324,418 research outputs found
The PilB-PilZ-FimX complex in the context of the inner membrane platform of the Type IV pilus.
No full textA) Model for interactions between the PilBND0/ND1-PilZ-FimXEAL complex and the PilM-PilNN-terminal complex. PilBND0/ND1 (green) and PilZ (yellow) are shown as determined in the PilB12-163-PilZ structure (present study). FimXEAL (blue) is placed as observed in interface 2 of the X. citri PilZΔ107-117-FimXGGDEF-EAL (this study), X. citri PilZ-FimXEAL [24] and the X. campestris pv. campestris PilZ-FimXEAL [30] complexes (see Fig 3). The homology model for X. citri PilM (see Materials and Methods) was oriented with respect to PilBND0/ND1 based on the V. vulnificus GspE-GspL [52] and V. cholerae EpsE-EpsL [57] crystal structures (see Figs 2E and S13). B) The PilBND0/ND1 domain is connected to the hexameric PilB core (made up of ND2 and ATPase domains) via a highly acidic and glycine-rich linker (approximately 30 residues; see Fig 2A). The homology model of the X. citri PilB hexameric core was built based on the G. metallireducens PilB core structure as described in Materials and Methods. C) Depiction of possible interactions of the PilB-PilZ-FimXEALcomplex with the proposed PilM-PilN dodecamer and PilC dimer based on the cryo-electron tomography model of the M. xanthus T4P [9]. The homology model of the PilC dimer (depicted in orange) was built as described in Materials and Methods. Coloring scheme for the other subunits is the same as in part A. Top and side views are shown. Note that the REC, PAS and GGDEF domains of FimX are not shown and that X. citri (this study) and P. aeruginosa FimX is a homodimer, probably due to interactions between N-terminal REC domains [29]. Here the model assumes a PilB-PilZ-FimX stoichiometry of 6:6:6. However, since the PilB hexamer may exhibit C2 symmetry, stoichiometries of 6:6:4 or 6:6:2 are also possible (see main text).</p
Residues important for PilB<sub>1-190</sub>-PilZ-FimX<sub>GGDEF-EAL</sub> ternary complex stability and T4P function.
No full textA) Size exclusion chromatography (Superdex 200 resin, 10/300 column) analysis of PilB1-190-PilZ-FimXGGDEF-EAL ternary complexes containing PilZ or PilB mutants. A set of mutants: PilZF28A, PilZD46A/E47A, PilZI10E, PilZW69A, PilZF49A/L51A, PilZF49E/L51E, PilZM117G, PilZΔM117, PilZΔ107–117 and PilB1-190_F101A/F108A (indicated in each chromatogram panel) were used to test their effect on ternary complex stability. In all the cases, a 1:1:1 PilB1-190:PilZ:FimXGGDEF-EAL molar ratio was used and the elution profile of the mixture is shown as a black line. All the mutants, except PilZF28A and PilZD46A/E47A, affect the stability of the FimXGGDEF-EAL-PilZ-PilB1-190 ternary complex. In all of the panels, the elution volume for the ternary complex is delimited by the vertical red broken lines. In the first panel, the elution profiles for wild type complex (black line) is shown together with the profiles for the three individual components FimXGGDEF-EAL (red line), PilB1-190 (blue line) and PilZ (green line). Each experiment was performed at least three times and representative results are shown. B) Top row: Fluorescence microscopy images of the edges of the twitching zones at the interstitial surface between the agar medium and the glass base of the microscopy chamber. For visualization, X. citri strains were transformed with the pBBR2-GFP plasmid. Wild-type cells, that are able to twitch, can separate from the main body of the colony and migrate on their own or in small groups, producing a rough, less organized boundary between the dense interior of the colony and the surrounding medium. Cells with mutations that compromise T4P function are not able to separate from the main body of the colony and so the colony border is much smoother and well-defined. Bottom row: Phage ΦXacm4-11 infection assays. Dark plaques are indicative of phage-induced bacterial lysis in a confluent culture background. For both twitching motility and bacteriophage infection assays: X. citri wild type (Xac_Wt), Xac_ΔpilZXAC1133 (ΔpilZ) and Xac_ΔpilZXAC1133 complemented with a plasmid (pURF047) directing the expression of the wild-type PilZ protein (ΔpilZc) or one of the following PilZ mutants: PilZI10E, PilZF49E, PilZF49E/L51E, PilZF49A/L51A,and PilZΔM117. S14 Fig shows that PilZI10E, PilZF49E, PilZF49E/L51E, PilZF49A/L51A, PilZΔM117 are all detected with anti-PilZ antibodies in these strains.</p
Structural biology of bacterial response regulator proteins and their complexes with cognate ligands
No full textTwo bacterial response regulator systems were studied in this thesis. NMR
spectroscopy and X-ray crystallography were used to determine the structures of both
domains of the antibiotic sensor TipAL, as well as of the c-di-GMP receptor PA4608,
in complex with cognate ligands. By comparison with structures of the free proteins,
we found that ligand binding induced biologically relevant structural rearrangements
in all proteins studied.
