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

Angelis D, Karasmanis EP, Bai X, Spiliotis ET - PLoS ONE (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Drexel University, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
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.

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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.(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 (Figure 6C–D), FCF molecules occupy two spatially distinct regions of SEPT7 and overlap with distinct moieties of GDP (guanosine base vs. phosphate chain).
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pone-0096390-g007: 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.(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 (Figure 6C–D), FCF molecules occupy two spatially distinct regions of SEPT7 and overlap with distinct moieties of GDP (guanosine base vs. phosphate chain).

Mentions: In the cluster of conformations with the lowest average binding energy (−5.87±0.55 kcal/mol), FCF is positioned deep in the nucleotide-binding pocket of SEPT7 with its phenol ring and urea moiety occupying the site of the guanine base and ribose ring of GTP. Similar to the conformation of the SEPT2-bound FCF, the nitrogen and oxygen atoms of FCF's urea moiety form hydrogen bonds with Thr-46 and Arg-246 of SEPT7 (Figure 7A), which correspond to Thr-52 and Arg-256 of SEPT2 (Figure 6A). In an identical fashion with Gly-241 of SEPT2, Gly-231 forms a strong hydrogen bond with the nitrogen atom of FCF's pyridine ring. Asp-178 of the conserved GTP-binding specificity motif (AKAD) of septins is predicted to form a hydrogen bond with FCF's pyridine ring, which also interacts with the conserved P-loop residue Gly-43 (Gly-49 in SEPT2). Similar to Glu-191 of SEPT2, Glu-184 is hydrogen-bound to a carbon atom from FCF's phenol ring. Strikingly, FCF interacts with six residues of SEPT7, which are completely conserved between SEPT7 and SEPT2 (Figure 8A), and are the same amino acids that mediate FCF binding to SEPT2.


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

Angelis D, Karasmanis EP, Bai X, Spiliotis ET - PLoS ONE (2014)

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.(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 (Figure 6C–D), FCF molecules occupy two spatially distinct regions of SEPT7 and overlap with distinct moieties of GDP (guanosine base vs. phosphate chain).
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pone-0096390-g007: 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.(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 (Figure 6C–D), FCF molecules occupy two spatially distinct regions of SEPT7 and overlap with distinct moieties of GDP (guanosine base vs. phosphate chain).
Mentions: In the cluster of conformations with the lowest average binding energy (−5.87±0.55 kcal/mol), FCF is positioned deep in the nucleotide-binding pocket of SEPT7 with its phenol ring and urea moiety occupying the site of the guanine base and ribose ring of GTP. Similar to the conformation of the SEPT2-bound FCF, the nitrogen and oxygen atoms of FCF's urea moiety form hydrogen bonds with Thr-46 and Arg-246 of SEPT7 (Figure 7A), which correspond to Thr-52 and Arg-256 of SEPT2 (Figure 6A). In an identical fashion with Gly-241 of SEPT2, Gly-231 forms a strong hydrogen bond with the nitrogen atom of FCF's pyridine ring. Asp-178 of the conserved GTP-binding specificity motif (AKAD) of septins is predicted to form a hydrogen bond with FCF's pyridine ring, which also interacts with the conserved P-loop residue Gly-43 (Gly-49 in SEPT2). Similar to Glu-191 of SEPT2, Glu-184 is hydrogen-bound to a carbon atom from FCF's phenol ring. Strikingly, FCF interacts with six residues of SEPT7, which are completely conserved between SEPT7 and SEPT2 (Figure 8A), and are the same amino acids that mediate FCF binding to SEPT2.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Drexel University, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus