15 research outputs found
Presentation_1_Crystal Structures of R-Type Bacteriocin Sheath and Tube Proteins CD1363 and CD1364 From Clostridium difficile in the Pre-assembled State.pdf
<p>Diffocins are high-molecular-weight phage tail-like bacteriocins (PTLBs) that some Clostridium difficile strains produce in response to SOS induction. Similar to the related R-type pyocins from Pseudomonas aeruginosa, R-type diffocins act as molecular puncture devices that specifically penetrate the cell envelope of other C. difficile strains to dissipate the membrane potential and kill the attacked bacterium. Thus, R-type diffocins constitute potential therapeutic agents to counter C. difficile-associated infections. PTLBs consist of rigid and contractile protein complexes. They are composed of a baseplate, receptor-binding tail fibers and an inner needle-like tube surrounded by a contractile sheath. In the mature particle, the sheath and tube structure form a complex network comprising up to 200 copies of a sheath and a tube protein each. Here, we report the crystal structures together with small angle X-ray scattering data of the sheath and tube proteins CD1363 (39 kDa) and CD1364 (16 kDa) from C. difficile strain CD630 in a monomeric pre-assembly form at 1.9 and 1.5 Å resolution, respectively. The tube protein CD1364 displays a compact fold and shares highest structural similarity with a tube protein from Bacillus subtilis but is remarkably different from that of the R-type pyocin from P. aeruginosa. The structure of the R-type diffocin sheath protein, on the other hand, is highly conserved. It contains two domains, whereas related members such as bacteriophage tail sheath proteins comprise up to four, indicating that R-type PTLBs may represent the minimal protein required for formation of a complete sheath structure. Comparison of CD1363 and CD1364 with structures of PTLBs and related assemblies suggests that several conformational changes are required to form complete assemblies. In the sheath, rearrangement of the flexible N- and C-terminus enables extensive interactions between the other subunits, whereas for the tube, such contacts are primarily established by mobile α-helices. Together, our results combined with information from structures of homologous assemblies allow constructing a preliminary model of the sheath and tube assembly from R-type diffocin.</p
Inhibition and Regulation of the Ergothioneine Biosynthetic Methyltransferase EgtD
Ergothioneine is
an emerging factor in cellular redox homeostasis
in bacteria, fungi, plants, and animals. Reports that ergothioneine
biosynthesis may be important for the pathogenicity of bacteria and
fungi raise the question as to how this pathway is regulated and whether
the corresponding enzymes may be therapeutic targets. The first step
in ergothioneine biosynthesis is catalyzed by the methyltransferase
EgtD that converts histidine into N-α-trimethylhistidine. This
report examines the kinetic, thermodynamic and structural basis for
substrate, product, and inhibitor binding by EgtD from <i>Mycobacterium
smegmatis</i>. This study reveals an unprecedented substrate
binding mechanism and a fine-tuned affinity landscape as determinants
for product specificity and product inhibition. Both properties are
evolved features that optimize the function of EgtD in the context
of cellular ergothioneine production. On the basis of these findings,
we developed a series of simple histidine derivatives that inhibit
methyltransferase activity at low micromolar concentrations. Crystal
structures of inhibited complexes validate this structure- and mechanism-based
design strategy
HheG, a Halohydrin Dehalogenase with Activity on Cyclic Epoxides
Halohydrin dehalogenases
(HHDHs) are of biotechnological interest
due to their promiscuous epoxide ring-opening activity with a set
of negatively charged nucleophiles, enabling the formation of C–C,
C–N, or C–O bonds. The recent discovery of HHDH-specific
sequence motifs aided the identification of a large number of halohydrin
dehalogenases from public sequence databases, enlarging the biocatalytic
toolbox substantially. During the characterization of 17 representatives
of these phylogenetically diverse enzymes, one HHDH, namely HheG from Ilumatobacter coccineus, was identified to convert
cyclic epoxide substrates. The enzyme exhibits significant activity
in the azidolysis of cyclohexene oxide and limonene oxide with turnover
numbers of 7.8 and 44 s<sup>–1</sup>, respectively. As observed
for other HHDHs, the cyanide-mediated epoxide ring-opening proceeded
with lower rates. Wild-type HheG displays modest enantioselectivity,
as the resulting azido- and cyanoalcohols of cyclohexene oxide ring-opening
were obtained in 40% enantiomeric excess. These biocatalytic findings
were further complemented by the crystal structure of the enzyme refined
to 2.3 Å. Analysis of HheG’s structure revealed a large
open cleft harboring the active site. This is in sharp contrast to
other known HHDH structures and aids in explaining the special substrate
scope of HheG
Supplementary Data Table S3 from Structural, mechanistic and functional insight into gliotoxin <i>bis</i>-thiomethylation in <i>Aspergillus fumigatus</i>
Supplementary Data Table S3 (Quantitaitve proteomic data
Directed Evolution of <i>Mycobacterium tuberculosis</i> β-Lactamase Reveals Gatekeeper Residue That Regulates Antibiotic Resistance and Catalytic Efficiency
<div><p>Directed evolution can be a powerful tool for revealing the mutational pathways that lead to more resistant bacterial strains. In this study, we focused on the bacterium <i>Mycobacterium tuberculosis,</i> which is resistant to members of the β-lactam class of antibiotics and thus continues to pose a major public health threat. Resistance of this organism is the result of a chromosomally encoded, extended spectrum class A β-lactamase, BlaC, that is constitutively produced. Here, combinatorial enzyme libraries were selected on ampicillin to identify mutations that increased resistance of bacteria to β-lactams. After just a single round of mutagenesis and selection, BlaC mutants were evolved that conferred 5-fold greater antibiotic resistance to cells and enhanced the catalytic efficiency of BlaC by 3-fold compared to the wild-type enzyme. All isolated mutants carried a mutation at position 105 (e.g., I105F) that appears to widen access to the active site by 3.6 Å while also stabilizing the reorganized topology. In light of these findings, we propose that I105 is a ‘gatekeeper’ residue of the active site that regulates substrate hydrolysis by BlaC. Moreover, our results suggest that directed evolution can provide insight into the development of highly drug resistant microorganisms.</p></div
Heterospecific expression of <i>M. tuberculosis</i> BlaC in <i>E. coli</i>.
