Identification of the antiepileptic racetam binding site in the synaptic vesicle protein 2A by molecular dynamics and docking simulations

Abstract

Synaptic vesicle protein 2A (SV2A) is an integral membrane protein necessary for the proper function of the central nervous system and is associated to the physiopathology of epilepsy. SV2A is the molecular target of the anti-epileptic drug levetiracetam and its racetam analogs. The racetam binding site in SV2A and the non-covalent interactions between racetams and SV2A are currently unknown; therefore, an in silico study was performed to explore these issues. Since SV2A has not been structurally characterized with X-ray crystallography or nuclear magnetic resonance, a three-dimensional (3D) model was built. The model was refined by performing a molecular dynamics simulation (MDS) and the interactions of SV2A with the racetams were determined by docking studies. A reliable 3D model of SV2A was obtained; it reached structural equilibrium during the last 15 ns of the MDS (50 ns) with remaining structural motions in the N-terminus and long cytoplasmic loop. The docking studies revealed that hydrophobic interactions and hydrogen bonds participate importantly in ligand recognition within the binding site. Residues T456, S665, W666, D670 and L689 were important for racetam binding within the trans-membrane hydrophilic core of SV2A. Identifying the racetam binding site within SV2A should facilitate the synthesis of suitable radio-ligands to study treatment response and possibly epilepsy progression.

Keywords: SV2A; brivaracetam; epilepsy; levetiracetam; seletracetam.

FIGURE 1

3D SV2A model. The I-Tasser server yielded a model of SV2A with 742 amino acids distributed in 12 TM domains consisting of α-helices, an undefined intra-luminal domain and two cytoplasmic domains: a relatively large N-terminus with a synaptotagmin 1 binding site (arrow) plus an ATP binding motif (arrow head) and a large loop between TM domains 6–7 with another ATP binding motif (two arrows heads).

FIGURE 2

Views of the simulation system, (A) after minimization and (B) after the MDS. Notice that SV2A rearranges within the lipid bilayer.

FIGURE 3

Root mean square deviation values for (A) the whole backbone and (B) the backbone of the TM region as a function of time. The gray bands correspond to the SD and the line within them represents the average value.

FIGURE 4

Radius of gyration values for (A) the whole backbone and (B) the backbone of the TM region as a function of time. The gray bands correspond to the SD and the line within them represents the average value.

FIGURE 5

Number of H-bonds as a function of time, (A) protein intra-molecular, (B) protein–water and (C) protein–lipid. The gray bands correspond to the SD and the line within them represents the average value.

FIGURE 6

Graphic RMSF representations. (A) RMSF of the atomic positions and (B) representation of protein flexibility as a gradient from low to high fluctuations.

FIGURE 7

Evolution of the total, hydrophobic and hydrophilic protein SAS; (A) exposed to water plus lipids, (B) exposed to water or (C) exposed to lipids as a function of time.

FIGURE 8

Solvent accessible surface area by residues exposed to the membrane inner core. The highest values correspond to amino acids totally exposed to membrane hydrophobic core.

FIGURE 9

PCA analysis. (A) Variance–covariance matrix of the SV2A α-carbon atoms for the 50 ns of the MDS. (B) Projection of the 50 ns trajectory onto the two principal eigenvectors obtained from the diagonalization of variance–covariance matrix; time along the trajectory is represented by a denser gradient.

FIGURE 10

Putative racetam binding site within the last SV2A conformer of the MDS. 3D models and schematic representations of the interactions between SV2A and (A) LEV, (B) BRIV, (C) SEL or (D) UCB30889 are shown. Hydrophobic interactions and H-bonds participate importantly in ligand recognition within the binding site (amino acids in circles).