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

Dimitrios Angelis,Elias T Spiliotis,Eva Pauline Karasmanis,Xiaobo Bai,Julie G Donaldson

doi:10.1371/journal.pone.0096390

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.

Highlights

Septins are a family of guanosine triphosphate (GTP) binding proteins, which are highly conserved in fungal and metazoan organisms [1,2]
All septins contain the GTP-specificity motif AKAD, which interacts with the guanine base of GTP, and the Walker A (GxxxxGKS/T) and B (DxxG) motifs that bind to the phosphate groups of GTP and coordinate with a magnesium ion for GTP hydrolysis, respectively [4,5]
Structural studies indicate that GTP-binding triggers conformational changes that destabilize the NC interface, while GTP hydrolysis appears to have the opposite effect on the G interface, which is more stable in its GDP- than GTP-bound state [11,12,13,14]

Summary

Introduction

Septins are a family of guanosine triphosphate (GTP) binding proteins, which are highly conserved in fungal and metazoan organisms [1,2]. In contrast to the monomeric small GTPases of the Ras superfamily, septins oligomerize via their GTP-binding domains, forming filamentous hetero-polymers [6,7]. Similar to microtubules and actin, septin filament assembly and disassembly involves nucleotide binding and hydrolysis, but how this occurs is not well understood. Septins polymerize by interacting with one another via the N- and C-termini (NC interface) and the nucleotide-binding pocket (G interface) of their GTP binding domains [6]. Structural studies indicate that GTP-binding triggers conformational changes that destabilize the NC interface, while GTP hydrolysis appears to have the opposite effect on the G interface, which is more stable in its GDP- than GTP-bound state [11,12,13,14]. Septin monomers hydrolyze GTP faster than septin dimers and oligomers, whose nucleotide-binding pocket is not readily accessible by GTP [12,15]. While more work is needed to fully understand how GTP-binding and hydrolysis affects the dynamics of septin filament assembly, pharmacological agents that stabilize or depolymerize septin filaments can be useful tools in understanding the mechanisms of septin assembly and function

Methods

Results

Conclusion