<p>(a) Serially diluted wt or Δ<i>tatC</i> cells expressing ssTorA-Bla or ssTorA-BlaC chimeras were spotted on Amp. (b) Western blot analysis of cytoplasmic (cyt) and periplasmic (per) fractions prepared from wt cells expressing ssTorA-BlaC, full-length BlaC, or BlaC lacking a signal peptide (ΔspBlaC). Arrow indicates BlaC. Samples prepared from an equivalent number of cells were loaded in each lane. Blots were probed with anti-FLAG antibodies.</p
Structural basis for enhanced BlaC-mediated resistance.
<p>(a) Active sites of wt BlaC (top), BlaC(I105F) (middle), or structural alignment of both (bottom). (b) Structural alignment of wt BlaC (yellow), BlaC(I105F) (cyan), and TEM-1 Bla (magenta). Arrow indicates aromatic residues of BlaC(I105F), and TEM-1 Bla.</p
Psoromic Acid is a Selective and Covalent Rab-Prenylation Inhibitor Targeting Autoinhibited RabGGTase
Post-translational attachment of geranylgeranyl isoprenoids
to
Rab GTPases, the key organizers of intracellular vesicular transport,
is essential for their function. Rab geranylgeranyl transferase (RabGGTase)
is responsible for prenylation of Rab proteins. Recently, RabGGTase
inhibitors have been proposed to be potential therapeutics for treatment
of cancer and osteoporosis. However, the development of RabGGTase
selective inhibitors is complicated by its structural and functional
similarity to other protein prenyltransferases. Herein we report identification
of the natural product psoromic acid (PA) that potently and selectively
inhibits RabGGTase with an IC<sub>50</sub> of 1.3 μM. Structure–activity
relationship analysis suggested a minimal structure involving the
depsidone core with a 3-hydroxyl and 4-aldehyde motif for binding
to RabGGTase. Analysis of the crystal structure of the RabGGTase:PA
complex revealed that PA forms largely hydrophobic interactions with
the isoprenoid binding site of RabGGTase and that it attaches covalently
to the N-terminus of the α subunit. We found that in contrast
to other protein prenyltransferases, RabGGTase is autoinhibited through
N-terminal <sub>α</sub>His2 coordination with the catalytic
zinc ion. Mutation of <sub>α</sub>His dramatically enhances
the reaction rate, indicating that the activity of RabGGTase is likely
regulated in vivo. The covalent binding of PA to the N-terminus of
the RabGGTase α subunit seems to potentiate its interaction
with the active site and explains the selectivity of PA for RabGGTase.
Therefore, psoromic acid provides a new starting point for the development
of selective RabGGTase inhibitors
Localization of R281 in a protein model of the myosin heavy chain.
<p>A, Positions of amino acids affected in NVM, HCM and DCM. The mutations were plotted on a model of chicken skeletal myosin subfragment-1 (PDB code 2MYS). Functional sites are indicated by arrows. 68 selected mutations causing HCM are indicated by yellow spheres and 4 mutations causing DCM by green spheres. The mutations were selected from the UniProt database (UniProtKB) and from two publications<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001362#pone.0001362-Woo1" target="_blank">[33]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001362#pone.0001362-Villard1" target="_blank">[34]</a>. The described NVM causing mutation is highlighted as a blue sphere and labeled according to the position in MYH7_HUMAN. B, Close-up view of the salt bridge between residues R281 and D325 in chicken skeletal myosin subfragment-1 (PDB code 2MYS). The amino acids are numbered according to MYH7_HUMAN. The sulfate molecule (yellow and orange) marks the ATPase active site for better orientation. The residues R281 and D325 are shown according to the CPK color scheme (grey, carbon atoms; red, oxygen atoms, i.e. acidic side chain; light blue, nitrogen atoms, i.e. basic side chain). The salt bridge between R281 and D325 is symbolized by dashed black lines that indicate potential hydrogen bonds. The helix attached to D325 is highlighted in red.</p
Genetic analysis.
<p>A, Linkage analysis. Two-point LOD scores for all chromosomes under the assumption of a reduced penetrance of 90% are shown. In the line below the diagram each red slash indicates the position of an analyzed marker on the corresponding chromosome. B, Genetic map of the candidate region 14cen-14q12. A part of the deCODE map is shown in the upper part. The critical interval is delimited by 14ptel and <i>D14S264</i>. The genomic organization of <i>MYH7</i> is delineated in the lower part. Exons are indicated by filled boxes and introns by horizontal lines. The translational start codon is located in exon 3. The positions of the intragenic markers <i>MYO</i> I in intron 1and <i>MYOII</i> in intron 24 are shown. The mutation was identified in exon 10. C, Mutation analysis. Chromatograms of the index patient III:4 and her unaffected son IV:2 are shown. The mutation site is marked by an arrow. Parts of the nucleotide sequence (from c.835 to c.849) and protein sequence (from 279 to 283) are given below.</p