Intracellular free flavin and its associated enzymes participate in oxygen and iron metabolism in Amphibacillus xylanus lacking a respiratory chain.

Amphibacillus xylanus is a recently identified bacterium which grows well under both aerobic and anaerobic conditions and may prove useful for biomass utilization. Amphibacillus xylanus, despite lacking a respiratory chain, consumes oxygen at a similar rate to Escherichia coli (130-140 µmol oxygen·min-1·g-1 dry cells at 37 °C), suggesting that it has an alternative system that uses a large amount of oxygen. Amphibacillus xylanus NADH oxidase (Nox) was previously reported to rapidly reduce molecular oxygen content in the presence of exogenously added free flavin. Here, we established a quantitative method for determining the intracellular concentrations of free flavins in A. xylanus, involving French pressure and ultrafiltration membranes. The intracellular concentrations of flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and riboflavin were estimated to be approximately 8, 3, and 1 µm, respectively. In the presence of FAD, the predominant free flavin species, two flavoproteins Nox (which binds FAD) and NAD(P)H oxidoreductase (Npo, which binds FMN), were identified as central free flavin-associated enzymes in the oxygen metabolic pathway. Under 8 µm free FAD, the catalytic efficiency (kcat/Km) of recombinant Nox and Npo for oxygen increased by approximately fivefold and ninefold, respectively. Nox and Npo levels were increased, and intracellular FAD formation was stimulated following exposure of A. xylanus to oxygen. This suggests that these two enzymes and free FAD contribute to effective oxygen detoxification and NAD(P)+ regeneration to maintain redox balance during aerobic growth. Furthermore, A. xylanus required iron to grow aerobically. We also discuss the contribution of the free flavin-associated system to the process of iron utilization.

Evaluation of a flat-field grazing incidence spectrometer for highly charged ion plasma emission in soft x-ray spectral region from 1 to 10 nm.

A flat-field grazing incidence spectrometer operating on the spectral region from 1 to 10 nm was built for research on physics of high temperature and high energy density plasmas. It consists of a flat-field grating with 2400 lines/mm as a dispersing element and an x-ray charged coupled device (CCD) camera as the detector. The diffraction efficiency of the grating and the sensitivity of the CCD camera were directly measured by use of synchrotron radiation at the BL-11D beamline of the Photon Factory (PF). The influence of contamination to the spectrometer also was characterized. This result enables us to evaluate the absolute number of photons in a wide range wavelength between 1 and 10 nm within an acquisition. We obtained absolutely calibrated spectra from highly charged ion plasmas of Gd, from which a maximum energy conversion efficiency of 0.26% was observed at a Nd:YAG laser intensity of 3 × 1012 W/cm2.

MT1-MMP recognition by ERM proteins and its implication in CD44 shedding.

Membrane type 1-matrix metalloproteinase (MT1-MMP) is a key enzyme involved in tumor cell invasion by shedding their cell-surface receptor CD44 anchored with F-actin through ezrin/radixin/moesin (ERM) proteins. We found the cytoplasmic tail of MT1-MMP directly binds the FERM domain of radixin, suggesting F-actin-based recruitment of MT1-MMP to CD44 for invasion. Our crystal structure shows that the central region of the MT1-MMP cytoplasmic tail binds subdomain A of the FERM domain, and makes an antiparallel ß-ß interaction with ß2A-strand. This binding mode is distinct from the previously determined binding mode of CD44 to subdomain C. We showed that radixin simultaneously binds both MT1-MMP and CD44, indicating ERM protein-mediated colocalization of MT1-MMP and its substrate CD44 and anchoring to F-actin. Our study implies that ERM proteins contribute toward accelerated CD44 shedding by MT1-MMP through ERM protein-mediated interactions between their cytoplasmic tails.

Structural mechanisms of human RecQ helicases WRN and BLM.

