X-ray crystallographic analysis of the complexes of enoyl acyl carrier protein reductase of Plasmodium falciparum with triclosan variants to elucidate the importance of different functional groups in enzyme inhibition

Triclosan, a well-known inhibitor of Enoyl Acyl Carrier Protein Reductase (ENR) from several pathogenic organisms, is a promising lead compound to design effective drugs. We have solved the X-ray crystal structures of Plasmodium falciparum ENR in complex with triclosan variants having different substituted and unsubstituted groups at different key functional locations. The structures revealed that 4 and 2' substituted compounds have more interactions with the protein, cofactor, and solvents when compared with triclosan. New water molecules were found to interact with some of these inhibitors. Substitution at the 2' position of triclosan caused the relocation of a conserved water molecule, leading to an additional hydrogen bond with the inhibitor. This observation can help in conserved water-based inhibitor design. 2' and 4' unsubstituted compounds showed a movement away from the hydrophobic pocket to compensate for the interactions made by the halogen groups of triclosan. This compound also makes additional interactions with the protein and cofactor which compensate for the lost interactions due to the unsubstitution at 2' and 4'. In cell culture, this inhibitor shows less potency, which indicates that the chlorines at 2' and 4' positions increase the ability of the inhibitor to cross multilayered membranes. This knowledge helps us to modify the different functional groups of triclosan to get more potent inhibitors

Two independently folding units of Plasmodium profilin suggest evolution via gene fusion

Gene fusion is a common mechanism of protein evolution that has mainly been discussed in the context of multidomain or symmetric proteins. Less is known about fusion of ancestral genes to produce small single-domain proteins. Here, we show with a domain-swapped mutant Plasmodium profilin that this small, globular, apparently single-domain protein consists of two foldons. The separation of binding sites for different protein ligands in the two halves suggests evolution via an ancient gene fusion event, analogous to the formation of multidomain proteins. Finally, the two fragments can be assembled together after expression as two separate gene products. The possibility to engineer both domain-swapped dimers and half-profilins that can be assembled back to a full profilin provides perspectives for engineering of novel protein folds, e.g., with different scaffolding functions

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

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

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

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

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

Molecular architecture of the recombinant human MCM2-7 helicase in complex with nucleotides and DNA

DNA replication is a key biological process that involves different protein complexes whose assembly is rigorously regulated in a successive order. One of these complexes is a replicative hexameric helicase, the MCM complex, which is essential for the initiation and elongation phases of replication. After the assembly of a double heterohexameric MCM2-7 complex at replication origins in G1, the 2 heterohexamers separate from each other and associate with Cdc45 and GINS proteins in a CMG complex that is capable of unwinding dsDNA during S phase. Here, we have reconstituted and characterized the purified human MCM2-7 (hMCM2-7) hexameric complex by co-expression of its 6 different subunits in insect cells. The conformational variability of the complex has been analyzed by single particle electron microscopy in the presence of different nucleotide analogs and DNA. The interaction with nucleotide stabilizes the complex while DNA introduces conformational changes in the hexamer inducing a cylindrical shape. Our studies suggest that the assembly of GINS and Cdc45 to the hMCM2-7 hexamer would favor conformational changes on the hexamer bound to ssDNA shifting the cylindrical shape of the complex into a right-handed spiral conformation as observed in the CMG complex bound to DNA