Structural determinants of DNA recognition by the NO sensor NsrR and related Rrf2-type [FeS]-transcription factors.

Several transcription factors of the Rrf2 family use an iron-sulfur cluster to regulate DNA binding through effectors such as nitric oxide (NO), cellular redox status and iron levels. [4Fe-4S]-NsrR from Streptomyces coelicolor (ScNsrR) modulates expression of three different genes via reaction and complex formation with variable amounts of NO, which results in detoxification of this gas. Here, we report the crystal structure of ScNsrR complexed with an hmpA1 gene operator fragment and compare it with those previously reported for [2Fe-2S]-RsrR/rsrR and apo-IscR/hyA complexes. Important structural differences reside in the variation of the DNA minor and major groove widths. In addition, different DNA curvatures and different interactions with the protein sensors are observed. We also report studies of NsrR binding to four hmpA1 variants, which indicate that flexibility in the central region is not a key binding determinant. Our study explores the promotor binding specificities of three closely related transcriptional regulators.

A structure-function study of ZraP and ZraS provides new insights into the two-component system Zra.

BACKGROUND: Zra belongs to the envelope stress response (ESR) two-component systems (TCS). It is atypical because of its third periplasmic repressor partner (ZraP), in addition to its histidine kinase sensor protein (ZraS) and its response regulator (ZraR) components. Furthermore, although it is activated by Zn2+, it is not involved in zinc homeostasis or protection against zinc toxicity. Here, we mainly focus on ZraS but also provide information on ZraP. METHODS: The purified periplasmic domain of ZraS and ZraP were characterized using biophysical and biochemical technics: multi-angle laser light scattering (MALLS), circular dichroism (CD), differential scanning fluorescence (DSF), inductively coupled plasma atomic emission spectroscopy (ICP-AES), cross-linking and small-angle X-ray scattering (SAXS). In-vivo experiments were carried out to determine the redox state of the cysteine residue in ZraP and the consequences for the cell of an over-activation of the Zra system. RESULTS: We show that ZraS binds one Zn2+ molecule with high affinity resulting in conformational changes of the periplasmic domain, consistent with a triggering function of the metal ion. We also demonstrate that, in the periplasm, the only cysteine residue of ZraP is at least partially reduced. Using SAXS, we conclude that the previously determined X-ray structure is different from the structure in solution. CONCLUSION: Our results allow us to propose a general mechanism for the Zra system activation and to compare it to the homologous Cpx system. GENERAL SIGNIFICANCE: We bring new input on the so far poorly described Zra system and notably on ZraS.

The Two-Component System ZraPSR Is a Novel ESR that Contributes to Intrinsic Antibiotic Tolerance in Escherichia coli.

During their lifecycle, bacteria are exposed to continuous changes in their environment, some of which are stressful and can be harmful. The cell envelope is the first line of defense against a hostile environment, but it is also the first target for damage. To deal with this problem, bacteria have evolved systems collectively called "envelope stress response," or ESR, dedicated to the detection and repair of damaged components. Here we decided to investigate whether the atypical two-component system ZraP-SR is a novel ESR. Based on the screening of more than 240 drugs using the Biolog technology, we show that the deletion of zraP or zraR confers increased susceptibility to five classes of antibiotics and to some environmental stress targeting the envelope. Using a microscopy approach, we also establish that ZraP and ZraR are required to maintain envelope integrity. So far, the ZraR regulator was only known to activate the transcription of zraP and zraSR. Using chromatin immunoprecipitation followed by sequencing and RT-qPCR, we have now identified 25 additional genes regulated by ZraR, the majority of which are involved in the response against stress. Taken together, our results demonstrate that ZraP-SR is a novel ESR.

Quaternary Structure of Fur Proteins, a New Subfamily of Tetrameric Proteins.

The ferric uptake regulator (Fur) belongs to the family of the DNA-binding metal-responsive transcriptional regulators. Fur is a global regulator found in all proteobacteria. It controls the transcription of a wide variety of genes involved in iron metabolism but also in oxidative stress or virulence factor synthesis. When bound to ferrous iron, Fur can bind to specific DNA sequences, called Fur boxes. This binding triggers the repression or the activation of gene expression, depending on the regulated genes. As a general view, Fur proteins are considered to be dimeric proteins both in solution and when bound to DNA. In this study, we have purified Fur from four pathogenic strains (Pseudomonas aeruginosa, Francisella tularensis, Yersinia pestis, and Legionella pneumophila) and compared them to Fur from Escherichia coli (EcFur), the best characterized of this family. By using a series of "in solution" techniques, including multiangle laser light scattering and small-angle X-ray scattering, as well as cross-linking experiments, we have shown that the Fur proteins can be classified into two groups, according to their quaternary structure. The group of dimers is represented by EcFur and YpFur and the group of very stable tetramers by PaFur, FtFur, and LpFur. Using PaFur as a case study, we also showed that the dissociation of the tetramers into dimers is necessary for binding of Fur to DNA, and that this dissociation requires the combined effect of metal ion binding and DNA proximity.

