Chlamydomonas reinhardtii centrin is a member of the EF-hand calcium-binding superfamily. It is found in the basal body complex and is important for flagellar motility. Like other members of the EF-hand family, centrin interacts with and modulates the function of other proteins in a calcium-dependent manner. To understand how C. reinhardtii centrin interacts with its protein targets, it has been crystallized in the presence of the model peptide melittin and X-ray diffraction data have been collected to 2.2 A resolution. The crystals are orthorhombic, with unit-cell parameters a = 52.1, b = 114.4, c = 34.8 A, and are likely to belong to space group P2(1)2(1)2.

In the presence of excess nickel, Escherichia coli NikR regulates cellular nickel uptake by suppressing the transcription of the nik operon, which encodes the nickel uptake transporter, NikABCDE. Previously published in vitro studies have shown that NikR is capable of binding a range of divalent transition metal ions in addition to Ni2+, including Co2+, Cu2+, Zn2+, and Cd2+. To understand how the high-affinity nickel binding site of NikR is able to accommodate these other metal ions, and to improve our understanding of NikR's mechanism of binding to DNA, we have determined structures of the metal-binding domain (MBD) of NikR in the apo form and in complex with Cu2+ and Zn2+ ions and compared them with the previously published structures with Ni2+. We observe that Cu2+ ions bind in a manner very similar to that of Ni2+, with a square planar geometry but with longer bond lengths. Crystals grown in the presence of Zn2+ reveal a protein structure similar to that of apo MBD with a disordered alpha3 helix, but with two electron density peaks near the Ni2+ binding site corresponding to two Zn2+ ions. These structural findings along with biochemical data on NikR support a hypothesis that ordering of the alpha3 helix is important for repressor activation.

The ribbon-helix-helix (RHH) superfamily of transcription factors uses a conserved three-dimensional structural motif to bind to DNA in a sequence-specific manner. This functionally diverse protein superfamily regulates the transcription of genes that are involved in the uptake of metals, amino-acid biosynthesis, cell division, the control of plasmid copy number, the lytic cycle of bacteriophages and, perhaps, many other cellular processes. In this Analysis, the structures of different RHH transcription factors are compared in order to evaluate the sequence motifs that are required for RHH-domain folding and DNA binding, as well as to identify conserved protein-DNA interactions in this superfamily.

S-nitrosylation is a post-translational protein modification that can alter the function of a variety of proteins. Despite the growing wealth of information that this modification may have important functional consequences, little is known about the structure of the moiety or its effect on protein tertiary structure. Here we report high-resolution x-ray crystal structures of S-nitrosylated and unmodified blackfin tuna myoglobin, which demonstrate that in vitro S-nitrosylation of this protein at the surface-exposed Cys-10 directly causes a reversible conformational change by "wedging" apart a helix and loop. Furthermore, we have demonstrated in solution and in a single crystal that reduction of the S-nitrosylated myoglobin with dithionite results in NO cleavage from the sulfur of Cys-10 and rebinding to the reduced heme iron, showing the reversibility of both the modification and the conformational changes. Finally, we report the 0.95-A structure of ferrous nitrosyl myoglobin, which provides an accurate structural view of the NO coordination geometry in the context of a globin heme pocket.

ST2 is an IL-1 receptor family member with transmembrane (ST2L) and soluble (sST2) isoforms. sST2 is a mechanically induced cardiomyocyte protein, and serum sST2 levels predict outcome in patients with acute myocardial infarction or chronic heart failure. Recently, IL-33 was identified as a functional ligand of ST2L, allowing exploration of the role of ST2 in myocardium. We found that IL-33 was a biomechanically induced protein predominantly synthesized by cardiac fibroblasts. IL-33 markedly antagonized angiotensin II- and phenylephrine-induced cardiomyocyte hypertrophy. Although IL-33 activated NF-kappaB, it inhibited angiotensin II- and phenylephrine-induced phosphorylation of inhibitor of NF-kappa B alpha (I kappa B alpha) and NF-kappaB nuclear binding activity. sST2 blocked antihypertrophic effects of IL-33, indicating that sST2 functions in myocardium as a soluble decoy receptor. Following pressure overload by transverse aortic constriction (TAC), ST2(-/-) mice had more left ventricular hypertrophy, more chamber dilation, reduced fractional shortening, more fibrosis, and impaired survival compared with WT littermates. Furthermore, recombinant IL-33 treatment reduced hypertrophy and fibrosis and improved survival after TAC in WT mice, but not in ST2(-/-) littermates. Thus, IL-33/ST2 signaling is a mechanically activated, cardioprotective fibroblast-cardiomyocyte paracrine system, which we believe to be novel. IL-33 may have therapeutic potential for beneficially regulating the myocardial response to overload.

