Evaluating FOXP2 homo-oligomerisation interfaces and their role in structure and DNA binding

Abstract

Forkhead box P2 (FOXP2) is a transcription factor involved in neural development, as well as the development of the gut, lung, and brain. Mutations in FOXP2 are associated with various diseases including developmental verbal dyspraxia, schizophrenia, and both breast cancer and prostate cancer, two of the most prevalent cancers worldwide. FOXP2 is a large multi-domain protein, containing a glutamine-rich region, zinc finger domain, leucine zipper domain, and DNA-binding forkhead domain. Like other FOXP proteins, FOXP2 is able to form homo-and hetero-oligomers, and FOXP subfamily members undergo oligomerisation-dependent DNA binding and transcriptional activity, a phenomenon that is unique to the FOXP subfamily of FOX transcription factors. Previous studies have suggested that only the leucine zipper is necessary for FOXP oligomerisation. However, the FOXP forkhead domain has the unique ability to form domain-swapped dimers, providing an additional interface for protein-protein interactions. The effects of a disruption to the domain-swapped dimer have only been studied on the FOXP2 forkhead domain. In this study of FOXP2, the R401D mutation of the leucine zipper and the A539P mutation of the forkhead domain were used to investigate the contributions of each interface to FOXP2 homo-oligomerisation, and to explore the effects of disruptions to each interface on FOXP2 structure and DNA binding. Constructs used in this study comprise the leucine zipper to the C-terminal end of the protein (leucinezipper-end). The effects of the mutations were established by comparisons of the mutants with wild-type FOXP2 (leucine zipper-end) and the combined effects of the mutations were investigated using the R401D/A539P FOXP2 (leucine zipper -end) construct. In silico predictions did not predict any binding sites other than the leucine zipper and forkhead domain of FOXP2 (leucine zipper -end), therefore these two domains formed the only interfaces for protein-protein and protein-DNA interactions. The A539P mutation alone had no apparent effects on the oligomeric state of the protein, since according to size exclusion chromatography both wild-type and A539P FOXP2 (leucine zipper -end) existed as a single large species of approximately hexameric size. R401D FOXP2 (leucine zipper -end) was observed to form an intermediate species of approximately trimeric size. The effects of the A539P mutation were apparent in R401D/A539P FOXP2 (leucine zipper -end), which existed as a mixture of the intermediate species and a small species of approximately dimeric size. Electrophoretic mobility shift assays showed that the same oligomeric states occurred upon DNA binding. In silico methods predicted that all FOXP2 (leucine zipper -end) variants shared similar secondary structures, and were predominantly disordered, which was confirmed with far-UV circular dichroism spectropolarimetry. The large proportion of disordered regions increases the propensity of FOXP2 (leucine zipper -end) for protein-protein interactions, and confers conformational freedom on the protein to facilitate any structural rearrangements required for protein-protein and protein-DNA interactions. Fluorescence anisotropy DNA-binding assays showed that although the R401D mutation decreased the DNA-binding affinity of FOXP2 (leucine zipper -end), the decrease was less significant than that observed for A539P FOXP2 (LeuZip -end). This could be due to the R401D mutation not preventing leucine zipper associations completely, since R401D FOXP2 (leucine zipper -end) was not monomeric. Despite adopting the same quaternary structure as the wild-type, A539P FOXP2 (leucine zipper -end) had a significantly weaker affinity for DNA, emphasising the importance of forkhead domain domain-swapping for DNA binding and revealing that the formation of a hexamer specifically was not significant for DNA binding. R401D/A539P FOXP2 (leucine zipper -end) had the weakest affinity for DNA, due to the combined effects of disruptions at both the leucine zipper and forkhead domain interfaces. Intrinsic tryptophan fluorescence spectroscopy of wild-type FOXP2 (leucine zipper -end) was the most quenched upon DNA binding, under non-reducing conditions. Since wild-type FOXP2 (leucine zipper -end) had the highest affinity for DNA, this could be due to the formation of additional DNA-protein contacts that were not present in the mutants, or due to the binding of more protein molecules to DNA resulting in an enhanced quenching effect. Thermal unfolding revealed that wild-type FOXP2 (leucine zipper -end) was the least stable variant, whereas R401D/A539P FOXP2 (leucine zipper -end) was the most stable, possibly indicating a more transient hexameric association and more stable dimeric association. Stern-Volmer constants showed that hexameric wild-type and A539P FOXP2 (leucine zipper -end) experienced the largest conformational changes upon DNA binding. Although the R401D/A539P mutant experienced a conformational change upon DNA binding, the change was not significant. In addition, although the forkhead domain itself is more structured upon DNA binding, far-UV circular dichroism spectropolarimetry showed an increase in global disorder upon DNA binding, under non-reducing conditions, possibly increasing the propensity for protein-protein interactions. These results suggest that the FOXP2 (leucine zipper -end) hexamer could dissociate – possibly into the more stable dimeric form – in the presence of DNA in order to efficiently locate the appropriate cis-regulatory element and then re-associate upon binding DNA. The possible existence of multiple oligomeric states could regulate FOXP2-controlled gene expression