Biophysical and physiological characterization of ZraP from Escherichia coli, the periplasmic accessory protein of the atypical ZraSR two-component system.

The ZraSR system belongs to the family of TCSs (two-component signal transduction systems). In Escherichia coli, it was proposed to participate in zinc balance and to protect cytoplasmic zinc overload by sequestering this metal ion into the periplasm. This system controls the expression of the accessory protein ZraP that would be a periplasmic zinc scavenger. ZraPSR is functionally homologous with CpxPAR that integrates signals of envelope perturbation, including misfolded periplasmic proteins. The auxiliary periplasmic regulator CpxP inhibits the Cpx pathway by interacting with CpxA. Upon envelope stress sensing, the inhibitory function of CpxP is relieved, resulting in CpxR activation. Similarly to CpxPAR, ZraPSR probably plays a role in envelope stress response as a zinc-dependent chaperone activity was demonstrated for ZraP in Salmonella. We have purified ZraP from E. coli and shown that it is an octamer containing four interfacial metal-binding sites contributing to dimer stability. These sites are located close to the N-terminus, whereas the C-terminus is involved in polymerization of the protein to form a tetramer of dimers. In vitro, ZraP binds copper with a higher affinity than zinc and displays chaperone properties partially dependent on zinc binding. In vivo, zinc-bound ZraP is a repressor of the expression of the zraPSR operon. However, we have demonstrated that none of the Zra proteins are involved in zinc or copper resistance. We propose an integrated mechanism in which zinc is a marker of envelope stress perturbation and ZraPSR TCS is a sentinel sensing and responding to zinc entry into the periplasm.

Response of CnrX from Cupriavidus metallidurans CH34 to nickel binding.

Resistance to high concentration of nickel ions is mediated in Cupriavidus metallidurans by the CnrCBA transenvelope efflux complex. Expression of the cnrCBA genes is regulated by the transmembrane signal transduction complex CnrYXH. Together, the metal sensor CnrX and the transmembrane antisigma factor CnrY control the availability of the extracytoplasmic function sigma factor CnrH. Release of CnrH from sequestration by CnrY at the cytoplasmic side of the membrane depends essentially on the binding of the agonist metal ion Ni(ii) to the periplasmic metal sensor domain of CnrX. CnrH availability leads to transcription initiation at the promoters cnrYp and cnrCp and to the expression of the genes in the cnrYXHCBA nickel resistance determinant. The first steps of signal propagation by CnrX rely on subtle metal-dependent allosteric modifications. To study the nickel-mediated triggering process by CnrX, we have altered selected residues, F66, M123, and Y135, and explored the physiological consequences of these changes with respect to metal resistance, expression of a cnrCBA-lacZ reporter fusion and protein production. M123C- and Y135F-CnrXs have been further characterized in vitro by metal affinity measurements and crystallographic structure analysis. Atomic-resolution structures of metal-bound M123C- and Y135F-CnrXs showed that Ni(ii) binds two of the three canonical conformations identified and that Ni(ii) sensing likely proceeds by conformation selection.

Metal sensing and signal transduction by CnrX from Cupriavidus metallidurans CH34: role of the only methionine assessed by a functional, spectroscopic, and theoretical study.

