RESUMEN
FOX proteins are a superfamily of transcription factors which share a DNA-binding domain referred to as the forkhead domain. Our focus is on the FOXP subfamily members, which are involved in language and cognition amongst other things. The FOXP proteins contain a conserved zinc finger and a leucine zipper motif in addition to the forkhead domain. The remainder of the sequence is predicted to be unstructured and includes an acidic C-terminal tail. In the present study, we aim to investigate how both the structured and unstructured regions of the sequence cooperate so as to enable FOXP proteins to perform their function. We do this by studying the effect of these regions on both oligomerisation and DNA binding. Structurally, the FOXP proteins appear to be comparatively globular with a high proportion of helical structure. The proteins multimerise via the leucine zipper, and the stability of the multimers is controlled by the unstructured interlinking sequence including the acid rich tail. FOXP2 is more compact than FOXP1, has a greater propensity to form higher order oligomers, and binds DNA with stronger affinity. We conclude that while the forkhead domain is necessary for DNA binding, the affinity of the binding event is attributable to the leucine zipper, and the unstructured regions play a significant role in the specificity of binding. The acid rich tail forms specific contacts with the forkhead domain which may influence oligomerisation and DNA binding, and therefore the acid rich tail may play an important regulatory role in FOXP transcription.
Asunto(s)
ADN/metabolismo , Factores de Transcripción Forkhead/química , Factores de Transcripción Forkhead/metabolismo , Biopolímeros/química , Biopolímeros/metabolismo , Cromatografía en Gel , Dicroismo Circular , Leucina Zippers , Unión Proteica , Dominios Proteicos , Espectrometría de Fluorescencia , Espectrofotometría Ultravioleta , Relación Estructura-ActividadRESUMEN
FOXP2 is a transcriptional repressor involved in development of the human brain and is the first gene product to be linked to the evolution of human speech. FOXP2 belongs to the FOX superfamily of proteins that share a common winged helix DNA binding domain - the forkhead domain. A divalent cation (Mg2+ or Ca2+) has been identified bound to a group of highly conserved residues in a number of FOX forkhead domain crystal structures. This work aims to investigate the role of the conserved divalent cation binding site by studying both the structure and DNA-binding function of the FOXP2 forkhead domain when in the presence and absence of either cation (Mg2+or Ca2+). The presence of the cations does not significantly alter the structure of the apo-FOXP2 forkhead domain. However, when in the presence of a cognate oligonucleotide sequence, differences are observed upon addition of divalent cation. These differences occur both in the structure and in the thermodynamic DNA binding signature of the FOXP2 forkhead domain. The incorporation of molecular dynamics simulations together with the experimental data provides us with sufficient insight so as to propose a possible role for divalent cations in the regulation of DNA binding to FOX transcription factors.