Purification, crystallization and preliminary X-ray crystallographic analysis of mammalian MSS4–Rab8 GTPase protein complex

The MSS4 (mammalian suppressor of Sec4) protein in complex with nucleotide-free Rab8 GTPase has been purified and crystallized in a form suitable for structure analysis and a complete data set has been collected to 2 Å resolution

A combinatorial approach to crystallization of PX–BAR unit of the human Sorting Nexin 9

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Purification and characterization of the recombinant proteins.

<p><b>A</b>, SDS-PAGE analysis showing the purity of the proteins used in this study. The gel was stained using Coomassie Brilliant Blue. The samples are 1. <i>Pf</i>-Frm1-FH1FH2, 2. <i>Pf</i>-Frm1-FH2, 3. <i>Pf</i>-Frm1-FH2Δlasso, 4. pig muscle actin, 5. <i>Pf</i>-Act1, 6. <i>Pb</i>-Act2, and 7. <i>Pf</i>-Pfn. The molecular weights of the standard proteins in kDa are indicated on the right. The differences in the molecular weights of the <i>Plasmodium</i> and pig actins are accounted for by the 6×His-tags present in the recombinant <i>Plasmodium</i> actins. <b>B</b>, Size-exclusion chromatograms showing a single peak for <i>Pf</i>-Frm1-FH1FH2 (red) and <i>Pf</i>-Frm1-FH2 (blue) and two peaks for <i>Pf</i>-Frm1-FH2Δlasso (green). The molecular weights of the peaks of <i>Pf</i>-Frm1-FH1FH2 and <i>Pf</i>-Frm1-FH2 correspond to the size of dimers, and the two peaks of <i>Pf</i>-Frm1-FH2Δlasso to a dimer and a monomer, as determined by MALS (vertical lines with colors corresponding to those of the chromatograms).</p

Effect of <i>Pf</i>-Pfn on actin polymerization kinetics in the presence of <i>Pf</i>-Frm1.

<p>The effect of <i>Pf</i>-Pfn added at a 1∶1 or 2∶1 molar ratio to actin on the kinetics of actin polymerization in the presence of the different <i>Pf</i>-Frm1 domains was tested by measuring the change in fluorescence upon incorporation of 5% pyrene-actin into growing actin polymers. The actin concentration used in all experiments was 5 µM. <b>A</b>, <i>Pf</i>-Pfn together with 10 nM <i>Pf</i>-Frm1-FH1FH2. <b>B</b>, <i>Pf</i>-Pfn together with 10 nM <i>Pf</i>-Frm1-FH2. <b>C</b>, <i>Pf</i>-Pfn together with 10 nM <i>Pf</i>-Frm1-FH2Δlasso.</p

Model for the dimerization of <i>Plasmodium</i> formin 1.

<p><b>A</b>, Sequence alignment between the <i>Pf</i>-Frm1 FH2 domain and the best template identified by Phyre for homology modeling (mouse mDia1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033586#pone.0033586-Nezami1" target="_blank">[43]</a>). <i>Pf</i>-Frm1-FH2 includes both the lasso and linker domains, while <i>Pf</i>-Frm1-FH2Δlasso has only the linker segment. <b>B</b>, Comparison of the dimer homology model of <i>Pf</i>-Frm1 (shown as cartoons; the dimerization in the model is built identical to that seen in the mDia1 template structure 3O4X <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033586#pone.0033586-Nezami1" target="_blank">[43]</a>) with the SAXS model built by using the core FH2 domains as rigid bodies (green) and building the linker, lasso, and FH1 segments by chain-like assemblies of dummy residues (cyan). The SAXS model was made with BUNCH, applying P2 symmetry, but no distance restraints. The Chi-value against the raw SAXS data is 1.4, reflecting a very good fit to the measurement; the fit is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033586#pone.0033586.s001" target="_blank">Figure S1B</a>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033586#pone.0033586.s001" target="_blank">Figure S1</a> also contains further rigid body refinement analysis of the homology model. <b>C</b>, A close-up view into the model of the lasso segment (green) in a <i>Pf</i>-Frm1 dimer; the FH2 domain monomers are indicated in blue and red. The orange segment represents the flexible linker region.</p

Solution structures of the <i>Pf</i>-Frm1 domains.