When CnrX, the periplasmic sensor protein in the CnrYXH transmembrane signal transduction complex of Cupriavidus metallidurans CH34, binds the cognate metal ions Ni(II) or Co(II), the ECF-type sigma factor CnrH is made available in the cytoplasm for the RNA-polymerase to initiate transcription at the cnrYp and cnrCp promoters. Ni(II) or Co(II) are sensed by a metal-binding site with a N3O2S coordination sphere with octahedral geometry, where S stands for the thioether sulfur of the only methionine (Met123) residue of CnrX. The M123A-CnrX derivative has dramatically reduced signal propagation in response to metal sensing while the X-ray structure of Ni-bound M123A-CnrXs showed that the metal-binding site was not affected by the mutation. Ni(II) remained six-coordinate in M123A-CnrXs, with a water molecule replacing the sulfur as the sixth ligand. H32A-CnrXs, the soluble model of the wild-type membrane-anchored CnrX, was compared to the double mutants H32A-M123A-CnrXs and H32A-M123C-CnrXs to spectroscopically evaluate the role of this unique ligand in the binding site of Ni or Co. The Co- and Ni-bound forms of the protein display unusually blue-shifted visible spectra. TD-DFT calculations using structure-based models allowed identification and assignment of the electronic transitions of Co-bound form of the protein and its M123A derivative. Among them, the signature of the S-Co transition is distinguishable in the shoulder at 530 nm. In vitro affinity measurements point out the crucial role of Met123 in the selectivity for Ni or Co, and in vivo data support the conclusion that Met123 is a trigger of the signal transduction.

GFP-like phototransformation mechanisms in the cytotoxic fluorescent protein KillerRed unraveled by structural and spectroscopic investigations.

KillerRed (KR) is a red fluorescent protein recognized as an efficient genetically encoded photosensitizer. KR generates reactive oxygen species via a complex process of photoreactions, ending up in photobleaching, the mechanism of which remains obscure. In order to clarify these mechanisms, we focus on a single mutant V44A (A44-KR) exhibiting the solely green component of KR. We report on the laser-induced structural transformations of A44-KR at cryogenic temperature, which we have investigated by combining UV-vis fluorescence/absorption spectroscopy with X-ray crystallography. Like the well-known GFP, A44-KR possesses a mixture of protonated (A) absorbing at 397 and deprotonated (B) absorbing at 515 nm chromophores, which are stressed by intense prolonged violet and blue laser sources. Both illuminations directly drive the B-chromophores toward a bleached trans isomerized form. A-type chromophores are sensitive only to violet illumination and are phototransformed either into a deprotonated green fluorescent form by decarboxylation of E218 or into a bleached form with a disordered p-hydroxybenzylidene. In crystallo spectroscopy at cryo-temperature allowed the identification and dissection of an exhaustive scheme of intermediates and end-products resulting from the phototransformation of A44-KR. This constitutes a framework for understanding the photochemistry of the photosensitizer KillerRed.

Spectroscopic characterization of the metal-binding sites in the periplasmic metal-sensor domain of CnrX from Cupriavidus metallidurans CH34.

CnrX, the dimeric metal sensor of the three-protein transmembrane signal transduction complex CnrYXH of Cupriavidus metallidurans CH34, contains one metal-binding site per monomer. Both Ni and Co elicit a biological response and bind the protein in a 3N2O1S coordination sphere with a nearly identical octahedral geometry as shown by the X-ray structure of CnrXs, the soluble domain of CnrX. However, in solution CnrXs is titrated by 4 Co-equiv and exhibits an unexpected intense band at 384 nm that was detected neither by single-crystal spectroscopy nor under anaerobiosis. The data from a combination of spectroscopic techniques (spectrophotometry, electron paramagnetic resonance, X-ray absorption spectroscopy) showed that two sites correspond to those identified by crystallography. The two extra binding sites accommodate Co(II) in an octahedral geometry in the absence of oxygen and are occupied in air by a mixture of low-spin Co(II) as well as EPR-silent Co(III). These extra sites, located at the N-terminus of the protein, are believed to participate to the formation of peroxo-bridged dimers. Accordingly, we hypothesize that the intense band at 384 nm relies on the formation of a binuclear µ-peroxo Co(III) complex. These metal binding sites are not physiologically relevant since they are not detected in full-length NccX, the closest homologue of CnrX. X-ray absorption spectroscopy demonstrates that NccX stabilizes Co(II) in two-binding sites similar to those characterized by crystallography in its soluble counterpart. Nevertheless, the original spectroscopic properties of the extra Co-binding sites are of interest because they are susceptible to be detected in other Co-bound proteins.

Structural basis for metal sensing by CnrX.

CnrX is the metal sensor and signal modulator of the three-protein transmembrane signal transduction complex CnrYXH of Cupriavidus metallidurans CH34 that is involved in the setup of cobalt and nickel resistance. We have determined the atomic structure of the soluble domain of CnrX in its Ni-bound, Co-bound, or Zn-bound form. Ni and Co ions elicit a biological response, while the Zn-bound form is inactive. The structures presented here reveal the topology of intraprotomer and interprotomer interactions and the ability of metal-binding sites to fine-tune the packing of CnrX dimer as a function of the bound metal. These data suggest an allosteric mechanism to explain how the complex is switched on and how the signal is modulated by Ni or Co binding. These results provide clues to propose a model for signal propagation through the membrane in the complex.

