Molecular recognition in complexes of TRF proteins with telomeric DNA

Abstract

Telomeres are specialized nucleoprotein assemblies that protect the ends of linear chromosomes. In humans and many other species, telomeres consist of tandem TTAGGG repeats bound by a protein complex known as shelterin that remodels telomeric DNA into a protective loop structure and regulates telomere homeostasis. Shelterin recognizes telomeric repeats through its two major components known as Telomere Repeat-Binding Factors, TRF1 and TRF2. These two homologous proteins are therefore essential for the formation and normal function of telomeres. Indeed, TRF1 and TRF2 are implicated in a plethora of different cellular functions and their depletion leads to telomere dysfunction with chromosomal fusions, followed by apoptotic cell death. More specifically, it was found that TRF1 acts as a negative regulator of telomere length, and TRF2 is involved in stabilizing the loop structure. Consequently, these proteins are of great interest, not only because of their key role in telomere maintenance and stability, but also as potential drug targets. In the current study, we investigated the molecular basis of telomeric sequence recognition by TRF1 and TRF2 and their DNA binding mechanism. We used molecular dynamics (MD) to calculate the free energy profiles for binding of TRFs to telomeric DNA. We found that the predicted binding free energies were in good agreement with experimental data. Further, different molecular determinants of binding, such as binding enthalpies and entropies, the hydrogen bonding pattern and changes in surface area, were analyzed to decompose and examine the overall binding free energies at the structural level. With this approach, we were able to draw conclusions regarding the consecutive stages of sequence-specific association, and propose a novel aspartate-dependent mechanism of sequence recognition. Finally, our work demonstrates the applicability of computational MD-based methods to studying protein-DNA interactions.

Figure 1. TRF-DNA complex structure and comparison of the TRF1/TRF2 DBD domains.

(A) A schematic representation of the system used for the calculation of the binding free energy profiles. The DNA dodecamer (5′-GGTTAGGGTTAG-3′) is aligned with the z axis, and the xy-distance between the centers of mass of TRF and DNA serves as a convenient reaction coordinate describing the binding process (for a precise definition of the reaction coordinate, see Methods and Fig. S1). The C-terminal recognition helix interacting with the DNA major groove is shown in green and the N-terminal linker binding within the minor groove is in purple. (B) Distribution of different types of amino acid residues in the TRF2 structure: hydrophobic residues are depicted in white, hydrophilic in green, basic in blue and acidic in red. (C) Differences in amino acid sequence between TRF1 and TRF2 mapped onto the TRF2 structure in a color coded manner: identical amino acids are marked in blue, green denotes conservative substitutions (little change) and red non-conservative substitutions (significant change). (D) Alignment of TRF1 and TRF2 sequences. Color-coding is consistent with panel C. The purple and green highlights denote the N-terminal linker and the C-terminal helix, respectively.

Figure 2. Free energy profiles for binding of TRF1 and TRF2 to telomeric DNA.

Dashed green lines show the boundaries between the three defined bound states and the unbound state (for the corresponding binding free energies, see Table 1).

Figure 3. Direct and water-mediated hydrogen bonds between the TRF proteins and DNA bases.

The pattern of direct and water-mediated hydrogen bonds between the TRF proteins and DNA bases. Only interactions between amino acid residues and nucleic bases are considered, as these base-specific contacts are potentially critical for sequence recognition. Filled circles at individual bases are scaled to reflect the probability that two residues are connected through a direct (orange) or water-mediated (cyan) hydrogen bond (for numeric probability values and hydrogen bond lifetimes, see Table S1 in File S1 and Table S5 in File S1). All contacts that are made with probability 0.05 are included. Grey percentage bars show the probability for a given base to be involved, at any given time, in either direct or water-mediated hydrogen bonds with the protein.

Figure 4. Detailed view of the interface between TRFs and DNA in the sequence-specific complex.

Four amino acid residues important for recognition of telomeric dsDNA are shown explicitly. See legend to Fig. 1A for base color-coding.

Figure 5. Protein entropy changes upon DNA binding.

Conformational entropy per atom for individual protein residues in the tightly-bound complex with telomeric DNA (narrow bars) and in the isolated state (wide bars, colored according to amino acid type), estimated using the quasi-harmonic approximation. Only the regions interacting directly with DNA in the tightly-bound complex are presented: the N-terminal linker (purple) and the C-terminal helix (green).

Figure 6. Spatial probability distributions for the critical residues of the TRF1 C-terminal helix.

High-probability isosurfaces indicate conformations available to the recognition residues in the unbound (A) and tightly-bound (B) state. Distributions for Lys-421, Asp-422 and Arg-425 are plotted in yellow, blue and orange, respectively.

Figure 7. Hydrogen bond profiles for individual TRF residues.

The probability of hydrogen bond formation between individual protein residues and DNA as a function of the distance from the DNA axis. Only the residues for which the probability exceeds 0.2 are presented. The protein structure is subdivided into three separate regions: the N-terminal linker (top), the middle region (middle) and the C-terminal helix (bottom).

Figure 8. Interaction energy profiles for individual protein residues.

Interaction energy (computed as a sum of electrostatic and van der Waals contributions) between the charged amino acid residues and their surroundings (DNA and solvent combined) as a function of the distance between the protein and the DNA axis. The protein structure is subdivided into three separate regions: the N-terminal linker (top), the middle region (middle) and the C-terminal helix (bottom). For a full set of amino acid interaction energies, see Fig. S7.

Figure 9. Solvent accessible surface of TRFs as a function of their distance from the DNA axis.

Hydrophobic (top), hydrophilic (middle) and overall (bottom) solvent accessible surface area of TRF1 and TRF2 as a function of the xy-distance between the protein and the DNA dodecamer.

Figure 10. Orientations of TRF1/TRF2 DBDs with respect to DNA as a function of xy-distance.

Two-dimensional free energy surfaces for the xy-distance and the three rigid-body rotation angles defining the orientation of TRF1 and TRF2 DBD domains with respect to the DNA dodecamer (aligned with the z axis). To calculate the angles, we first obtained a rotation matrix which gives the best-fit of the instantaneous protein structure to the initial one in the tightly-bound complex with DNA. The Rotation matrix was then expressed as a product of three matrices representing rotations about the x, y and z axis and the rotation angles were derived from these matrices.