A computational and experimental study of the structure of the C-terminal domain of the wildtype and mutant FOXL1 protein

Abstract

Forkhead box (FOX) proteins are transcription factors that play a significant role during embryonic development and throughout adulthood with functions in regulating cell differentiation, proliferation, and apoptosis. Consequently, mutated or unregulated FOX proteins have been linked with numerous human genetic diseases. Recently, a deletion of five residues (GIPFL) in the C-terminal domain of the FOXL1 protein has been linked to another human genetic disease. This manuscript presents attempts to determine the three-dimensional structure of the C-terminal domain of FOXL1 protein, both with the mutation (FOXL1MUT) and without (FOXL1CTERM), in order to uncover the structural features that prevent the proper functioning of the mutant. This structural information was obtained using both computational and experimental methods. Computationally, coarse-grain molecular dynamic (MD) simulations were performed using replica exchange MD to provide a prediction of the folded structure. Experimentally, the C-terminal domain of FOXL1 and its mutant was expressed, enriched, and then structurally characterized using circular dichroism. Bioinformatics analysis of FOXL1 revealed that the mutation occurred in the most ordered and evolutionary-conserved portion of the C-terminal domain of FOXL1, suggesting that this mutation could severely affect the structure and function of FOXL1CTERM. This is in agreement with the replica exchange molecular dynamics simulations results, which predicted that FOXL1CTERM was more folded and structured than FOXL1MUT. Moreover, the simulations showed that the mutation in the FOXL1MUT system disrupted its structure and hydrophobic core, causing the mutant to have an increased amount of randomly coiled structure. The computational results are supported by preliminary experimental data. Circular dichroism results indicated that FOXL1CTERM has a predominantly helical structure while FOXL1MUT was partially helical with some randomly coiled regions.