Low-temperature switching by photoinduced protonation in photochromic fluorescent proteins.

We have studied the photoswitching behaviour of a number of photochromic fluorescent proteins at cryo-temperature. Spectroscopic investigations at the ensemble level showed that EYFP, Dronpa and IrisFP all exhibit reversible photoswitching at 100 K, albeit with a low quantum yield. The photophysics of the process were studied in more details in the case of EYFP. The data suggest that photoinduced protonation of the chromophore is responsible for off-switching at cryo-temperature, and thus is possible in the absence of significant conformational freedom. This finding is consistent with the hypothesis that chromophore protonation may precede large amplitude conformational changes such as cis-trans isomerisation during off-photoswitching at room temperature. However, our data suggest that low-barrier photoinduced protonation pathways may in fact compete with room-temperature off-switching reactions in photochromic fluorescent proteins. The occurrence of reversible photoswitching at low-temperature is of interest to envisage cryo-nanoscopy experiments using genetically encoded fluorophores.

Raman-assisted X-ray crystallography for the analysis of biomolecules.

In this chapter, we describe Raman microspectrophotometry applied to crystals of biomolecules. Raman spectra collected in crystallo provide structural information highly complementary to X-ray diffraction, relate the crystalline state to the solution state, and allow the identification of ligand-bound or intermediate states of macromolecules. Nonresonant Raman spectroscopy is particularly suitable to the study of macromolecular crystals, and therefore applies to a wide range of noncolored crystalline proteins. Practical issues related to the investigation of crystals by Raman microspectrophotometry are reviewed, and the current limitations are highlighted.

DHR51, the Drosophila melanogaster homologue of the human photoreceptor cell-specific nuclear receptor, is a thiolate heme-binding protein.

Heme has been recently described as a regulating ligand for the activity of the human nuclear receptors (NR) REV-ERBalpha and REV-ERBbeta and their Drosophila homologue E75. Here, we report the cloning, expression in Escherichia coli, purification, and screening for the heme-binding ability of 11 NR ligand-binding domains of Drosophila melanogaster (DHR3, DHR4, DHR39, DHR51, DHR78, DHR83, HNF4, TLL, ERR, FTZ-F1, and E78), of unknown structure. One of these NRs, DHR51, homologous to the human photoreceptor cell-specific nuclear receptor (PNR), specifically binds heme and exhibits a UV-visible spectrum identical to that of heme-bound E75-LBD. EPR and UV-visible absorption spectroscopy indicates that, like in E75, the heme contains a hexa-coordinated low spin ferric iron. One of its axial ligands is a tightly bound cysteine, while the other one is a histidine. A dissociation constant of 0.5 microM for the heme was measured by isothermal titration calorimetry. We show that DHR51 binds NO and CO and discuss the possibility that DHR51 may be either a gas or a heme sensor.

Ultrafast heme-residue bond formation in six-coordinate heme proteins: implications for functional ligand exchange.

A survey is presented of picosecond kinetics of heme-residue bond formation after photolysis of histidine, methionine, or cysteine, in a broad range of ferrous six-coordinate heme proteins. These include human neuroglobin, a bacterial heme-binding superoxide dismutase (SOD), plant cytochrome b 559, the insect nuclear receptor E75, horse heart cytochrome c and the heme domain of the bacterial sensor protein Dos. We demonstrate that the fastest and dominant phase of binding of amino acid residues to domed heme invariably takes place with a time constant in the narrow range of 5-7 ps. Remarkably, this is also the case in the heme-binding SOD, where the heme is solvent-exposed. We reason that this fast phase corresponds to barrierless formation of the heme-residue bond from a configuration close to the bound state. Only in proteins where functional ligand exchange occurs, additional slower rebinding takes place on the time scale of tens of picoseconds after residue dissociation. We propose that the presence of these slower phases reflects flexibility in the heme environment that allows external ligands (O2, CO, NO, . . .) to functionally replace the internal residue after thermal dissociation of the heme-residue bond.

Drosophila nuclear receptor E75 is a thiolate hemoprotein.

