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.

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 Zn, it is not involved in zinc homeostasis or protection against zinc toxicity.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.