Molecular Models of STAT5A Tetramers Complexed to DNA Predict Relative Genome-Wide Frequencies of the Spacing between the Two Dimer Binding Motifs of the Tetramer Binding Sites

Abstract

STAT proteins bind DNA as dimers and tetramers to control cellular development, differentiation, survival, and expansion. The tetramer binding sites are comprised of two dimer-binding sites repeated in tandem. The genome-wide distribution of the spacings between the dimer binding sites shows a distinctive, non-random pattern. Here, we report on estimating the feasibility of building possible molecular models of STAT5A tetramers bound to a DNA double helix with all possible spacings between the dimer binding sites. We found that the calculated feasibility estimates correlated well with the experimentally measured frequency of tetramer-binding sites. This suggests that the feasibility of forming the tetramer complex was a major factor in the evolution of this DNA sequence variation.

Fig 1. The domain structure of STAT proteins.

(A) Names and positions of the six domains on the primary sequence: NTD (N-terminal domain), CCD (4-helix bundle coiled-coil domain), DBD (DNA-binding domain), LD (linker or connector domain), SH2, and TAD (transactivation domain). We treated the DBD and LD as one combined domain, as SCOP [17] does for STAT3β. There is a short flexible 13-residue linker between the NTD and CCD, which partly overlaps with the NTD. There is also a phospho-tyrosine containing segment (PTS) between the SH2 and TAD, which we combined with the TAD. See Methods for more detail. (B) The sequence of mouse STAT5A (NCBI Accn# CAA88419.1). The residues are highlighted according to the domains to which they belong, using the same coloring scheme used in (A). The 13-residue linker is boxed. The phospho-tyrosine residue (Y694) and the two following residues (V695 and K696) are shown in red. (C) STAT5A domains in the modeled structure of the STAT5A core, which includes the CCD, DBD, LD, SH2 and the residues 694–696 of the PTS. Dotted line represents the connecting residues (685–693) that were not included in the model.

Fig 2. A flow chart of the model building procedure.

Fig 3

Distances between the N-terminal Cα atoms of two core monomers D-A’ (blue), A-D’ (green), and A-A’ or D-D’ (red) at the CTCD values indicated on the x-axis. The distance between the monomers D-D’ is the same as that between the monomers A-A’. The dotted line is at the distance of 136 Å, which is an expected upper limit of distances that can be spanned by an NDD with two 13-mer peptides. See Methods for the procedure used to calculate these distances.

Fig 4. Two examples of the modeled tetramer structure.

Left panels (NDD on-axis): The plan view (A) and elevation (B) of the tetramer model at the CTCD of 16; the horizontal white line is the 2-fold symmetry axis. Right panels (NDD off-axis): The plan view (C) and elevation (D) of the tetramer model at the CTCD of 21; the 2-fold axis is vertical in (C) and along the viewing direction in (D). In each panel, DNA is white; the core monomers are green (core A), blue (core D), magenta (A’), and red (D’); and NDDs are yellow. The linkers between the C-terminals of the NDD and the N-terminals of the core are indicated as dotted yellow lines.

Fig 5

Computed scaled feasibility measures of the tetramer formation vs. CTCD using two NDDs (A) or at least one NDD (B). The red and blue points are for the ‘eclipsed’ and the ‘staggered’ tetramers, respectively, with the NDD off- and on-axis, respectively. The red and blue points were scaled so as to put them on the same scale as the experimental counts (shown in thin blue line) of tetramer binding sites in the mouse genome. For this figure, we used the DNA with 10.5 bps per helical turn. The results using the 10.0 bp DNA are given in Fig 6.

Fig 6

Computed scaled feasibility measures of the tetramer formation vs. CTCD using two NDDs (A) or at least one NDD (B) using the DNA with 10.0 bps per helical turn.

Fig 7. Histograms of the end-to-end distances of 13-residue peptides in protein structures in PDB, using only those peptides with the Blosum62 score greater than 8 (blue), 9 (red) or 10 (green) when compared to the linker sequence (shown in Box in Fig 1B) in STAT5A.