Exploring the interaction of SV2A with racetams using homology modelling, molecular dynamics and site-directed mutagenesis
Lee J. Daniels V. Sands Z.A. Lebon F. Shi J. Biggin P.C. Verma C.
Lee J.
Daniels V.
Sands Z.A.
Lebon F.
Shi J.
Biggin P.C.
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Alternative Title
Abstract
The putative Major Facilitator Superfamily (MFS) transporter, SV2A, is the target for levetiracetam (LEV), which is a successful antiepileptic 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 antiepileptic compounds. Since there is no structure for SV2A, homology modelling can provide insight into the ligandbinding 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 LEVanalogue 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 ongoing design of improved antiepileptic compounds. © 2015 Lee et al.
Keywords
carrier proteins and binding proteins, etiracetam, radioligand, SV2A protein, ucb 30889, unclassified drug, anticonvulsive agent, etiracetam, membrane protein, nerve protein, piracetam, protein binding, SV2A protein, human, amino acid sequence, Article, binding site, controlled study, drug protein binding, drug structure, human, human cell, hydrophobicity, ligand binding, molecular dynamics, nonhuman, protein conformation, radioassay, sequence alignment, sequence analysis, site directed mutagenesis, structural homology, analogs and derivatives, chemistry, genetics, metabolism, molecular genetics, protein secondary structure, sequence homology, Mus, Amino Acid Sequence, Anticonvulsants, Humans, Membrane Glycoproteins, Molecular Dynamics Simulation, Molecular Sequence Data, Mutagenesis, Site-Directed, Nerve Tissue Proteins, Piracetam, Protein Binding, Protein Structure, Secondary, Sequence Homology, Amino Acid
Source Title
PLoS ONE
Publisher
Series/Report No.
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Rights
Attribution 4.0 International
Date
2015
DOI
10.1371/journal.pone.0116589
Type
Article