Affinity of ucb 30889 for rat recombinant SV2A (wildtype and mutants) labeled with [³H]ucb 30889 (n = 3).

<p>Affinity of ucb 30889 for rat recombinant SV2A (wildtype and mutants) labeled with [³H]ucb 30889 (n = 3).</p

Looking to the future

<div><p>The putative Major Facilitator Superfamily (MFS) transporter, SV2A, is the target for levetiracetam (LEV), which is a successful anti-epileptic drug. Furthermore, SV2A knock out mice display a severe seizure phenotype and die after a few weeks. Despite this, the mode of action of LEV is not known at the molecular level. It would be extremely desirable to understand this more fully in order to aid the design of improved anti-epileptic compounds. Since there is no structure for SV2A, homology modelling can provide insight into the ligand-binding site. However, it is not a trivial process to build such models, since SV2A has low sequence identity to those MFS transporters whose structures are known. A further level of complexity is added by the fact that it is not known which conformational state of the receptor LEV binds to, as multiple conformational states have been inferred by tomography and ligand binding assays or indeed, if binding is exclusive to a single state. Here, we explore models of both the inward and outward facing conformational states of SV2A (according to the alternating access mechanism for MFS transporters). We use a sequence conservation analysis to help guide the homology modelling process and generate the models, which we assess further with Molecular Dynamics (MD). By comparing the MD results in conjunction with docking and simulation of a LEV-analogue used in radioligand binding assays, we were able to suggest further residues that line the binding pocket. These were confirmed experimentally. In particular, mutation of D670 leads to a complete loss of binding. The results shed light on the way LEV analogues may interact with SV2A and may help with the on-going design of improved anti-epileptic compounds.</p></div

The ligand binding sites in the Inward-apo model of SV2A (A) and the Outward-apo model (B) from simulation (60 ns)

<p>. The ligand (black stick) was docked to a snapshot the apo-model after 80 ns simulation. Key residues identified by mutagenesis are highlighted as stick representations. Schematic interaction maps of the docked ligand, generated via MOE with an interaction cut-off of 6 Å are shown for the Inward (C) and Outward (D) models. Residues starred are conserved hydrophobic residues common to both the Inward and Outward ligand binding pockets. (E) Affinity of ucb 30889 for recombinant rat SV2A (wt and mutants). A concentration range of ucb 30889 was incubated with 5 nM of [³H]ucb 30889 during 120 min at 4°C. B0 is the binding of [³H]ucb 30889 in the absence of any competing compound. Data are representative of three independent experiments. pIC₅₀ values were calculated from untransformed raw data by non-linear regression using a model describing a sigmoidal dose-response curve with variable slope and are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116589#pone.0116589.t003" target="_blank">Table 3</a>. The position of the mutants with respect to the ligand in the Outward model is shown in (F).</p

(A) Conservation pattern of residues (M, A, V, I, L, C, Y, W and F) as ascertained by an alignment of 758 sequences form a BLAST search against rat SV2A

<p>. The degree of conservation is coloured from blue to red as a function of percentage. The position of the TM helices as predicted from the consensus prediction (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116589#pone.0116589.s001" target="_blank">S1 Fig</a>.) are indicated. (B) Chemical structure of ucb 30889, a commonly used radio-ligand that is an analogue of LEV.</p

Conservation of residues in an alignment of 758 sequences from a BLAST search of SV2A.

<p>Conservation of residues in an alignment of 758 sequences from a BLAST search of SV2A.</p

(A) Change in RMSDs from 80 ns of the Inward-ucb 30889 simulation (green) and (B) the Outward-ucb 30889 simulation (red lines) from the initial snapshot from the respective apo simulations

<p>. Helix conservation in the (C) Inward-ucb 30889 and (D) Outward-ucb 30889 simulations.</p