The RecQ family DNA helicases Werner syndrome protein (WRN) and Bloom syndrome protein (BLM) play a key role in protecting the genome against deleterious changes. In humans, mutations in these proteins lead to rare genetic diseases associated with cancer predisposition and accelerated aging. WRN and BLM are distinguished from other helicases by possessing signature tandem domains toward the C terminus, referred to as the RecQ C-terminal (RQC) and helicase-and-ribonuclease D-C-terminal (HRDC) domains. Although the precise function of the HRDC domain remains unclear, the previous crystal structure of a WRN RQC-DNA complex visualized a central role for the RQC domain in recognizing, binding and unwinding DNA at branch points. In particular, a prominent hairpin structure (the ß-wing) within the RQC winged-helix motif acts as a scalpel to induce the unpairing of a Watson-Crick base pair at the DNA duplex terminus. A similar RQC-DNA interaction was also observed in the recent crystal structure of a BLM-DNA complex. I review the latest structures of WRN and BLM, and then provide a docking simulation of BLM with a Holliday junction. The model offers an explanation for the efficient branch migration activity of the RecQ family toward recombination and repair intermediates.

Structure of the RecQ C-terminal domain of human Bloom syndrome protein.

Bloom syndrome is a rare genetic disorder characterized by genomic instability and cancer predisposition. The disease is caused by mutations of the Bloom syndrome protein (BLM). Here we report the crystal structure of a RecQ C-terminal (RQC) domain from human BLM. The structure reveals three novel features of BLM RQC which distinguish it from the previous structures of the Werner syndrome protein (WRN) and RECQ1. First, BLM RQC lacks an aromatic residue at the tip of the ß-wing, a key element of the RecQ-family helicases used for DNA-strand separation. Second, a BLM-specific insertion between the N-terminal helices exhibits a looping-out structure that extends at right angles to the ß-wing. Deletion mutagenesis of this insertion interfered with binding to Holliday junction. Third, the C-terminal region of BLM RQC adopts an extended structure running along the domain surface, which may facilitate the spatial positioning of an HRDC domain in the full-length protein.

Redox-controlled, reversible rearrangement of a tris(2-pyridylthio)methyl ligand on nickel to an isomer with an "N,S-confused" 2-pyridylthiolate arm.

Interchange between the nickel +2 and +3 oxidation states precisely controls the reversible rearrangement of the tris(2-pyridylthio)methanide (tptm) ligand in the organometallic nickel(II) complex [{Ni(µ-Br)-(tptm)}(2)] (2). Oxidation of 2 first gives the corresponding Ni(III) complex [{Ni(µ-Br)(tptm)}(2)][PF(6)](2) (4). However, in solution the tptm ligand in 4 slowly undergoes a rearrangement, in which the N and S atoms of one of the pyridylthiolate arms exchange Ni and C bonding partners, thereby resulting in an "N,S-confused" isomer of tptm in the product, [NiBr(bpttpm)]PF(6) (5; bpttpm= bis(2-pyridylthio)(2-thiopyridinium)-methyl). Reduction of 5 reverses this ligand rearrangement and 2 is reformed quantitatively. The individual steps involved in these unusual ligand rearrangements were investigated by a number of methods, including voltammetric analysis, and a mechanism for this process is proposed. X-ray crystal structure determinations of the key compounds 2, 4 and 5 have been obtained.

Carbene-carbanion equilibrium for tris(2-pyridylthio)methanido Fe(II) complexes.

A reaction of FeCl(2) with tris(2-pyridylthio)methane (tptmH) produced the carbanion complex [Fe(tptm)(CH(3)CN)(2)](FeCl(4)){(C(2)H(5))(3)NH}. When FeI(2) was used instead of FeCl(2), the carbene complex [FeI(pyt)(bptmd)] (pyt = 2-pyridinethiolate, bptmd = bis(2-pyridylthio)methylidene) was isolated. The carbene forms [FeX(pyt)(bptmd)](n+) (n = 1 for X = CH(3)CN, n = 0 for X = I) were observed for [Fe(tptm)(CH(3)CN)(2)](FeCl(4)){(C(2)H(5))(3)NH} and [FeI(pyt)(bptmd)] in chloroform, whereas the carbene-carbanion equilibrium was observed in acetonitrile by NMR measurements. The thermodynamic parameters were evaluated by variable temperature (1)H NMR measurements using the diamagnetic salt [Fe(tptm)(CH(3)CN)(2)]PF(6) for [Fe(tptm)(CH(3)CN)(2)](+)⇆ [Fe(pyt)(bptmd)(CH(3)CN)](+) + CH(3)CN (ΔH = 23 kJ mol(-1), ΔS = 55 J mol(-1) K(-1)).

Solution structure of the HRDC domain of human Bloom syndrome protein BLM.

