Combining modelling and mutagenesis studies of synaptic vesicle protein 2A to identify a series of residues involved in racetam binding

Abstract

<jats:p>LEV (levetiracetam), an antiepileptic drug which possesses a unique profile in animal models of seizure and epilepsy, has as its unique binding site in brain, SV2A (synaptic vesicle protein 2A). Previous studies have used a chimaeric and site-specific mutagenesis approach to identify three residues in the putative tenth transmembrane helix of SV2A that, when mutated, alter binding of LEV and related racetam derivatives to SV2A. In the present paper, we report a combined modelling and mutagenesis study that successfully identifies another 11 residues in SV2A that appear to be involved in ligand binding. Sequence analysis and modelling of SV2A suggested residues equivalent to critical functional residues of other MFS (major facilitator superfamily) transporters. Alanine scanning of these and other SV2A residues resulted in the identification of residues affecting racetam binding, including Ile273 which differentiated between racetam analogues, when mutated to alanine. Integrating mutagenesis results with docking analysis led to the construction of a mutant in which six SV2A residues were replaced with corresponding SV2B residues. This mutant showed racetam ligand-binding affinity intermediate to the affinities observed for SV2A and SV2B.</jats:p>

Synaptic Vesicle Glycoprotein 2A Ligands in the Treatment of Epilepsy and Beyond

“…The subsequent complexes were then simulated using molecular dynamics in an attempt to further characterize SV2A–racetam binding site interactions. Despite the short timescale simulations and the challenge and caveats of modeling the extramembranous domains, the studies support the putative binding pocket observed by Shi et al [85] and Lee et al [86], and, furthermore, identified additional hydrophobic and hydrogen bond interactions with T456, S665 and L689, which may be important for ligand recognition within the putative racetam binding site.…”

“…These predictions were subsequently borne out by SDM performed on W454A and D670A in conjunction with binding affinity assays [86]. Taken together with the studies of Shi et al [85], the outward-open conformation of SV2A (i.e. with the putative binding site exposed to the interior of the synaptic vesicle) presents a levetiracetam-binding pocket that is in greater agreement with the SDM experiments.…”

“…Although the sequence identity between SV2A and other MFS proteins is very low, typically <20 %, homology models have been constructed using available MFS x-ray structures as templates. In an early study Shi et al [85] selected three residues—F658, G659 and V661—based on their alignment with functionally relevant residues in LacY for site-directed mutagenesis (SDM) studies with levetiracetam and structurally-related analogs such as ucb 30889, collectively referred to as racetams, to elucidate residues that are important for racetam binding. Furthermore, Shi et al [85] constructed an SV2A homology model using the LacY crystal structure as a template, and in conjunction with racetam docking studies they proposed additional residues for SDM studies.…”

“…In an early study Shi et al [85] selected three residues—F658, G659 and V661—based on their alignment with functionally relevant residues in LacY for site-directed mutagenesis (SDM) studies with levetiracetam and structurally-related analogs such as ucb 30889, collectively referred to as racetams, to elucidate residues that are important for racetam binding. Furthermore, Shi et al [85] constructed an SV2A homology model using the LacY crystal structure as a template, and in conjunction with racetam docking studies they proposed additional residues for SDM studies. In total, through binding affinity experiments, 14 mutants were found to be implicated in racetam binding (F277A, W300A, W300F, Y462A, K694A, G303A, F658A, V661A, I663A, W666A, N667A, S294A, M301A, G659A) and were proposed to encompass the SV2A racetam binding site.…”

Synaptic Vesicle Protein 2 (SV2) Isoforms

“…The importance of SV receptors is more evident when this cellular membrane receptor is bound to neurotransmissors, drugs and neurotoxins, such as tetanus toxin and botulinium neurotoxin (BoNTA), especially the type A (BoNTA), that in therapeutic doses and for endocytosis lets to improve conditions as epilepsy and cancer in a more specific manner (Arnon et al, 2001;Karsenty et al, 2009;de Groot et al, 2010;Montal, 2010;Blum et al, 2012;Shi et al, 2011) and lead to apoptosis (Choi et al, 2007). BoNTA has been successfully used in the treatment of refractory detrusor overactivity.…”