The thioredoxin system helps maintain a reducing environment in cells, but thioredoxin functions as more than simply an antioxidant. Thioredoxin functions depend on the protein's redox state, as determined by two conserved cysteines. Key biologic activities of thioredoxin include antioxidant, growth control, and antiapoptotic properties, resulting from interaction with target molecules including transcription factors. Mechanisms by which thioredoxin regulates cell growth include binding to signaling molecules such as apoptosis signal-regulating kinase-1 (ASK-1) and thioredoxin-interacting protein (Txnip). The molecular interplay between thioredoxin, ASK-1, and Txnip potentially influences cell growth and survival in diverse human diseases such as cancer, diabetes, and heart disease. In this review, we focus on the structure of thioredoxin and its functional regulation of cell growth through the interactions with signaling molecules.

Metal ion homeostasis is critical to the survival of all cells. Regulation of nickel concentrations in Escherichia coli is mediated by the NikR repressor via nickel-induced transcriptional repression of the nickel ABC-type transporter, NikABCDE. Here, we report two crystal structures of nickel-activated E. coli NikR, the isolated repressor at 2.1 A resolution and in a complex with its operator DNA sequence from the nik promoter at 3.1 A resolution. Along with the previously published structure of apo-NikR, these structures allow us to evaluate functional proposals for how metal ions activate NikR, delineate the drastic conformational changes required for operator recognition, and describe the formation of a second metal-binding site in the presence of DNA. They also provide a rare set of structural views of a ligand-responsive transcription factor in the unbound, ligand-induced, and DNA-bound states, establishing a model system for the study of ligand-mediated effects on transcription factor function.

NikR is a metal-responsive transcription factor that controls nickel uptake in Escherichia coli by regulating expression of a nickel-specific ATP-binding cassette (ABC) transporter. We have determined the first two structures of NikR: the full-length apo repressor at a resolution of 2.3 A and the nickel-bound C-terminal regulatory domain at a resolution of 1.4 A. NikR is the only known metal-responsive member of the ribbon-helix-helix family of transcription factors, and its structure has a quaternary arrangement consisting of two dimeric DNA-binding domains separated by a tetrameric regulatory domain that binds nickel. The position of the C-terminal regulatory domain enforces a large spacing between the contacts that each NikR DNA-binding domain can make with the nik operator. The regulatory domain of NikR contains four nickel-binding sites at the tetramer interface, each exhibiting a novel square-planar coordination by three histidines and one cysteine side chain.

A crystal structure of the anaerobic Ni-Fe-S carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum has been determined to 2.8-Å resolution. The CODH family, for which the R. rubrum enzyme is the prototype, catalyzes the biological oxidation of CO at an unusual Ni-Fe-S cluster called the C-cluster. The Ni-Fe-S C-cluster contains a mononuclear site and a four-metal cubane. Surprisingly, anomalous dispersion data suggest that the mononuclear site contains Fe and not Ni, and the four-metal cubane has the form [NiFe3S4] and not [Fe4S4]. The mononuclear site and the four-metal cluster are bridged by means of Cys531 and one of the sulfides of the cube. CODH is organized as a dimer with a previously unidentified [Fe4S4] cluster bridging the two subunits. Each monomer is comprised of three domains: a helical domain at the N terminus, an α/β (Rossmann-like) domain in the middle, and an α/β (Rossmann-like) domain at the C terminus. The helical domain contributes ligands to the bridging [Fe4S4] cluster and another [Fe4S4] cluster, the B-cluster, which is involved in electron transfer. The two Rossmann domains contribute ligands to the active site C-cluster. This x-ray structure provides insight into the mechanism of biological CO oxidation and has broader significance for the roles of Ni and Fe in biological systems.

We have prepared ionic liquids by mixing either iron(II) chloride or iron(III) chloride with 1-butyl-3-methylimidazolium chloride (BMIC). Iron(II) chloride forms ionic liquids from a mole ratio of 1 FeCl(2)/3 BMIC to almost 1 FeCl(2)/1 BMIC. Both Raman scattering and ab initio calculations indicate that FeCl(4)(2-) is the predominant iron-containing species in these liquids. Iron(III) chloride forms ionic liquids from a mole ratio of 1 FeCl(3)/1.9 BMIC to 1.7 FeCl(3)/1 BMIC. When BMIC is in excess, Raman scattering indicates the presence of FeCl(4-). When FeCl(3) is in excess, Fe(2)Cl(7-) begins to appear and the amount of Fe(2)Cl(7-) increases with increasing amounts of FeCl(3). Ionic liquids were also prepared from a mixture of FeCl(2) and FeCl(3) and are discussed. Finally, we have used both Hartree-Fock and density functional theory methods to compute the optimized structures and vibrational spectra for these species. An analysis of the results using an all-electron basis set, 6-31G, as well as two different effective core potential basis sets, LANL2DZ and CEP-31G is presented.