The putative multidrug resistance gene tipA of the soil bacterium Streptomyces
lividans codes for the transcription factor TipAL, which comprises two domains: a
DNA-binding domain named TipAN, and a ligand-binding domain named TipAS,
which is capable of recognizing and binding a diverse group of macrocyclic
thiopeptide antibiotics. After antibiotic binding, TipAL binds to promoter DNA and
activates transcription. In order to elucidate the specificity and flexibility of antibiotic
recognition as well as the mechanism of transcriptional activation, the two domains of
TipAL were studied separately.
We have determined the structures of TipAS with bound promothiocin A or
nosiheptide antibiotics by NMR spectroscopy. The N-terminal part of the TipAS
sequence, which is flexible and unstructured in free TipAS, forms three new helices in
both complexes, burying the bound antibiotics. Considering that the newly formed
helices form the connection between the TipAS and TipAN domains, we propose that
the formation of additional secondary structure forms the basis of transcriptional
activation by TipAL after ligand binding. The TipAS complexes with promothiocin A
and nosiheptide are similar, but differ in the dynamics of the newly formed helices;
the smaller ligand, promothiocin A, appears to leave more room for movement of
TipAS.
The structure of TipAN in complex with a fragment of tipA promoter DNA was
solved by X-ray crystallography. TipAN binds to the symmetric promoter as a dimer,
which is held together by a long, antiparallel coiled coil. In contrast to homologous
proteins, TipAN does not bend and twist bound DNA, which is a prerequisite for
transcriptional activation by other proteins of the same family. This indicates that the
activated TipAS domain is required for transcriptional activation by TipAL.
C-di-GMP is a second messenger molecule that appears to be ubiquitous in, and
unique to, the bacterial kingdom. It generally controls the switch from motile, singlecell
lifestyles to surface-attached, multicellular communities such as biofilms. The
natural receptors of c-di-GMP are the PilZ domain proteins, which include PA4608 in
Pseudomonas aeruginosa. We have solved the NMR solution structure of PA4608 in
complex with c-di-GMP. C-di-GMP binds to the protein as an intercalated dimer, displacing the C-terminal 310 helix found in the apo form. The N-terminal part of
PA4608, which contains the highly conserved RxxxR motif and which is flexible and
unstructured before ligand binding, contacts the distal side of c-di-GMP
Superposition of PilB<sub>12-163</sub>-PilZ and PilZ-FimX<sub>EAL</sub> structures.
