Forkhead box protein (FOXP2)

Shane Canfield '20 and Yoshio Wagner '21

Contents:

I. Introduction

The FOXP2 protein, encoded by the FOXP2 gene, is a transcriptional repressor that is responsible for the regulation of many genes. The protein is involved in other important roles such as early brain and neuron development, as well as synaptic plasticity which results in changes in synaptic strength that, over time, helps contribute to memory (Fisher and Scharff 2009). Another and more understood role of FOXP2 is its development of speech and language in vertebrates. Interestingly, FOXP2 was the first gene to be linked to the evolution of human speech (Morris et al. 2018).

FOXP2 is a transcription factor, and more importantly , classified in the Forkhead box family (FOX). Involved in many protein-protein interactions, FOXP2 is involved in regulation of gene expression. Within these protein-protein interactions, the protein binds to the genes in DNA through a specific binding motif called the forkhead domain. FOXP2 regulates up to 300-400 different promoters. Mutations in this forkhead domain are what contributes most to the human diseases seen from a faulty FOXP2 (Lehmann et al. 2003).

II. The Asymmetric Unit

The of FOXP2 consists of six copies of the FOXP2 forkhead domain and 2 DNA segments. There are two separate ways in which the forkhead domains interact with DNA. The first consists of the simple monomeric binding, or one forkhead box bound to DNA. The other is the forkhead dimer , where two forkheads bind together and stick to DNA . These dimers also exhibit base swapping, a feature that will be addressed later. There is a slight difference in binding strength between the monomeric and dimer where the monomeric binds more tightly than the dimers. In total there are two monomeric subunits and two homodimers.

III. The Winged Helix Structure

The winged helix binding domain consists of five (including a ) , and three . The order of the secondary structures from N to C terminus is H1, S1, H2, H4, H3, S2, S3, and H5 respectively. Featured in all proteins of the FOX family, H4 (3-10 helix) is especially challenging to locate because it connects H2 and H3. H5 is vestigial of a �wing� found in other FOX proteins that makes extensive DNA contacts.

IV. Binding Domains

FOXP2 has several conserved binding domains. The discovered domains include a glutamine-rich area, zinc finger , leucine zipper, and of course the forkhead domains. Often called the , the forkhead binding domain is a DNA binding domain specific to FOX proteins.

The helix responsible for the binding of a conserved sequence is H3. H3 binds and recognizes 5�CAAATT3�. H3 binds by wedging itself into

The winged-helix employs the help of other residues to wedge itself even further into the major groove of DNA, using (Optional ). The Asn550 forms a bidentate or water-mediated bond with Ade10. Furthermore, His552 and Arg553 form a water-mediated bond with Thy10� and Thy11� respectively . The main and side chain atoms of Arg553, His554, Ser557, and Leuc558 makes van der Waals contact with Cyt8, Gua8�, Thy9�, Thy10�, Thy11�, Ade12� and Ade13�. The aromatic residues Tyr509 from H1, Leu527, and Tyr531 from H2, and Trp573 from S3 interact with both H3 and the sugar-phosphate backbone. Overall, the winged-helix generously employs many van der Waals, but very few general H-bonds to DNA. These abundant van der Waals interactions are likely the reason that FOXP2 can bind to so many different DNA sites.

On top of the use of all these van der Waals interactions, FOXP2 uses a leucine zipper to bind DNA with high affinity as well. The leucine zipper is essential for the strong silencing abilities of FOXP2.

V. Dimerization

The monomeric forkhead domains form an equilibrium with a ; they do this through domain swapping � the two monomers swap helix and strands S2 and S3. The other forms of FOX proteins have similar structures between H2 and H3, but one amino acid that differentiates FOXP from most FOX proteins is an Alanine at position . Most FOX proteins have a Proline at this site. This change helps form the dimerization, as the interactions allow a to form. Furthermore, a Leucine Zipper around 50 residues to the N terminal of the winged-helix also help to mediate the dimerization. The deletion of this binding sequence inhibits the ability for FOXP2 to form both homo and heterodimers (hetero-dimerization is possible with other FOXP proteins). The dimer is stabilized by internal .

These dimers likely work to loop interchromosomal DNA. The positive charges on the amino acids neutralize the otherwise negative charge of the DNA. This ability to bind to two DNA strands further illustrates the ability of the protein to control the repression of many different silencers through combinatorial control.

VI. A Critical Mutation

The R553H mutation is a mutation that shows a strong correlation to severe congenital speech disorders. makes a water-mediated hydrogen bond with Thy11� and several van der Waals interactions with Thy11�, Ade12�, Ade13�. These interactions are critical for the functionality of the Foxhead binding domain because of R553�s ability to allow for the binding of the Winged-Helix to the DNA.

VII. References

Lehmann, O. J., Sowden, J. C., Carlsson, P., Jordan, T., & Bhattacharya, S. S. (2003). Fox's in development and disease doi://doi.org/10.1016/S0168-9525(03)00111-2

Li, S., Weidenfeld, J., & Morrisey, E. E. (2003). Transcriptional and DNA Binding Activity of the Foxp1/2/4 Family I s Modulated by Heterotypic and Homotypic Protein Interactions. Molecular and Cellular Biology, 24(2), 809-822. doi:10.1128/mcb.24.2.809-822.2004 .

Stroud, J. C., Wu, Y., Bates, D. L., Han, A., Nowick, K., Paabo, S., . . . Chen, L. (2006). Structure of the Forkhead Domain of FOXP2 Bound to DNA. Structure of the Forkhead Domain of FOXP2 Bound to DNA, 14(1), 159-166. doi:10.1016/ j.str.2005.10.005

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