<p><b>A</b>, Raw synchrotron SAXS data for <i>Pf</i>-Frm1-FH1FH2 (red), <i>Pf</i>-Frm1-FH2 (blue), and <i>Pf</i>-Frm1-FH2Δlasso (green). <b>B</b>, Distance distribution functions derived from the SAXS data; coloring as in panel A. <b>C</b>, SANS data for <i>Pf</i>-Frm1-FH1FH2. A radius of gyration (R_g) of 5.7 nm and a maximum particle dimension (D_max) of 18 nm can be estimated from the data. <b>D</b>, Averaged <i>ab initio</i> dummy atom models for <i>Pf</i>-Frm1-FH1FH2 (red) and <i>Pf</i>-Frm1-FH2 (blue) created by DAMMIN. The extensions at the extremities most likely correspond to the FH1 domain. <b>E</b>, Averaged <i>ab initio</i> models for <i>Pf</i>-Frm1-FH2 created by DAMMIN (blue) and GASBOR (cyan). The two figures are related by a 90° rotation about the X-axis. Both methods produce very similar models that fit the data. <b>F</b>, Averaged <i>ab initio</i> model for <i>Pf</i>-Frm1-FH2Δlasso (green) created by DAMMIN, superimposed on the structure of <i>Pf</i>-Frm1-FH1FH2 (red). Note that <i>Pf</i>-Frm1-FH2Δlasso is highly elongated, lacking a compact domain in the middle. <b>G</b>, SRCD spectra for the <i>Pf</i>-Frm1 variants. Coloring as in panel A. <i>Pf</i>-Frm1-FH2 has the highest relative helical content; see text for details on spectral deconvolution.</p

Properties of the three formin versions derived from SEC/MALS and SAXS.

<p>The given volume is that of the respective averaged <i>ab initio</i> dummy bead model. The Chi-value describes the fit between the experimental scattering data and the model; with values close to unity reflecting a good fit.</p>*<p>MW = molecular weight.</p>**<p>R_g = radius of gyration.</p>***<p>D_max = maximum particle dimension.</p

Effect of <i>Pf</i>-Frm1 on actin polymerization kinetics.

<p>The effect of <i>Pf</i>-Frm1 on the kinetics of actin polymerization was tested by measuring the change in fluorescence upon incorporation of 5% pyrene-actin into growing actin polymers. The actin concentration used in all experiments was 5 µM. The initial rates (ΔF/s) were calculated as the slope of the linear part (120 s) of the fluorescence curves. In order to facilitate comparison, the samples containing only actin were set to the value 1. <b>A–B</b>, Different concentrations of <i>Pf</i>-Frm1-FH1FH2. <b>C–D</b>, Different concentrations of <i>Pf</i>-Frm1-FH2. <b>E–F</b>, Different concentrations of <i>Pf</i>-Frm1-FH2Δlasso.</p

A FRET-based high-throughput screening platform for the discovery of chemical probes targeting the scaffolding functions of human tankyrases

Abstract Tankyrases catalyse poly-ADP-ribosylation of their binding partners and the modification serves as a signal for the subsequent proteasomal degradation of these proteins. Tankyrases thereby regulate the turnover of many proteins involved in multiple and diverse cellular processes, such as mitotic spindle formation, telomere homeostasis and Wnt/β-catenin signalling. In recent years, tankyrases have become attractive targets for the development of inhibitors as potential therapeutics against cancer and fibrosis. Further, it has become clear that tankyrases are not only enzymes, but also act as scaffolding proteins forming large cellular signalling complexes. While many potent and selective tankyrase inhibitors of the poly-ADP-ribosylation function exist, the inhibition of tankyrase scaffolding functions remains scarcely explored. In this work we present a robust, simple and cost-effective high-throughput screening platform based on FRET for the discovery of small molecule probes targeting the protein–protein interactions of tankyrases. Validatory screening with the platform led to the identification of two compounds with modest binding affinity to the tankyrase 2 ARC4 domain, demonstrating the applicability of this approach. The platform will facilitate identification of small molecules binding to tankyrase ARC or SAM domains and help to advance a structure-guided development of improved chemical probes targeting tankyrase oligomerization and substrate protein interactions

Crystal structures of <i>Plasmodium</i> actin I and II.

<p>(<b>A</b>) <i>P. berghei</i> actin II (<i>Pb</i>ActII; yellow) superimposed on α-actin (1eqy <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004091#ppat.1004091-McLaughlin1" target="_blank">[39]</a>; cyan). (<b>B</b>) <i>P. falciparum</i> actin I (<i>Pf</i>ActI; red) superimposed on α-actin. In both (<b>A</b>) and (<b>B</b>), ATP, subdomains 1–4, and several regions discussed in the text are indicated. Both N and C termini reside in subdomain 1; the N terminus is visible at the front, and the C-terminal helix is on the back side. Note that the C-terminal helix is not visible in actin I. The C-terminal part and the nearby hydrophobic cluster with Trp357 are shown in the zoomed view on the right and the region involved in intra-filament contacts in subdomain 3 in the box at the lower left corner. The blue and pink dots in (<b>B</b>) indicate the approximate positions of the structural elements shown in detail in (<b>C</b>) and (<b>D</b>), respectively. (<b>C</b>) Lys 207 and Glu188 are at an intimate distance in actin I. A similar salt bridge is present between the corresponding residues in latrunculin-bound α-actin, but the hydrogen-bonding distance is longer without the drug. (<b>D</b>) The proline-rich loop with Gly115 in actin I superimposed on that of α-actin. Note the bending of the loop in actin I, due to the more flexible glycine residue.</p