Drosophila E75 is a member of the nuclear receptor superfamily. These eukaryotic transcription factors are involved in almost all physiological processes. They regulate transcription in response to binding of rigid hydrophobic hormone ligands. As it is the case for many nuclear receptors, the E75 hormone ligand was originally unknown. Recently, however, it was shown that the ligand binding domain (LBD) of E75 contains a tightly bound heme prosthetic group and is gas responsive. Here we have used site-directed mutagenesis along with UV-visible and electron paramagnetic resonance (EPR) spectroscopies to characterize and assign the heme iron axial ligands in E75. The F370Y mutation and addition of hemin to the growth medium during expression of the protein in Escherichia coli were necessary to produce good yields of heme-enriched E75 LBD. EPR studies revealed the presence of several species containing a strongly iron bound thiolate. The involvement of cysteines 396 and 468 in heme binding was subsequently shown by single and double mutations. Using a similar approach, we have also established that the sixth iron ligand of a well-defined coordination conformation, which accounts for approximately half of the total species, is histidine 574. The other iron coordination pairs are discussed. We conclude that E75 is a new example of a thiolate hemoprotein and that it may be involved in hormone synthesis regulation.

Crystal structure of a novel tetrameric complex of agonist-bound ligand-binding domain of Biomphalaria glabrata retinoid X receptor.

Nuclear receptors form an important class of transcription regulators in metazoans. To learn more about the evolution of these proteins, we have initiated structural studies on nuclear receptor ligand-binding domains from various animals. Here we present the crystal structure of the ligand-binding domain (LBD) of the retinoid X receptor (RXR) from the mollusc Biomphalaria glabrata. The structure reveals a novel tetrameric association in which each monomer is complexed to the human RXR ligand 9-cis retinoic acid and to a human co-activator-derived peptide. The ligand and the co-activator peptide are bound in essentially the same manner as observed in previously reported human RXR LBD structures, suggesting that the mechanisms of RXR-mediated transcription regulation are very similar in mollusc and human. The structure shows further that binding of ligand and co-activator peptide does not necessarily lead to the typical holo-conformation in which helix 12 (H12) folds back and packs against the LBD. Within a canonical dimer, only one monomer is in this closed agonist conformation. The other monomer is in an open conformation with H12 protruding from the LBD core, occupying the H12 interaction groove of another open monomer in an adjacent dimer in a domain swapping fashion, thus resulting in a tetrameric association. Additional tetramer interfaces are formed between H11 of the closed LBD and H6 of the open LBD. This novel holo-tetramer configuration may have a biological role in activating genes whose promoters are poorly recognised by dimers but much more efficiently by the corresponding tetramers.

Binding of the 2F5 monoclonal antibody to native and fusion-intermediate forms of human immunodeficiency virus type 1 gp41: implications for fusion-inducing conformational changes.

We investigated how the broadly neutralizing monoclonal antibody 2F5 affects the human immunodeficiency virus type 1 envelope glycoprotein as it undergoes receptor-induced conformational changes and show that 2F5 binds both native and fusion-intermediate conformations, suggesting inhibition of a late step in virus entry. We also demonstrate conformational changes in the C heptad of gp41.

Dissection of human immunodeficiency virus type 1 entry with neutralizing antibodies to gp41 fusion intermediates.

Human immunodeficiency virus type 1 (HIV-1) entry requires conformational changes in the transmembrane subunit (gp41) of the envelope glycoprotein (Env) involving transient fusion intermediates that contain exposed coiled-coil (prehairpin) and six-helix bundle structures. We investigated the HIV-1 entry mechanism and the potential of antibodies targeting fusion intermediates to block Env-mediated membrane fusion. Suboptimal temperature (31.5 degrees C) was used to prolong fusion intermediates as monitored by confocal microscopy. After transfer to 37 degrees C, these fusion intermediates progressed to syncytium formation with enhanced kinetics compared with effector-target (E/T) cell mixtures that were incubated only at 37 degrees C. gp41 peptides DP-178, DP-107, and IQN17 blocked fusion more efficiently (5- to 10-fold-lower 50% inhibitory dose values) when added to E/T cells at the suboptimal temperature prior to transfer to 37 degrees C. Rabbit antibodies against peptides modeling the N-heptad repeat or the six-helix bundle of gp41 blocked fusion and viral infection at 37 degrees C only if preincubated with E/T cells at the suboptimal temperature. Similar fusion inhibition was observed with human six-helix bundle-specific monoclonal antibodies. Our data demonstrate that antibodies targeting gp41 fusion intermediates are able to bind to gp41 and arrest fusion. They also indicate that six-helix bundles can form prior to fusion and that the lag time before fusion occurs may include the time needed to accumulate preformed six-helix bundles at the fusion site.