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Molecular recognition in complexes of TRF proteins with telomeric DNA.

Wieczór M, Tobiszewski A, Wityk P, Tomiczek B, Czub J - PLoS ONE (2014)

Bottom Line: More specifically, it was found that TRF1 acts as a negative regulator of telomere length, and TRF2 is involved in stabilizing the loop structure.We found that the predicted binding free energies were in good agreement with experimental data.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physical Chemistry, Faculty of Chemistry, Gdansk University of Technology, Gdansk, Poland.

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.

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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.
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pone-0089460-g001: 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.

Mentions: The DNA-binding domain of the TRF proteins is a tri-helical bundle with the C-terminal helix fitting into the DNA major groove and the N-terminal end, in the linker region, binding to the minor groove (Fig. 1). The NMR [17], [18] and X-ray [19] structures of the TRF1 and TRF2 DBDs (which for simplicity will be referred to as TRF1 and TRF2) bound to ds telomeric DNA have been solved, providing insight into the molecular basis of recognition and binding specificity. DNA-binding constants determined for TRF1 and TRF2 and their several mutants by means of surface plasmon resonance allowed to identify a number of residues important for specific interactions with telomeric DNA [18]. However, macromolecular binding is essentially a dynamic process, and its overall thermodynamic and kinetic description (e.g., experimental binding and rate constants) cannot be rigorously explained at the molecular level using only few microscopic snapshots from structural studies. Rather, such an explanation requires adequate sampling of the equilibrium ensemble along the binding pathway and including the effect of thermally-induced structural fluctuations of the individual residues and solvent at the flexible recognition interface. So far, however, no such detailed information on the binding mechanism of the TRF proteins to DNA is available, which necessarily limits our understanding of the origins of telomere structural stability. Moreover, certain G-triplet specific intercalators were found to disrupt the specific interaction of TRFs with telomeric DNA, which in turn was shown to trigger apoptosis [20], [21]. This suggests that a deeper insight into the molecular determinants of specificity in the TRF-DNA complexes could facilitate further development of potential therapeutics.


Molecular recognition in complexes of TRF proteins with telomeric DNA.

Wieczór M, Tobiszewski A, Wityk P, Tomiczek B, Czub J - PLoS ONE (2014)

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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3935891&req=5

pone-0089460-g001: 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.
Mentions: The DNA-binding domain of the TRF proteins is a tri-helical bundle with the C-terminal helix fitting into the DNA major groove and the N-terminal end, in the linker region, binding to the minor groove (Fig. 1). The NMR [17], [18] and X-ray [19] structures of the TRF1 and TRF2 DBDs (which for simplicity will be referred to as TRF1 and TRF2) bound to ds telomeric DNA have been solved, providing insight into the molecular basis of recognition and binding specificity. DNA-binding constants determined for TRF1 and TRF2 and their several mutants by means of surface plasmon resonance allowed to identify a number of residues important for specific interactions with telomeric DNA [18]. However, macromolecular binding is essentially a dynamic process, and its overall thermodynamic and kinetic description (e.g., experimental binding and rate constants) cannot be rigorously explained at the molecular level using only few microscopic snapshots from structural studies. Rather, such an explanation requires adequate sampling of the equilibrium ensemble along the binding pathway and including the effect of thermally-induced structural fluctuations of the individual residues and solvent at the flexible recognition interface. So far, however, no such detailed information on the binding mechanism of the TRF proteins to DNA is available, which necessarily limits our understanding of the origins of telomere structural stability. Moreover, certain G-triplet specific intercalators were found to disrupt the specific interaction of TRFs with telomeric DNA, which in turn was shown to trigger apoptosis [20], [21]. This suggests that a deeper insight into the molecular determinants of specificity in the TRF-DNA complexes could facilitate further development of potential therapeutics.

Bottom Line: More specifically, it was found that TRF1 acts as a negative regulator of telomere length, and TRF2 is involved in stabilizing the loop structure.We found that the predicted binding free energies were in good agreement with experimental data.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physical Chemistry, Faculty of Chemistry, Gdansk University of Technology, Gdansk, Poland.

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.

Show MeSH
Related in: MedlinePlus