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A Single Amino Acid in the Hinge Loop Region of the FOXP Forkhead Domain is Significant for Dimerisation

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Abstract

The forkhead box (FOX) proteins are a family of transcription factors that interact with DNA via a winged helix motif that forms part of the forkhead domain. The FOXP (FOXP1–4) subfamily is unique in the family in that the forkhead domains of these proteins are able to dimerise via domain swapping. In this event, structural elements are exchanged via extension of the hinge loop region. Despite the high sequence homology among the FOXP subfamily members, the stability of their forkhead domain dimers varies, with FOXP3 forming the most stable dimer. An amino acid difference is observed in the hinge region of the FOXP subfamily where a tyrosine in all members is replaced with a phenylalanine in FOXP3. In this work, the role of phenylalanine at this position in the hinge region was investigated. This was done by creating the Y540F variant of the FOXP2 forkhead domain. The effect of the Y540F mutation on the structure, dimerisation propensity and DNA binding ability of the FOXP subfamily was investigated. The mutation altered the structure of the protein by decreasing the disorder of the backbone as measured by circular dichroism spectroscopy and by altering the local environment of the hinge region as measured by tryptophan fluorescence. The propensity of the forkhead domain to form a dimer was improved ~9.5 fold by the mutation. This was attributed to increased hydrophobicity at the dimer interface as well as altered tension in the hinge loop region. DNA binding assays indicated that the affinity for DNA was decreased by the mutation. Taken together, these findings suggest that domain swapping may modulate DNA binding.

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Abbreviations

CD:

Circular dichroism

EMSA:

Electrophoretic mobility shift assay

FHD:

Forkhead domain

FOX:

Forkhead box family

IPEX:

Immune dysregulation, polyendocrinopathy, enteropathy and X-linked

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Acknowledgments

We extend our gratitude to Lin Chen, University of Southern California, Los Angeles, CA for the generous donation of the pET-30 LIC plasmid encoding FOXP2 FHD. This work was supported by the University of the Witwatersrand and the South African National Research Foundation Grants 80681, 89515 and 64788 from the South African Chairs Initiative of the Department of Science and Technology and National Research Foundation. Any opinion, findings and conclusions or recommendations expressed in this material are those of the author(s) and therefore the National Research Foundation, the Department of Science and Technology does not accept any liability with regard thereto.

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Authors and Affiliations

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

    Kershia Perumal, Heini W. Dirr & Sylvia Fanucchi

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  1. Kershia Perumal
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  2. Heini W. Dirr
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  3. Sylvia Fanucchi
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Correspondence to Sylvia Fanucchi.

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Perumal, K., Dirr, H.W. & Fanucchi, S. A Single Amino Acid in the Hinge Loop Region of the FOXP Forkhead Domain is Significant for Dimerisation. Protein J 34, 111–121 (2015). https://doi.org/10.1007/s10930-015-9603-4

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