Exploring the Interaction of SV 2 A with Racetams Using Homology Modelling , Molecular Dynamics and Site-Directed Mutagenesis

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 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 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 ongoing design of improved anti-epileptic compounds. PLOS ONE | DOI:10.1371/journal.pone.0116589 February 18, 2015 1 / 15 OPEN ACCESS Citation: Lee J, Daniels V, Sands ZA, Lebon F, Shi J, Biggin PC (2015) Exploring the Interaction of SV2A with Racetams Using Homology Modelling, Molecular Dynamics and Site-Directed Mutagenesis. PLoS ONE 10(2): e0116589. doi:10.1371/journal. pone.0116589 Academic Editor: Chandra Verma, Bioinformatics Institute, SINGAPORE Received: August 13, 2014 Accepted: December 11, 2014 Published: February 18, 2015 Copyright: © 2015 Lee et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: Joanna Lee is a BBSRC-funded student in receipt of additional financial support from UCB BioPharma SPRL. Zara Sands, Florence Lebon and Jiye Shi are all employees of UCB BioPharma SPRL. During the course of the research reported herein, Veronique Daniels was an employee of UCB BioPharma SPRL and her research was supported by a grant from the Walloon Region (Belgium) – DGO6 (Convention 6536). There are no patents or products in development or marketed products to