The crystal structure of human forkhead box N1 in complex with DNA reveals the structural basis for forkhead box family specificity

Joseph A Newman,Hazel Aitkenhead,Angeline E Gavard,Ioanna A Rota,Adam E Handel,Georg A Hollander,Opher Gileadi

doi:10.1074/jbc.ra119.010365

Abstract

Forkhead box N1 (FOXN1) is a member of the forkhead box family of transcription factors and plays an important role in thymic epithelial cell differentiation and development. FOXN1 mutations in humans and mice give rise to the “nude” phenotype, which is marked by athymia. FOXN1 belongs to a subset of the FOX family that recognizes an alternative forkhead-like (FHL) consensus sequence (GACGC) that is different from the more widely recognized forkhead (FKH) sequence RYAAAYA (where R is purine, and Y is pyrimidine). Here, we present the FOXN1 structure in complex with DNA containing an FHL motif at 1.6 Å resolution, in which the DNA sequence is recognized by a mixture of direct and water-mediated contacts provided by residues in an α-helix inserted in the DNA major groove (the recognition helix). Comparisons with the structure of other FOX family members revealed that the FKH and FHL DNA sequences are bound in two distinct modes, with partially different registers for the protein DNA contacts. We identified a single alternative rotamer within the recognition helix itself as an important determinant of DNA specificity and found protein sequence features in the recognition helix that could be used to predict the specificity of other FOX family members. Finally, we demonstrate that the C-terminal region of FOXN1 is required for high-affinity DNA binding and that FOXN1 has a significantly reduced affinity for DNA that contains 5′-methylcytosine, which may have implications for the role of FOXN1 in thymic involution.

Highlights

Forkhead box N1 (FOXN1) is a member of the forkhead box family of transcription factors and plays an important role in thymic epithelial cell differentiation and development
We present the FOXN1 structure in complex with DNA containing an FHL motif at 1.6 Å resolution, in which the DNA sequence is recognized by a mixture of direct and water-mediated contacts provided by residues in an ␣-helix inserted in the DNA major groove
FOXN1 is a FOX family transcription factor that primarily functions as an activator regulating the development of epithelial cells in skin and thymus

Summary

Results and discussion

The structure of the forkhead domain (residues 270 –366) of human FOXN1 was determined in the presence and absence of DNA at 1.6 and 2.7 Å resolution, respectively. The third base pair (C–G) is recognized by FOXN1 via a direct hydrogen bond to the guanine N7 donated by the ND1 of His321, together with close van der Waals contacts to the cytosine C5, which, if replaced by a thymine, would cause steric clashes with the side chain of Ser318 and the main chain carbonyl of Gly314 (Fig. 2C) Given that this position forms one of two CpG sites on the motif, we expect that FOXN1 would, by the same mechanism, be unable to bind to 5-methylcytosine at this position. The sixth position of the FKH motif is commonly cytosine, thymine is possible In both FOXN1 (position 5) and FKH recognition, this is achieved via hydrogen bonds from Arg320 to the complementary guanine or adenine, in the latter the distance between the guanidinium group and nucleobase is too far to make a bidentate interaction, the more relaxed specificity (Fig. 4, bottom right panel). In agreement with this notion, constructs containing the C terminus appeared to behave as multimers in gel filtration, the likely disordered nature of the C terminus would complicate this analysis and its interpretation

Structural determinants of FHL binding

Experimental procedures

Crystallization and structure determination