC4 forms a tight complex with DNA Ligase IV while XLF interacts directly with XRCC4. Both XRCC4 and XLF form homodimers that can polymerize as heterotypic filaments independently of DNA Ligase IV. Emerging structural and in vitro biochemical data suggest that XRCC4 and XLF together generate a filamentous structure that promotes bridging between DNA molecules. Here, we show that ablating XRCC4's affinity for XLF results in DNA repair deficits including a surprising deficit in VDJ coding, but not signal end joining. These data are consistent with a model whereby XRCC4/XLF complexes hold DNA ends together—stringently required for coding end joining, but dispensable for signal end joining. Finally, DNA-PK phosphorylation of XRCC4/XLF complexes disrupt DNA bridging in vitro, suggesting a regulatory role for DNA-PK's phosphorylation of XRCC4/XLF complexes.

The biogenesis of respiratory complexes is a multistep process that requires finely tuned coordination of subunit assembly, metal cofactor insertion, and membrane-anchoring events. The dissimilatory nitrate reductase of the bacterial anaerobic respiratory chain is a membrane-bound heterotrimeric complex nitrate reductase A (NarGHI) carrying no less than eight redox centers. Here, we identified different stable folding assembly intermediates of the nitrate reductase complex and analyzed their redox cofactor contents using electron paramagnetic resonance spectroscopy. Upon the absence of the accessory protein NarJ, a global defect in metal incorporation was revealed. In addition to the molybdenum cofactor, we show that NarJ is required for specific insertion of the proximal iron-sulfur cluster (FS0) within the soluble nitrate reductase (NarGH) catalytic dimer. Further, we establish that NarJ ensures complete maturation of the b-type cytochrome subunit NarI by a proper timing for membrane anchoring of the NarGH complex. Our findings demonstrate that NarJ has a multifunctional role by orchestrating both the maturation and the assembly steps.All biological systems require the biogenesis of functional respiratory or photosynthetic complexes for their viability. In bacteria, bioenergetic electron transfer chains are associated to the inner membrane. Biogenesis of these complexes is an intricate process that requires several steps such as the synthesis of the different subunits, their assembly, the incorporation of various types of metal or organic cofactors, and the anchoring of the complex to the membrane. In the case of exported metalloproteins, the assembly and cofactor incorporation steps need to be accomplished prior to translocation of the inner membrane via the twin arginine translocase apparatus (1-3). Importantly, accessory proteins are often involved in biogenesis of metalloproteins (4 -9). Although it is most likely that all these events occur in a coordinate fashion to yield a final functional multimeric metalloprotein, information about how this coordination is performed is scarce.The well studied and characterized Escherichia coli dissimilatory quinol-nitrate oxidoreductase of the anaerobic respiratory chain, referred to as the nitrate reductase A (NarGHI) 4 (10, 11), can be considered as a suitable model for deciphering the biogenesis pathway of multimeric metalloproteins. NarGHI is a non-exported membrane-bound respiratory complex composed of three subunits that bind eight redox centers: (i) a catalytic subunit (NarG) containing a molybdenum-bis-molybdopterin guanine dinucleotide cofactor (Moco) and a proximal [4Fe-4S] cluster (FS0) (12, 13), (ii) an electron transfer subunit (NarH) carrying one [3Fe-4S] cluster (FS4) and three [4Fe-4S] clusters (FS1 to FS3) (14, 15), and (iii) a quinol-oxidizing membrane-bound subunit (NarI) containing two b-type hemes (b D and b P ) (11,16,17). The NarJ protein encoded by the narGHJI operon plays an essential role in nitrate reductase activity, enabling Moco insertion...

