Lee J.Daniels V.Sands Z.A.Lebon F.Shi J.Biggin P.C.Verma C.BIOLOGY2019-11-072019-11-072015Lee J., Daniels V., Sands Z.A., Lebon F., Shi J., Biggin P.C., Verma C. (2015). Exploring the interaction of SV2A with racetams using homology modelling, molecular dynamics and site-directed mutagenesis. PLoS ONE 10 (2) : e0116589. ScholarBank@NUS Repository. https://doi.org/10.1371/journal.pone.011658919326203https://scholarbank.nus.edu.sg/handle/10635/161744The 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.Attribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/carrier proteins and binding proteinsetiracetamradioligandSV2A proteinucb 30889unclassified druganticonvulsive agentetiracetammembrane proteinnerve proteinpiracetamprotein bindingSV2A protein, humanamino acid sequenceArticlebinding sitecontrolled studydrug protein bindingdrug structurehumanhuman cellhydrophobicityligand bindingmolecular dynamicsnonhumanprotein conformationradioassaysequence alignmentsequence analysissite directed mutagenesisstructural homologyanalogs and derivativeschemistrygeneticsmetabolismmolecular geneticsprotein secondary structuresequence homologyMusAmino Acid SequenceAnticonvulsantsHumansMembrane GlycoproteinsMolecular Dynamics SimulationMolecular Sequence DataMutagenesis, Site-DirectedNerve Tissue ProteinsPiracetamProtein BindingProtein Structure, SecondarySequence Homology, Amino AcidArticle