No full textA)Left: Cartoon representations of the PilZ-FimXEAL-c-di-GMP (FimXEAL colored in blue, PilZ colored in orange and c-di-GMP (stick model) colored in red) and PilB12-163-PilZ (PilB12-163 colored in green and PilZ colored in yellow) complexes. Right: Superposition of PilB12-163-PilZ and PilZ-FimXEAL complexes using PilZ as reference. B) Structural alignment of the PilZ structures from A showing the common interface residues in both complexes as sticks. Note that in this figure, the interaction interface (interface 1) between FimXEAL and PilZ is as described previously[24]. An alternative mode of interaction (interface 2) is proposed and tested as described in the main text and detailed in Figs S6, 2F and 4. (TIF)</p
Modulation of the conductance of a 2,2′-bipyridine-functionalized peptidic ion channel by Ni
No full textAn alpha-helical amphipathic peptide with the sequence H(2)N-(LSSLLSL)(3)-CONH(2) was obtained by solid phase synthesis and a 2,2'-bipyridine was coupled to its N-terminus, which allows complexation of Ni(2+). Complexation of the 2,2'-bipyridine residues was proven by UV/Vis spectroscopy. The peptide helices were inserted into lipid bilayers (nano black lipid membranes, nano-BLMs) that suspend the pores of porous alumina substrates with a pore diameter of 60 nm by applying a potential difference. From single channel recordings, we were able to distinguish four distinct conductance states, which we attribute to an increasing number of peptide helices participating in the conducting helix bundle. Addition of Ni(2+) in micromolar concentrations altered the conductance behaviour of the formed ion channels in nano-BLMs considerably. The first two conductance states appear much more prominent demonstrating that the complexation of bipyridine by Ni(2+) results in a considerable confinement of the observed multiple conductance states. However, the conductance levels were independent of the presence of Ni(2+). Moreover, from a detailed analysis of the open lifetimes of the channels, we conclude that the complexation of Ni(2+) diminishes the frequency of channel events with larger open times
Size exclusion chromatography of PilZ-FimXGGDEF-EAL and PilB1-190-PilZ binary complexes.
No full textSize exclusion chromatography (Superdex 200, 10/300 column) analysis of interactions of PilZ mutants (PilZF49E/L51E, PilZI10E and PilZD46A/E47A) with FimXGGDEF-EAL (A) and PilB1-190 (B). In each chromatogram, the elution profile of the PilZ–FimXGGDEF-EAL (1.5:1molar ratio, black line) (A) and PilB1-190 –PilZ (1:1 molar ratio, black line) mixtures (B) are shown on the left and the SDS-PAGE analysis of representative fractions are shown on the right. Where indicated, c-di-GMP was added to the PilZ–FimXGGDEF-EAL mixture (2-fold excess of c-di-GMP to FimXGGDEF-EAL (continuous red line in A)). Note that the addition of c-di-GMP to the PilZwt (wild type PilZ)–FimXGGDEF-EAL and PilZD46A/E47A –FimXGGDEF-EAL mixture results in a shift in its elution profile. The elution profiles for FimXGGDEF-EAL alone is shown in blue in A. The elution profiles for PilB1-190 and PilZ mutants on their own are shown in blue and red respectively in B. In these experiments, FimXGGDEF-EAL and PilB1-190 have N-terminal 6xHis-tags. Each experiment was performed at least three times and representative results are shown. (TIF)</p
Evaluation of proxies for seismic site conditions in large urban areas: The example of Santiago de Chile.
