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A Phosphomimetic Study Implicates Ser557 in Regulation of FOXP2 DNA Binding

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Abstract

FOXP2 is a transcription factor expressed in multiple tissues during embryonic development. FOXP2 regulates transcription by binding to DNA at its DNA binding domain, the forkhead domain (FHD) through the recognition helix. Ser557 is a residue located within the recognition helix that has the potential to become phosphorylated posttranslationally. In this study we investigated whether phosphorylation of Ser557 can influence the structure and DNA binding of the FOXP2 FHD. We did this by constructing S557E, a phosphomimetic mutant, and comparing its behaviour to the wild type. The mutation did not affect the secondary or tertiary structure of the protein although it did decrease the propensity of the FOXP2 FHD to form dimers. Most notably, the mutation showed significantly reduced DNA binding compared to the wild type as detected using electrophoretic mobility shift assays. Molecular docking was also performed in which the wild type, phosphomimetic mutant and phosphorylated wild-type were docked to DNA and their interactions with DNA were compared. These results indicated that the wild type forms more interactions with the DNA and that the phosphomimetic mutant as well as the phosphorylated wild type did not associate as favourably with the DNA. This indicates that phosphorylation of Ser557 could disrupt DNA binding likely due to electrostatic and steric hindrance. This suggests that phosphorylation of Ser557 in the FOXP2 FHD could act as a control mechanism for FOXP2 and ultimately could be involved in regulation of transcription.

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Acknowledgements

This work was supported by the University of the Witwatersrand, South African National Research Foundation Grants 80681 and 68898 to S.F. and H.W.D. respectively, the South African Research Chairs Initiative of the Department of Science and Technology Grant 64788 to H.W.D., and the Medical Research Council of South Africa.

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  1. Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, 2050, South Africa

    Ashleigh Blane, Heini W. Dirr & Sylvia Fanucchi

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  1. Ashleigh Blane
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Correspondence to Sylvia Fanucchi.

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Blane, A., Dirr, H.W. & Fanucchi, S. A Phosphomimetic Study Implicates Ser557 in Regulation of FOXP2 DNA Binding. Protein J 37, 311–323 (2018). https://doi.org/10.1007/s10930-018-9777-7

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