In silico docking of forchlorfenuron (FCF) to septins suggests that FCF interferes with GTP binding.

Septins are GTP-binding proteins that form cytoskeleton-like filaments, which are essential for many functions in eukaryotic organisms. Small molecule compounds that disrupt septin filament assembly are valuable tools for dissecting septin functions with high temporal control. To date, forchlorfenuron (FCF) is the only compound known to affect septin assembly and functions. FCF dampens the dynamics of septin assembly inducing the formation of enlarged stable polymers, but the underlying mechanism of action is unknown. To investigate how FCF binds and affects septins, we performed in silico simulations of FCF docking to all available crystal structures of septins. Docking of FCF with SEPT2 and SEPT3 indicated that FCF interacts preferentially with the nucleotide-binding pockets of septins. Strikingly, FCF is predicted to form hydrogen bonds with residues involved in GDP-binding, mimicking nucleotide binding. FCF docking with the structure of SEPT2-GppNHp, a nonhydrolyzable GTP analog, and SEPT7 showed that FCF may assume two alternative non-overlapping conformations deeply into and on the outer side of the nucleotide-binding pocket. Surprisingly, FCF was predicted to interact with the P-loop Walker A motif GxxxxGKS/T, which binds the phosphates of GTP, and the GTP specificity motif AKAD, which interacts with the guanine base of GTP, and highly conserved amino acids including a threonine, which is critical for GTP hydrolysis. Thus, in silico FCF exhibits a conserved mechanism of binding, interacting with septin signature motifs and residues involved in GTP binding and hydrolysis. Taken together, our results suggest that FCF stabilizes septins by locking them into a conformation that mimics a nucleotide-bound state, preventing further GTP binding and hydrolysis. Overall, this study provides the first insight into how FCF may bind and stabilize septins, and offers a blueprint for the rational design of FCF derivatives that could target septins with higher affinity and specificity

FCF binding induces a shift in the thermal denaturation profile of SEPT2.

<p>(A) Curve plots show the fluorescence intensity (relative fluorescence units; RFU) of SYPRO ORANGE as a function of temperature for purified recombinant SEPT2 protein in the presence of DMSO carrier and increasing concentrations of FCF. (B) The negative first derivative of the SYPRO ORANGE fluorescence intensity (RFU) was plotted against temperature. The melting curves illustrate the transition temperatures during the denaturation of SEPT2. Insert shows the median melting temperature of SEPT2 from three independent experiments in the presence of increasing FCF concentrations. Error bars represent the highest and lowest values obtained from three independent experiments.</p

FCF is predicted to bind to the outer side or deep into the nucleotide-binding pocket of SEPT7 in a similar fashion to its interaction with SEPT2-GppNHp.

<p>(A) Ribbon and stick diagrams show the orientation and atomic interactions of a representative pose of FCF bound to SEPT7 (PDB: 3T5D) from the cluster of conformations (57 out of 250) with the lowest binding free energy. Red text denotes amino acids and their corresponding protomers that interact with both FCF and GDP. (B) Ribbon and stick representations depict the position and atomic bonds of a representative pose of FCF bound to SEPT7 (PDB: 3T5D) from the cluster of conformations (37 out of 250) with the second lowest binding free energy. Red text denotes amino acids and their corresponding protomers that interact with both FCF and GDP. (C–D) Ribbon representations show the two lowest energy conformation of FCF superimposed with the nucleotide-binding pocket of SEPT7 in the absence (C) and presence of GDP (D). Similar to the dominant conformations of FCF with the nucleotide-binding pocket of SEPT2-GppNHp (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096390#pone-0096390-g006" target="_blank">Figure 6C–D</a>), FCF molecules occupy two spatially distinct regions of SEPT7 and overlap with distinct moieties of GDP (guanosine base vs. phosphate chain).</p

FCF increases the thermal stability of SEPT3 and SEPT7.

<p>Differential scanning fluorimetry was performed for purified recombinant His-SEPT3 (A–B), His-SEPT7(29-298) (C–D) and GST-Aurora B (D–E) in the presence of DMSO carrier and FCF (500 µM). Curves show the raw data of fluorescence intensity of SYPRO ORANGE (A, C, E) and the negative first derivative of SYPRO ORANGE fluorescence (B, D, F) plotted against temperature.</p

FCF is predicted to interact preferentially with the nucleotide-binding pockets of SEPT2 and SEPT3.