Bloom syndrome is a rare genetic disorder characterized by severe growth retardation and cancer predisposition. The disease is caused by a loss of function of the Bloom syndrome protein (BLM), a member of the RecQ family of DNA helicases. Here we report on the first 3D structure of a BLM fragment, a solution structure of the C-terminal helicase-and-ribonuclease D-C-terminal (HRDC) domain from human BLM. The structure reveals unique features of BLM HRDC that are distinct from the HRDC domain of Werner syndrome protein. In particular, BLM HRDC retains many acidic residues exposed to the solvent, which makes the domain surface extensively electronegative. Consistent with this, fluorescence polarization assays showed an inability of isolated BLM HRDC to interact with DNA substrates. Analyses employing ultracentrifugation, gel-filtration, CD spectroscopy and dynamic light scattering showed that the BLM HRDC domain exists as a stable monomer in solution. The results show that BLM HRDC is a compact, robust and acidic motif which may play a distinct role apart from DNA binding.

Structural basis for the specific inhibition of heterotrimeric Gq protein by a small molecule.

Heterotrimeric GTP-binding proteins (G proteins) transmit extracellular stimuli perceived by G protein-coupled receptors (GPCRs) to intracellular signaling cascades. Hundreds of GPCRs exist in humans and are the targets of a large percentage of the pharmaceutical drugs used today. Because G proteins are regulated by GPCRs, small molecules that directly modulate G proteins have the potential to become therapeutic agents. However, strategies to develop modulators have been hampered by a lack of structural knowledge of targeting sites for specific modulator binding. Here we present the mechanism of action of the cyclic depsipeptide YM-254890, which is a recently discovered Gq-selective inhibitor. YM-254890 specifically inhibits the GDP/GTP exchange reaction of alpha subunit of Gq protein (Galphaq) by inhibiting the GDP release from Galphaq. X-ray crystal structure analysis of the Galphaqbetagamma-YM-254890 complex shows that YM-254890 binds the hydrophobic cleft between two interdomain linkers connecting the GTPase and helical domains of the Galphaq. The binding stabilizes an inactive GDP-bound form through direct interactions with switch I and impairs the linker flexibility. Our studies provide a novel targeting site for the development of small molecules that selectively inhibit each Galpha subunit and an insight into the molecular mechanism of G protein activation.

Structural basis for DNA strand separation by the unconventional winged-helix domain of RecQ helicase WRN.

The RecQ family of DNA helicases including WRN (Werner syndrome protein) and BLM (Bloom syndrome protein) protects the genome against deleterious changes. Here we report the cocrystal structure of the RecQ C-terminal (RQC) domain of human WRN bound to a DNA duplex. In the complex, the RQC domain specifically interacted with a blunt end of the duplex and, surprisingly, unpaired a Watson-Crick base pair in the absence of an ATPase domain. The beta wing, an extended hairpin motif that is characteristic of winged-helix motifs, was used as a "separating knife" to wedge between the first and second base pairs, whereas the recognition helix, a principal component of helix-turn-helix motifs that are usually embedded within DNA grooves, was unprecedentedly excluded from the interaction. Our results demonstrate a function of the winged-helix motif central to the helicase reaction, establishing the first structural paradigm concerning the DNA structure-specific activities of the RecQ helicases.

The PHCCEx domain of Tiam1/2 is a novel protein- and membrane-binding module.

Tiam1 and Tiam2 (Tiam1/2) are guanine nucleotide-exchange factors that possess the PH-CC-Ex (pleckstrin homology, coiled coil and extra) region that mediates binding to plasma membranes and signalling proteins in the activation of Rac GTPases. Crystal structures of the PH-CC-Ex regions revealed a single globular domain, PHCCEx domain, comprising a conventional PH subdomain associated with an antiparallel coiled coil of CC subdomain and a novel three-helical globular Ex subdomain. The PH subdomain resembles the beta-spectrin PH domain, suggesting non-canonical phosphatidylinositol binding. Mutational and binding studies indicated that CC and Ex subdomains form a positively charged surface for protein binding. We identified two unique acidic sequence motifs in Tiam1/2-interacting proteins for binding to PHCCEx domain, Motif-I in CD44 and ephrinB's and the NMDA receptor, and Motif-II in Par3 and JIP2. Our results suggest the molecular basis by which the Tiam1/2 PHCCEx domain facilitates dual binding to membranes and signalling proteins.

