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Exploring the interaction of SV2A with racetams using homology modelling, molecular dynamics and site-directed mutagenesis.

ABSTRACT: 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 ligand-binding 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 on-going design of improved anti-epileptic compounds.

SUBMITTER: Lee J

PROVIDER: S-EPMC4333566 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Exploring the interaction of SV2A with racetams using homology modelling, molecular dynamics and site-directed mutagenesis.

Lee Joanna J Daniels Veronique V Sands Zara A ZA Lebon Florence F Shi Jiye J Biggin Philip C PC

PloS one 20150218 2

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 mode ...[more]

PMID: 25692762

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Project description:BackgroundAminopeptidase B (Ap-B; EC 3.4.11.6) catalyzes the cleavage of basic residues at the N-terminus of peptides and processes glucagon into miniglucagon. The enzyme exhibits, in vitro, a residual ability to hydrolyze leukotriene A4 into the pro-inflammatory lipid mediator leukotriene B4. The potential bi-functional nature of Ap-B is supported by close structural relationships with LTA4 hydrolase (LTA4H ; EC 3.3.2.6). A structure-function analysis is necessary for the detailed understanding of the enzymatic mechanisms of Ap-B and to design inhibitors, which could be used to determine the complete in vivo functions of the enzyme.ResultsThe rat Ap-B cDNA was expressed in E. coli and the purified recombinant enzyme was characterized. 18 mutants of the H325EXXHX18E348 Zn2+-binding motif were constructed and expressed. All mutations were found to abolish the aminopeptidase activity. A multiple alignment of 500 sequences of the M1 family of aminopeptidases was performed to identify 3 sub-families of exopeptidases and to build a structural model of Ap-B using the x-ray structure of LTA4H as a template. Although the 3D structures of the two enzymes resemble each other, they differ in certain details. The role that a loop, delimiting the active center of Ap-B, plays in discriminating basic substrates, as well as the function of consensus motifs, such as RNP1 and Armadillo domain are discussed. Examination of electrostatic potentials and hydrophobic patches revealed important differences between Ap-B and LTA4H and suggests that Ap-B is involved in protein-protein interactions.ConclusionAlignment of the primary structures of the M1 family members clearly demonstrates the existence of different sub-families and highlights crucial residues in the enzymatic activity of the whole family. E. coli recombinant enzyme and Ap-B structural model constitute powerful tools for investigating the importance and possible roles of these conserved residues in Ap-B, LTA4H and M1 aminopeptidase catalytic sites and to gain new insight into their physiological functions. Analysis of Ap-B structural model indicates that several interactions between Ap-B and proteins can occur and suggests that endopeptidases might form a complex with Ap-B during hormone processing.

Project description:BackgroundDefensins comprise a group of antimicrobial peptides, widely recognized as important elements of the innate immune system in both animals and plants. Cationicity, rather than the secondary structure, is believed to be the major factor defining the antimicrobial activity of defensins. To test this hypothesis and to improve the activity of the newly identified avian beta-defensin Apl_AvBD2 by enhancing the cationicity, we performed in silico site directed mutagenesis, keeping the predicted secondary structure intact. Molecular dynamics (MD) simulation studies were done to predict the activity. Mutant proteins were made by in vitro site directed mutagenesis and recombinant protein expression, and tested for antimicrobial activity to confirm the results obtained in MD simulation analysis.ResultsMD simulation revealed subtle, but critical, structural variations between the wild type Apl_AvBD2 and the more cationic in silico mutants, which were not detected in the initial structural prediction by homology modelling. The C-terminal cationic 'claw' region, important in antimicrobial activity, which was intact in the wild type, showed changes in shape and orientation in all the mutant peptides. Mutant peptides also showed increased solvent accessible surface area and more number of hydrogen bonds with the surrounding water molecules. In functional studies, the Escherichia coli expressed, purified recombinant mutant proteins showed total loss of antimicrobial activity compared to the wild type protein.ConclusionThe study revealed that cationicity alone is not the determining factor in the microbicidal activity of antimicrobial peptides. Factors affecting the molecular dynamics such as hydrophobicity, electrostatic interactions and the potential for oligomerization may also play fundamental roles. It points to the usefulness of MD simulation studies in successful engineering of antimicrobial peptides for improved activity and other desirable functions.

Dataset Information

Exploring the interaction of SV2A with racetams using homology modelling, molecular dynamics and site-directed mutagenesis.

Publications

Exploring the interaction of SV2A with racetams using homology modelling, molecular dynamics and site-directed mutagenesis.

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