No full textCharacterizing the local site response in large cities is an important step towards seismic hazard assessment. To this regard, single station seismic noise measurements were carried out at 146 sites in the northern part of Santiago de Chile. This extensive survey allowed the fundamental resonance frequency of the sedimentary cover, derived from horizontal-to-vertical (H/V) spectral ratios, to be mapped. By inverting the spectral ratios under the constraint of the thickness of the sedimentary cover, known from previous gravimetric measurements, local S-wave velocity profiles have been retrieved. After interpolation between the individual profiles, the resulting high resolution 3D S-wave velocity model allows the entire area, as well as deeper parts of the basin, to be represented in great detail. Since one lithology shows a great scatter in the velocity values only a very general correlation between S-wave velocity in the uppermost 30 m (View the MathML source
) and local geology is found. Local S-wave velocity profiles can serve as a key factor in seismic hazard assessment, since they allow an estimate of the amplification potential of the sedimentary cover. Mapping the intensity distribution of the 27 February 2010 Maule, Chile, event (Mw = 8.8) the results indicate that local amplification of the ground motion might partially explain the damage distribution and encourage the use of the low cost seismic noise techniques for the study of seismic site effects
Entwicklung und Herstellung pilz- und termitenfester Holzspanplatten
No full textS.185-189Die verfahrenstechnischen Möglichkeiten zur Herstellung von pilz-und termitenfesten Holzspanplatten unter Verwendung von Pentachlorphenol als Schutzmittel werden dargelegt. Durch Einbringung von rund 1,5% Pentachlorphenol als wässerige Lösung des Natriumsalzes in Holzspanplatten konnten ausreichend pilzfeste Erzeugnisse gewonnen werden. Der Suspendierung des freien Pentachlorphenols in den wässerig-kolloiden Kunstharz-Bindemittellösungen wird yerfahrenstechnisch der Vorzug gegeben. Nach dieser Arbeitsweise wurden bei einem Zusatz von 1,5% Pentachlorphenol unter Verwendung von Harnstoff-Formaldehydkunstharzen als Bindemittel pilz-und termitenfeste Platten erhalten.12Nr.
Expression, crystallization and preliminary crystallographic analysis of PilZ(XAC1133) from Xanthomonas axonopodis pv. citri
No full textProteins containing PilZ domains are widespread in Gram-negative bacteria and have recently been shown to be involved in the control of biofilm formation, adherence, aggregation, virulence-factor production and motility. Furthermore, some PilZ domains have recently been shown to bind the second messenger bis(3'-> 5') cyclic diGMP. Here, the cloning, expression, purification and crystallization of PilZ(XAC1133), a protein consisting of a single PilZ domain from Xanthomonas axonopodis pv. citri, is reported. The closest PilZ(XAC1133) homologues in Pseudomonas aeruginosa and Neisseria meningitidis control type IV pilus function. Recombinant PilZ(XAC1133) containing selenomethionine was crystallized in space group P6(1). The unit-cell parameters were a = 62.125, b = 62.125, c = 83.543 angstrom. These crystals diffracted to 1.85 angstrom resolution and a MAD data set was collected at a synchrotron source. The calculated Matthews coefficient suggested the presence of two PilZ(XAC1133) molecules in the asymmetric unit
Crystal structure of the PilB<sub>12-163</sub>-PilZ complex.
No full textA) Cartoon representation of the asymmetric unit of the PilB12-163-PilZ crystal that contains two copies of each subunit. PilB12-163 chains are colored in green (chain A) and blue (chain B) and PilZ chains are colored in yellow (chain C) and magenta (chain D). B) Sub-domains and secondary structure elements of PilB12-163. Left: The ND0 sub-domain is shown in orange and the ND1 sub-domain is shown in green. Right: Topology diagram for PilB12-163. C) Surface representation of X. citri PilZ with the conserved motifs MI-MV in the PA2960/XAC1133 orthologous group colored, as described in Guzzo et al. (2009). D) Superposition of X. citri PilZ crystal structures. PilZ crystal structure on its own (PDB: 3CNR), PilZ within the PilZ-FimXEAL complex (PDB: 4FOU) and PilZ within the PilB12-163-PilZ complex (this study). One important difference between the three PilZ structures is that the last 10 residues of PilZ (residues 107–117) are well structured in the PilB12-163-PilZ complex, but unstructured in PilZ on its own and in the FimXEAL-PilZ complex. E) 2F0-FC electron density map (contoured at 1.0 σ) for the 1.7 Å PilB12-163-PilZ structure in the region around PilZ residue M117 and the hydrophobic pocket made up of conserved PilB and PilZ residues. F) Surface representation of the PilB12-163-PilZ complex with PilB12-163 colored in green and PilZ colored in yellow except for residues 107–117 (the conserved motif MV) in red. (TIF)</p
- …