Crystallographic characterization of the membrane-targeting domains of the Rac-specific guanine nucleotide-exchange factors Tiam1 and Tiam2.

T-lymphoma invasion and metastasis 1 and 2 (Tiam1 and Tiam2) are guanine nucleotide-exchange factors that specifically activate Rac GTPase by facilitating the dissociation of GDP. Translocation of Tiam1 and Tiam2 from the cytoplasm to the plasma membrane is an essential step in Rac activation and is mediated by the conserved PH-CC-Ex (pleckstrin-homology, coiled-coil and extra region) region in the N-terminal region. Here, the purification, crystallization and X-ray data collection of the Tiam1 and Tiam2 PH-CC-Ex regions are reported. The regions are shown to exist as a monomer in solution as a folded globular domain. The Tiam2 PH-CC-Ex domain crystallizes in space group P4(1)2(1)2 or P4(3)2(1)2 with four molecules in the asymmetric unit. An X-ray diffraction data set has been collected to 3.2 A resolution.

Crystallographic characterization of the radixin FERM domain bound to the cytoplasmic tail of membrane-type 1 matrix metalloproteinase (MT1-MMP).

ERM proteins play a role in the cross-linking found between plasma membranes and actin filaments. The N-terminal FERM domains of ERM proteins are responsible for membrane association through direct interaction with the cytoplasmic tails of integral membrane proteins. During cell migration and movement, membrane-type 1 matrix metalloproteinase (MT1-MMP) on plasma membranes sheds adhesion molecule CD44 in addition to degrading the extracellular matrix. Here, the interaction between the radixin FERM domain and the MT1-MMP cytoplasmic tail is reported and preliminary crystallographic characterization of crystals of the radixin FERM domain bound to the cytoplasmic tail of MT1-MMP is presented. The crystals belong to space group P6(1)22, with unit-cell parameters a = b = 122.7, c = 128.3 A, and contain one complex in the crystallographic asymmetric unit. The diffraction data were collected to a resolution of 2.4 A.

Structural basis for CD44 recognition by ERM proteins.

CD44 is an important adhesion molecule that functions as the major hyaluronan receptor which mediates cell adhesion and migration in a variety of physiological and pathological processes. Although full activity of CD44 requires binding to ERM (ezrin/radixin/moesin) proteins, the CD44 cytoplasmic region, consisting of 72 amino acid residues, lacks the Motif-1 consensus sequence for ERM binding found in intercellular adhesion molecule (ICAM)-2 and other adhesion molecules of the immunoglobulin superfamily. Ultracentrifugation sedimentation studies and circular dichroism measurements revealed an extended monomeric form of the cytoplasmic peptide in solution. The crystal structure of the radixin FERM domain complexed with a CD44 cytoplasmic peptide reveals that the KKKLVIN sequence of the peptide forms a beta strand followed by a short loop structure that binds subdomain C of the FERM domain. Like Motif-1 binding, the CD44 beta strand binds the shallow groove between strand beta5C and helix alpha1C and augments the beta sheet beta5C-beta7C from subdomain C. Two hydrophobic CD44 residues, Leu and Ile, are docked into a hydrophobic pocket with the formation of hydrogen bonds between Asn of the CD44 short loop and loop beta4C-beta5C from subdomain C. This binding mode resembles that of NEP (neutral endopeptidase 24.11) rather than ICAM-2. Our results reveal a characteristic versatility of peptide recognition by the FERM domains from ERM proteins, suggest a possible mechanism by which the CD44 tail is released from the cytoskeleton for nuclear translocation by regulated intramembrane proteolysis, and provide a structural basis for Smad1 interactions with activated CD44 bound to ERM protein.

Structural basis of the cytoplasmic tail of adhesion molecule CD43 and its binding to ERM proteins.

