Electrostatic hot spot on DNA-binding domains mediates phosphate desolvation and the pre-organization of specificity determinant side chains

Abstract

A major obstacle towards elucidating the molecular basis of transcriptional regulation is the lack of a detailed understanding of the interplay between non-specific and specific protein-DNA interactions. Based on molecular dynamics simulations of C(2)H(2) zinc fingers (ZFs) and engrailed homeodomain transcription factors (TFs), we show that each of the studied DNA-binding domains has a set of highly constrained side chains in preset configurations ready to form hydrogen bonds with the DNA backbone. Interestingly, those domains that bury their recognition helix into the major groove are found to have an electrostatic hot spot for Cl(-) ions located on the same binding cavity as the most buried DNA phosphate. The spot is characterized by three protein hydrogen bond donors, often including two basic side chains. If bound, Cl(-) ions, likely mimicking phosphates, steer side chains that end up forming specific contacts with bases into bound-like conformations. These findings are consistent with a multi-step DNA-binding mechanism in which a pre-organized set of TF side chains assist in the desolvation of phosphates into well defined sites, prompting the re-organization of specificity determining side chains into conformations suitable for the recognition of their cognate sequence.

Figure 1.

EGR–DNA complex and protein–DNA interactions. (A) Cartoon of EGR bound to DNA [PDB code: 1AAY (39)]. Fingers I, II and III are color red, green and blue, respectively. (B) Schematics diagram showing key side chain–base interactions between EGR and its target DNA site at positions −1, +3 and +6 of binding helix.

Figure 2.

Increasing bound-like behavior of EGR specificity determinant residue Arg₊₆ in FI as a function of ion concentration. Change in RMSD of Arg₊₆ with respect to its bound conformation as a function of time and increasing number of counter ions in the simulation box: (A) 20 ions; (B) 16 ions; (C) 12 ions; (D) 8 ions; (E) 4 ions; (F) No ions. The histograms show the distribution of the RMSDs in each simulation, reflecting the increasing amount of bound-like behavior of Arg₊₆ as a function of ion concentration.

Figure 3.

Differential ion dependence of bound-like behavior of key side chains in EGR fingers I and III. The effective ionic strength in millimolar corresponds to the number of ions × 11.5. The conformations are considered bound-like if the RMSD from the crystal structure conformation is under 2 Å. (A) Fraction of bound-like conformations for buried Arg₊₆ in finger I. (B) Fraction of bound-like conformations for buried Arg₋₁ in finger III. (C) Fraction of bound-like conformations for exposed Arg₋₁ in finger I. (D) Fraction of bound-like conformations for exposed Arg₊₆ in finger III. Error bars are the direct standard deviation from three or more independent four nanosecond MD simulations. Note that in the co-crystal both Arg₋₁s and Arg₊₆s have identical structures. However, side chain dynamics is more bound-like if side chain is found buried in the complex relative to if it is found exposed to solvent.

Figure 4.

Ions occupy the phosphate binding site in the protein–DNA complex. Distribution of counter ion positions in fingers I–III of EGR and in the engrailed homeodomain relative to the phosphorus atom position in their corresponding co-crystals. The cartoon representations of each domain are on the right: DNA phosphorus atom shown in orange spheres; Cys, His residues and binding motif residues are shown as sticks; CPK colored sticks show the crystal conformation; and, colored sticks show a snapshot of the position of key side chains. A blue sphere shows for each domain the position of the Cl⁻ in the simulation that had the largest residence time using a 3 Å clustering radius. (A) Finger I (red), for comparison we also show the distribution with respect to a second phosphate binding site; (B) Finger II (green); (C) Finger III (blue); (D) Engrailed homeodomain, phosphorus atom is shown in yellow sphere. DNA is shown as yellow sticks. The two phosphate backbone contacting residues are Leu26, Arg31 and Arg53 are shown in light blue sticks. The contacts are depicted as pink dashed lines. For comparison we also show the distribution with respect to a second phosphate binding site. We find only one ion hot spot per domain, corresponding to the locus of the phosphate that buries the largest amount of SASA upon binding.