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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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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).
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pone-0089460-g002: 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).

Mentions: In an attempt to characterize and compare the affinity of TRF1 and TRF2 for telomeric DNA, we used the umbrella sampling (US) method to compute the free energy profiles for the binding of both proteins to the TTAGGG tandem repeat sequence (see Fig. 1A, Fig. S1 and Methods for details). The obtained profiles, shown in Fig. 2, share some common features, the most prominent of which is the overall shape of the free energy landscape that is dominated by electrostatic attraction between the positively charged proteins and the negatively charged DNA molecule. Another similarity is the absence of an activation barrier along the association pathway. This suggests that binding does not require major structural rearrangements of either the proteins or DNA, and the energetic cost of interface desolvation is insignificant in relation to the energy of association. Barrierless free energy profiles also indicate that the attractive interactions between the binding partners compensate for the entropic penalty associated with the loss of translational freedom (proportional to ) and thus may promote the kinetics of binding. Any possible loss of rotational freedom at intermediate xy-distances is also compensated by the electrostatic attraction (in fact, only slight deviations from the uniform distribution of the rotation angles are observed; see further discussion below). For both proteins, the minimum corresponding to a sequence-specific complex is located ∼1.8 nm from the DNA axis. The depth and width of this minimum are however substantially different: TRF1 binds to DNA more tightly and is therefore confined within a deep and narrow potential well, while TRF2 has a markedly broader and more shallow minimum region and can fluctuate more freely in the direction perpendicular to the DNA axis. This difference suggests that the precise nature of the binding mechanism might depend on the type of the protein.


Molecular recognition in complexes of TRF proteins with telomeric DNA.

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

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).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0089460-g002: 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).
Mentions: In an attempt to characterize and compare the affinity of TRF1 and TRF2 for telomeric DNA, we used the umbrella sampling (US) method to compute the free energy profiles for the binding of both proteins to the TTAGGG tandem repeat sequence (see Fig. 1A, Fig. S1 and Methods for details). The obtained profiles, shown in Fig. 2, share some common features, the most prominent of which is the overall shape of the free energy landscape that is dominated by electrostatic attraction between the positively charged proteins and the negatively charged DNA molecule. Another similarity is the absence of an activation barrier along the association pathway. This suggests that binding does not require major structural rearrangements of either the proteins or DNA, and the energetic cost of interface desolvation is insignificant in relation to the energy of association. Barrierless free energy profiles also indicate that the attractive interactions between the binding partners compensate for the entropic penalty associated with the loss of translational freedom (proportional to ) and thus may promote the kinetics of binding. Any possible loss of rotational freedom at intermediate xy-distances is also compensated by the electrostatic attraction (in fact, only slight deviations from the uniform distribution of the rotation angles are observed; see further discussion below). For both proteins, the minimum corresponding to a sequence-specific complex is located ∼1.8 nm from the DNA axis. The depth and width of this minimum are however substantially different: TRF1 binds to DNA more tightly and is therefore confined within a deep and narrow potential well, while TRF2 has a markedly broader and more shallow minimum region and can fluctuate more freely in the direction perpendicular to the DNA axis. This difference suggests that the precise nature of the binding mechanism might depend on the type of the protein.

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