CD43/leukosialin/sialophorin is the major adhesion molecule in most hematopoietic cells and belongs to the sialomucin superfamily. In leukocyte emigration and activation, the exclusion of CD43 from the immunological synapse is an essential step. While the exclusion requires binding of the cytoplasmic region to ERM (ezrin/radixin/moesin) proteins, the detailed specific nature of the interaction between CD43 and ERM proteins is obscure. We have characterized the conformational properties of the CD43 cytoplasmic region, consisting of 124 amino acid residues, by hydrodynamic and spectroscopic measurements. Sedimentation equilibrium and velocity studies of ultracentrifugation revealed that the CD43 cytoplasmic peptide exists in a monomeric and extended form in solution. The crystal structure of the complex between the radixin FERM (4.1 and ERM) domain and the CD43 juxtamembrane region peptide reveals that the nonpolar region of the peptide binds subdomain C of the FERM domain. CD43 lacks the Motif-1 sequence for FERM binding found in the FERM-intercellular adhesion molecule-2 complex but possesses two conserved leucine residues that dock into the hydrophobic pocket of subdomain C without forming a 3(10)-helix. The FERM-binding site on CD43 is overlapped with the functional nuclear localization signal sequence. Our structure suggests that regulation of ERM binding may be coupled with regulated intramembrane proteolysis of CD43 followed by the nuclear transfer of the cytoplasmic peptide.

Synthesis and characterization of tris(2-pyridylthio)methanido Zn complex with a Zn-C bond and DFT calculation of its one-electron oxidized species.

Tris(2-pyridylthio)methane (tptmH) reacts with ZnCl(2) producing the Zn-C containing complex of [ZnCl(tptm)], whose cyclic voltammogram shows an irreversible oxidation peak at 0.2 V vs. E(0')(Fc(+/0)). DFT calculations suggested that 1e(-) oxidation should occur at the tptm ligand resulting in the cleavage of the Zn-C bond, leading to decomposition of the complex.

Crystallization and preliminary crystallographic analysis of the catalytic domain of human flap endonuclease 1 in complex with a nicked DNA product: use of a DPCS kit for efficient protein-DNA complex crystallization.

Flap endonuclease 1 (FEN1) is a structure-specific nuclease that removes the RNA/DNA primer associated with Okazaki fragments in DNA replication. Here, crystals of the complex between the catalytic domain of human FEN1 and a DNA product have been obtained. For efficient crystallization screening, a DNA-protein complex crystallization screening (DPCS) kit was designed based on commercial crystallization kits. The crystal was found to belong to space group P2(1), with unit-cell parameters a = 61.0, b = 101.3, c = 106.4 A, beta = 106.4 degrees. The asymmetric unit is predicted to contain two complexes in the crystallographic asymmetric unit. A diffraction data set was collected to a resolution of 2.75 A.

Structural basis of PSGL-1 binding to ERM proteins.

P-selectin glycoprotein ligand-1 (PSGL-1), an adhesion molecule with O-glycosylated extracellular sialomucins, is involved in leukocyte inflammatory responses. On activation, ezrin-radixin-moesin (ERM) proteins mediate the redistribution of PSGL-1 on polarized cell surfaces to facilitate binding to target molecules. ERM proteins recognize a short binding motif, Motif-1, conserved in cytoplasmic tails of adhesion molecules, whereas PSGL-1 lacks Motif-1 residues important for binding to ERM proteins. The crystal structure of the complex between the radixin FERM domain and a PSGL-1 juxtamembrane peptide reveals that the peptide binds the groove of FERM subdomain C by forming a beta-strand associated with strand beta5C, followed by a loop flipped out towards the solvent. The Motif-1 3(10) helix present in the FERM-ICAM-2 complex is absent in PSGL-1 given the absence of a critical Motif-1 alanine residue, and PSGL-1 reduces its contact area with subdomain C. Non-conserved positions are occupied by large residues Met9 and His8, which stabilize peptide conformation and enhance groove binding. Non-conserved residues play an important role in compensating for loss of binding energy resulting from the absence of conserved residues important for binding.

Crystallographic characterization of the radixin FERM domain bound to the cytoplasmic tail of adhesion molecule CD44.

CD44 is an important adhesion molecule that specifically binds hyaluronic acid and regulates cell-cell and cell-matrix interactions. Increasing evidence has indicated that CD44 is assembled in a regulated manner into the membrane-cytoskeletal junction, a process that is mediated by ERM (ezrin/radixin/moesin) proteins. Crystals of a complex between the radixin FERM domain and the C-terminal cytoplasmic region of CD44 have been obtained. The crystal of the radixin FERM domain bound to the CD44 cytoplasmic tail peptide belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 62.70, b = 66.18, c = 86.22 A, and contain one complex in the crystallographic asymmetric unit. An intensity data set was collected to a resolution of 2.1 A.