Understanding when and how metal cofactor insertion occurs into a multisubunit metalloenzyme is of fundamental importance. Molybdenum cofactor insertion is a tightly controlled process that involves specific interactions between the proteins that promote cofactor delivery, enzyme-specific chaperones, and the apoenzyme. In the assembly pathway of the multisubunit molybdoenzyme, membrane-bound nitrate reductase A from Escherichia coli, a NarJassisted molybdenum cofactor (Moco) insertion step, must precede membrane anchoring of the apoenzyme. Here, we have shown that the NarJ chaperone interacts at two distinct binding sites of the apoenzyme, one interfering with its membrane anchoring and another one being involved in molybdenum cofactor insertion. The presence of the two NarJ-binding sites within NarG is required to ensure productive formation of active nitrate reductase. Our findings supported the view that enzyme-specific chaperones play a central role in the biogenesis of multisubunit molybdoenzymes by coordinating subunits assembly and molybdenum cofactor insertion.Molybdoenzymes are involved in numerous metabolic reactions in the carbon, nitrogen, and sulfur cycles and crucial for all forms of life (1). With the exception of nitrogenase, the active site of molybdoenzymes contains a molybdenum cofactor (Moco) 3 that has an ubiquitous basic structure composed of a molybdenum atom coordinated to one or two molecules of a tricyclic pyranopterin (2, 3). The past few years have seen spectacular advances in our understanding of the molecular mechanisms of Moco biosynthesis, a highly conserved biosynthetic pathway (4 -8). In contrast, information concerning biogenesis of molybdoenzymes is scarce. Molybdoenzyme biogenesis, the process that ensures productive formation of active molybdoenzymes, generally involves both metal cofactor insertion and multisubunit assembly. In prokaryotes, the Moco insertion process is a cytoplasmic post-translational event (9) often assisted by enzyme-specific chaperones (10 -13).Dissimilatory nitrate reductase A from Escherichia coli (NarGHI) is one of the best studied multisubunit molybdoenzymes (14) and can be considered as a model system for studying the biogenesis process in prokaryotic enzymes. NarGHI is a heterotrimeric enzyme comprising a Moco and an iron-sulfur-containing catalytic subunit (NarG, 139 kDa), an iron-sulfur-containing subunit (NarH, 58 kDa) and a quinol-oxidizing membrane-bound heme b subunit (NarI, 26 kDa) (14, 15). NarGH is located in the cytoplasm, anchored to the cytoplasmic membrane by NarI. When liberated from the membrane, the NarGH complex retains its activity using artificial electron donors such as benzyl viologen. Finally, the enzyme-specific chaperone NarJ plays an essential role for nitrate reductase A activity, facilitating Moco insertion into NarG (11).As observed for other known molybdoenzymes (16 -19), the crystal structure of the NarGHI complex (20, 21) reveals that Moco is an extended molecule deeply buried into the enzyme complex at the NarG-H ...

The maturation of Escherichia coli nitrate reductase A requires the incorporation of the Mo-(bis-MGD) cofactor to the apoprotein. For this process, the NarJ chaperone is strictly required (Blasco, F., Dos Santos, J. P., Magalon, A., Frixon, C., Guigliarelli, B., Santini, C. L., and Giordano, G. (1998) Mol. Microbiol. 28, 435-447). We report the first description of protein interactions between molybdenum cofactor biosynthetic proteins (MogA, MoeA, MobA, and MobB) and the aponitrate reductase (NarG) using a bacterial two-hybrid approach. Two conditions have to be satisfied to allow the visualization of the interactions, (i) the presence of an active and mature molybdenum cofactor and (ii) the presence of the NarJ chaperone and of the NarG structural partner subunit, NarH. Formation of tungsten-substituted cofactor prevents the interaction between NarG and the four biosynthetic proteins. Our results suggested that the final stages of molybdenum cofactor biosynthesis occur on a complex made up by MogA, MoeA, MobA, and MobB, which is also in charge with the delivery of the mature cofactor onto the aponitrate reductase A in a NarJ-assisted process.

A novel class of molecular chaperones co‐ordinates the assembly and targeting of complex metalloproteins by binding to an amino‐terminal peptide of the cognate substrate. We have previously shown that the NarJ chaperone interacts with the N‐terminus of the NarG subunit coming from the nitrate reductase complex, NarGHI. In the present study, NMR structural analysis revealed that the NarG(1–15) peptide adopts an α‐helical conformation in solution. Moreover, NarJ recognizes and binds the helical NarG(1–15) peptide mostly via hydrophobic interactions as deduced from isothermal titration calorimetry analysis. NMR and differential scanning calorimetry analysis revealed a modification of NarJ conformation during complex formation with the NarG(1–15) peptide. Isothermal titration calorimetry and BIAcore experiments support a model whereby the protonated state of the chaperone controls the time dependence of peptide interaction. Structured digital abstract • http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7557484: NarJT (uniprotkb:http://www.ebi.uniprot.org/entry/P0AF26) and NarG (uniprotkb:http://www.ebi.uniprot.org/entry/P09152) bind (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407) by isothermal titration calorimetry (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0065) • http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7557456: NarJT (uniprotkb:http://www.ebi.uniprot.org/entry/P0AF26) and NarG (uniprotkb:http://www.ebi.uniprot.org/entry/P09152) bind (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407) by nuclear magnetic resonance (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0077)