<p>(A and B) Ribbon and stick representations show the most energetically favorable conformations of FCF bound to SEPT2 (A) and SEPT3 (B) compared to the crystal structures of the GDP-bound SEPT2 (PDB: 2QNR) and SEPT3 (PDB: 3SOP). Ribbon representations show the position of FCF and GDP with respect to the alpha helices, beta strands, P-loop, and the switch I and switch II regions of the nucleotide-binding pocket of septins. Stick representations depict the amino acids and atomic bonds (dotted lines) underlying the septin interactions with FCF and GDP. Red text denotes amino acids and their corresponding protomers that interact with both FCF and GDP. (C) Alignment of the amino acid sequences of SEPT2 and SEPT3. Common residues that interact with both FCF and GDP are shaded in red and all other amino acids that interact with FCF are shaded in gray. Identical and similar amino acids are shown in green and pink fonts, respectively. Sequence mismatches and insertions/deletions are denoted in blue and brown, respectively.</p

FCF does not affect the thermostability of SEPT2 dimers in the presence of GDP, but increases SEPT2 stability in the presence of GmpCpp.

<p>Recombinant His-tagged SEPT2 dimers were purified using size exclusion chromatography and SEPT2 dimer stability was assayed by differential scanning fluorimetry. (A and B) Curves show the negative first derivative of SYPRO ORANGE fluorescence plotted against temperature for SEPT2 dimers in the presence of DMSO (control), FCF (100 µM), GDP (100 µM), and FCF plus GDP (100 µM each). Plot (B) shows the median melting temperature of SEPT2 from three independent experiments. Error bars represent the highest and lowest values obtained from these experiments. (C and D) Curves show the negative first derivative of SYPRO ORANGE fluorescence plotted against temperature for SEPT2 dimers in the presence of DMSO (control), FCF (100 µM), GmpCpp (100 µM), and FCF plus CmpCpp (100 µM each). Plot (D) shows the median melting temperature of SEPT2 from three independent experiments. Error bars represent the highest and lowest values obtained from these independent experiments.</p

<i>In silico</i> FCF interacts with highly conserved septin residues and signature motifs.

<p>(A) Sequence alignment highlights the conserved amino acids (shaded in red) that mediate the interaction of FCF with the deep end of the nucleotide binding pockets of SEPT2 (PDB: 3FTQ) and SEPT7 (PDB: 3T5D). Identical and similar amino acids are shown in green and pink fonts, respectively. Sequence mismatches and insertions/deletions are denoted in blue and brown, respectively. (B and C) Sequence alignments of the Walker A motif GxxxxGKS/T and the immediately following threonine, an invariant septin residue, highlight the amino acids (shaded in gray) that interact with FCF at the deep end (B) and outer side (C) of the nucleotide-binding pockets of SEPT2 (PDB: 3FTQ), SEPT3 (PDB: 3SOP) and SEPT7 (PDB: 3T5D). (D) Sequence alignments of the GTP-binding specificity motif AKAD and the following threonine residue highlight the amino acids (shaded in gray) that interact with FCF at the deep end of the nucleotide-binding pockets of SEPT2 (PDB: 3FTQ), SEPT3 (PDB: 3SOP) and SEPT7 (PDB: 3T5D).</p

<i>In silico</i> binding of FCF to two distinct sites of the nucleotide-binding pocket of SEPT2-GppNHp.

<p>(A) Ribbon and stick diagrams show the orientation and atomic interactions of a representative pose of FCF bound to the GppNHp-bound crystal structure of SEPT2 (PDB: 3FTQ) from the cluster of conformations (113 out of 250) with the lowest binding free energy. Red text denotes amino acids and their corresponding protomers that interact with both FCF and GDP. (B) Ribbon and stick representations depict the position and atomic bonds of a representative pose of FCF bound to the GppNHp-bound crystal structure of SEPT2 (PDB: 3FTQ) from the cluster of conformations (73 out of 250) with the second lowest binding free energy. Red letters denote amino acids that interact with both FCF and GDP. (C and D) Ribbon representations show the two conformations of FCF superimposed with the structure of SEPT2 in the absence (C) and presence of GppNHp (D). Note the lack of overlap between the two FCF conformations, which overlap with distinct portions of the GppNHp molecules (guanosine base vs. gamma phosphate).</p

<i>In silico</i> FCF exhibits differential binding preference for septins.

<p>Histograms show the distribution of binding free energies of 250 conformations of FCF bound to the crystal structures of SEPT2 (A), SEPT3 (B), SEPT2-GppNHp (C), SEPT7 (D), and the SEPT2 (E) and SEPT6 (F) subunits of the SEPT2/6/7 complex. Computational simulations of FCF-septin binding were performed using the AutoDock program after removing the guanosine nucleotides from the septin crystal structures. Each bar represents a cluster of FCF-bound septin conformations of similar docking poses and binding free energies. The number of conformations from each cluster was plotted against the binding free energy of the most energetically preferable conformation (lowest binding free energy).</p

Summary of septin amino acids that interact with guanine nucleosides and FCF.

<p>*Numbers correspond to the docked poses within the cluster of conformations shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096390#pone-0096390-g003" target="_blank">Figure 3</a>.</p><p>**Parentheses indicate the corresponding protomer of each amino acid.</p