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.
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October 2011
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Conference Article|
September 21 2011
Combining modelling and mutagenesis studies of synaptic vesicle protein 2A to identify a series of residues involved in racetam binding
Jiye Shi;
Jiye Shi
1
*UCB Celltech, Branch of UCB Pharma S.A., 208 Bath Road, Slough SL1 3WE, U.K.
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Dina Anderson;
†UCB Celltech, Branch of UCB Pharma S.A., Granta Park, Cambridge CB1 6GS, U.K.
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Berkley A. Lynch;
Berkley A. Lynch
3
†UCB Celltech, Branch of UCB Pharma S.A., Granta Park, Cambridge CB1 6GS, U.K.
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Jean-Gabriel Castaigne;
Jean-Gabriel Castaigne
4
†UCB Celltech, Branch of UCB Pharma S.A., Granta Park, Cambridge CB1 6GS, U.K.
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Patrik Foerch;
Patrik Foerch
‡UCB Pharma S.A., Chemin du Foriest, B-1420 Braine-l'Alleud, Belgium
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Florence Lebon
Florence Lebon
5
‡UCB Pharma S.A., Chemin du Foriest, B-1420 Braine-l'Alleud, Belgium
5To whom correspondence should be addressed (email florence.lebon@ucb.com).
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Publisher: Portland Press Ltd
Received:
May 27 2011
Online ISSN: 1470-8752
Print ISSN: 0300-5127
© The Authors Journal compilation © 2011 Biochemical Society
2011
Biochem Soc Trans (2011) 39 (5): 1341–1347.
Article history
Received:
May 27 2011
Citation
Jiye Shi, Dina Anderson, Berkley A. Lynch, Jean-Gabriel Castaigne, Patrik Foerch, Florence Lebon; Combining modelling and mutagenesis studies of synaptic vesicle protein 2A to identify a series of residues involved in racetam binding. Biochem Soc Trans 1 October 2011; 39 (5): 1341–1347. doi: https://doi.org/10.1042/BST0391341
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