In Silico Docking of Forchlorfenuron (FCF) to Septins Suggests that FCF Interferes with GTP Binding

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.

Off-Target Effects of the Septin Drug Forchlorfenuron on Nonplant Eukaryotes

“…Thus, some evidence points to direct FCF-septin interactions that modify interactions between septin polymers. Moreover, the thermal stabilities of individually purified human septins are altered by the presence of FCF (28), consistent with direct binding. Finally, in silico modeling demonstrates that FCF fits well within the septin GTP-binding pocket (28).…”

“…Moreover, the thermal stabilities of individually purified human septins are altered by the presence of FCF (28), consistent with direct binding. Finally, in silico modeling demonstrates that FCF fits well within the septin GTP-binding pocket (28). In general, the available evidence suggests that GTP binding and hydrolysis play structural roles in promoting higherorder septin assembly (11,29).…”

“…If FCF indeed competes with GTP for occupancy of the septin nucleotide-binding pocket (28), it likely does so during de novo septin synthesis and folding, because preassembled yeast septins display only negligible in vivo exchange of bound GTP (65). Accordingly, in order to explain the rapid appearance of ectopic septin structures upon exposure of yeast cells to FCF, only a small number of FCF-bound septin proteins suffice to nucleate ectopic polymerization by GTP-bound septins that were synthesized prior to the introduction of FCF.…”

Septin Dynamics Are Essential for Exocytosis

“…Here, to elucidate the role of septins in exocytosis, we used acute down-regulation of the major ubiquitously expressed septin, septin-2, by transient siRNA. In addition, we used the inhibitor of septin organization, forchlorfenuron (FCF), which specifically impairs assembly and disassembly of septin hetero-oligomers (27)(28)(29) without affecting actin or tubulin polymerization (27,30). Both FCF and knockdown of septin-2 decreased the rate of exocytosis in cultured cells.…”

Production and analysis of a mammalian septin hetero-octamer complex

“…Plasmids pCDF-SEPT6/7 (co-expression plasmid encoding SEPT6 and SEPT7-StrepII), 63 pET15b-SEPT2 (mouse SEPT2 with a Thrombin cleavable N-terminal His6 fusion) 63 and pET28-SEPT3 (used for PCR amplification in construction of SEPT3/SEPT2 co-expression vector) 82 were previously described. pET15b-SEPT2 was also modified to include a C-terminal fusion to mCherry, pET15b-SEPT2-mCh.…”