David Lascoux

SufE Transfers Sulfur from SufS to SufB for Iron-Sulfur Cluster Assembly

Iron-sulfur (Fe-S) clusters are key metal cofactors of metabolic, regulatory, and stress response proteins in most organisms. The unique properties of these clusters make them susceptible to disruption by iron starvation or oxidative stress. Both iron and sulfur can be perturbed under stress conditions, leading to Fe-S cluster defects. Bacteria and higher plants contain a specialized system for Fe-S cluster biosynthesis under stress, namely the Suf pathway. In Escherichia coli the Suf pathway consists of six proteins with functions that are only partially characterized. Here we describe how the SufS and SufE proteins interact with the SufBCD protein complex to facilitate sulfur liberation from cysteine and donation for Fe-S cluster assembly. It was previously shown that the cysteine desulfurase SufS donates sulfur to the sulfur transfer protein SufE. We have found here that SufE in turn interacts with the SufB protein for sulfur transfer to that protein. The interaction occurs only if SufC is present. Furthermore, SufB can act as a site for Fe-S cluster assembly in the Suf system. This provides the first evidence of a novel site for Fe-S cluster assembly in the SufBCD complex.

Structural and functional characterization of 2-oxo-histidine in oxidized PerR protein

In Bacillus subtilis, PerR is a metal-dependent sensor of hydrogen peroxide. PerR is a dimeric zinc protein with a regulatory site that coordinates either Fe(2+) (PerR-Zn-Fe) or Mn(2+) (PerR-Zn-Mn). Though most of the peroxide sensors use cysteines to detect H(2)O(2), it has been shown that reaction of PerR-Zn-Fe with H(2)O(2) leads to the oxidation of one histidine residue. Oxidation of PerR leads to the incorporation of one oxygen atom into His37 or His91. This study presents the crystal structure of the oxidized PerR protein (PerR-Zn-ox), which clearly shows a 2-oxo-histidine residue in position 37. Formation of 2-oxo-histidine is demonstrated and quantified by HPLC-MS/MS. EPR experiments indicate that PerR-Zn-H37ox retains a significant affinity for the regulatory metal, whereas PerR-Zn-H91ox shows a considerably reduced affinity for the metal ion. In spite of these major differences in terms of metal binding affinity, oxidation of His37 and/or His91 in PerR prevents DNA binding.

Mechanistic studies of the SufS-SufE cysteine desulfurase: evidence for sulfur transfer from SufS to SufE.

SufS is a cysteine desulfurase of the suf operon shown to be involved in iron–sulfur cluster biosynthesis under iron limitation and oxidative stress conditions. The enzyme catalyzes the conversion of L‐cysteine to L‐alanine and sulfide through the intermediate formation of a protein‐bound cysteine persulfide in the active site. SufE, another component of the suf operon, has been previously shown to bind tightly to SufS and to drastically stimulate its cysteine desulfurase activity. Working with Escherichia coli proteins, we here demonstrate that a conserved cysteine residue in SufE at position 51 is essential for the SufS/SufE cysteine desulfurase activity. Mass spectrometry has been used to demonstrate (i) the ability of SufE to bind sulfur atoms on its cysteine 51 and (ii) the direct transfer of the sulfur atom from the cysteine persulfide of SufS to SufE. A reaction mechanism is proposed for this novel two‐component cysteine desulfurase.

The Crystal Structure of the Epstein–Barr Virus Protease Shows Rearrangement of the Processed C Terminus

Epstein-Barr virus (EBV) belongs to the gamma-herpesvirinae subfamily of the Herpesviridae. The protease domain of the assemblin protein of herpesviruses forms a monomer-dimer equilibrium in solution. The protease domain of EBV was expressed in Escherichia coli and its structure was solved by X-ray crystallography to 2.3A resolution after inhibition with diisopropyl-fluorophosphate (DFP). The overall structure confirms the conservation of the homodimer and its structure throughout the alpha, beta, and gamma-herpesvirinae. The substrate recognition could be modelled using information from the DFP binding, from a crystal contact, suggesting that the substrate forms an antiparallel beta-strand extending strand beta5, and from the comparison with the structure of a peptidomimetic inhibitor bound to cytomegalovirus protease. The long insert between beta-strands 1 and 2, which was disordered in the KSHV protease structure, was found to be ordered in the EBV protease and shows the same conformation as observed for proteases in the alpha and beta-herpesvirus families. In contrast to previous structures, the long loop located between beta-strands 5 and 6 is partially ordered, probably due to DFP inhibition and a crystal contact. It also contributes to substrate recognition. The protease shows a specific recognition of its own C terminus in a binding pocket involving residue Phe210 of the other monomer interacting across the dimer interface. This suggests conformational changes of the protease domain after its release from the assemblin precursor followed by burial of the new C terminus and a possible effect onto the monomer-dimer equilibrium. The importance of the processed C terminus was confirmed using a mutant protease carrying a C-terminal extension and a mutated release site, which shows different solution properties and a strongly reduced enzymatic activity.

The SUF iron–sulfur cluster biosynthetic machinery: Sulfur transfer from the SUFS–SUFE complex to SUFA

Iron–sulfur cluster biosynthesis depends on protein machineries, such as the ISC and SUF systems. The reaction is proposed to imply binding of sulfur and iron atoms and assembly of the cluster within a scaffold protein followed by transfer of the cluster to recipient apoproteins. The SufA protein from Escherichia coli, used here as a model scaffold protein is competent for binding sulfur atoms provided by the SufS–SufE cysteine desulfurase system covalently as shown by mass spectrometry. Investigation of site‐directed mutants and peptide mapping experiments performed on digested sulfurated SufA demonstrate that binding exclusively occurs at the three conserved cysteines (cys50, cys114, cys116). In contrast, it binds iron only weakly (K a = 5 × 105 M−1) and not specifically to the conserved cysteines as shown by Mössbauer spectroscopy. [Fe–S] clusters, characterized by Mössbauer spectroscopy, can be assembled during reaction of sulfurated SufA with ferrous iron in the presence of a source of electrons.

Type III secretion proteins PcrV and PcrG from Pseudomonas aeruginosa form a 1:1 complex through high affinity interactions

BackgroundPseudomonas aeruginosa, an increasingly prevalent opportunistic pathogen, utilizes a type III secretion system for injection of toxins into host cells in order to initiate infection. A crucial component of this system is PcrV, which is essential for cytotoxicity and is found both within the bacterial cytoplasm and localized extracellularly, suggesting that it may play more than one role in Pseudomonas infectivity. LcrV, the homolog of PcrV in Yersinia, has been proposed to participate in effector secretion regulation by interacting with LcrG, which may act as a secretion blocker. Although PcrV also recognizes PcrG within the bacterial cytoplasm, the roles played by the two proteins in type III secretion in Pseudomonas may be different from the ones suggested for their Yersinia counterparts.ResultsIn this work, we demonstrate by native mass spectrometry that PcrV and PcrG expressed and purified from E. coli form a 1:1 complex in vitro. Circular dichroism results indicate that PcrG is highly unstable in the absence of PcrV; in contrast, both PcrV alone and the PcrV:PcrG complex have high structural integrity. Surface plasmon resonance measurements show that PcrV interacts with PcrG with nanomolar affinity (15.6 nM) and rapid kinetics, an observation which is valid both for the full-length form of PcrG (residues 1–98) as well as a form which lacks the C-terminal 24 residues, which are predicted to have low secondary structure content.ConclusionsPcrV is a crucial component of the type III secretion system of Pseudomonas, but the way in which it participates in toxin secretion is not understood. Here we have characterized the interaction between PcrV and PcrG in vitro, and shown that PcrG is highly unstable. However, it associates readily with PcrV through a region located within its first 74 amino acids to form a high affinity complex. The fact that PcrV associates and dissociates quickly from an unstable molecule points to the transient nature of a PcrV:PcrG complex. These results are in agreement with analyses from pcrV deletion mutants which suggest that PcrV:PcrG may play a different role in effector secretion than the one described for the LcrV:LcrG complex in Yersinia.

Is the cytoplasmic loop of MerT, the mercuric ion transport protein, involved in mercury transfer to the mercuric reductase?

In MerT, the mercury transporter, a first cysteine pair, located in the first trans‐membrane helix, receives mercury from the periplasm. Then, a second cysteine pair, housed in a cytoplasmic loop connecting the second and the third trans‐membrane helices, is thought to transfer the metal to another cysteine pair located in the N‐terminal extension of the mercuric reductase. We found that a 23‐amino acid synthetic peptide corresponding to the cytoplasmic loop can bind one mercury atom per molecule and that this mercury atom can be transferred specifically to MerAa. The solution structure of Hg‐bound ppMerT has been solved by 1H NMR spectroscopy.