Molecular basis and quantitative assessment of TRF1 and TRF2 protein interactions with TIN2 and Apollo peptides

Abstract

Shelterin is a six-protein complex (TRF1, TRF2, POT1, RAP1, TIN2, and TPP1) that also functions in smaller subsets in regulation and protection of human telomeres. Two closely related proteins, TRF1 and TRF2, make high-affinity contact directly with double-stranded telomeric DNA and serve as a molecular platform. Protein TIN2 binds to TRF1 and TRF2 dimer-forming domains, whereas Apollo makes interaction only with TRF2. To elucidate the molecular basis of these interactions, we employed molecular dynamics (MD) simulations of TRF1_TRFH-TIN2_TBM and TRF2_TRFH-TIN2_TBM/Apollo_TBM complexes and of the isolated proteins. MD enabled a structural and dynamical comparison of protein-peptide complexes including H-bond interactions and interfacial residues that may regulate TRF protein binding to the given peptides, especially focusing on interactions described in crystallographic data. Residues with a selective function in both TRF1_TRFH and TRF2_TRFH and forming a stable hydrogen bond network with TIN2_TBM or Apollo_TBM peptides were traced. Our study revealed that TIN2_TBM forms a well-defined binding mode with TRF1_TRFH as compared to TRF2_TRFH, and that the binding pocket of TIN2_TBM is deeper for TRF2_TRFH protein than Apollo_TBM. The MD data provide a basis for the reinterpretation of mutational data obtained in crystallographic work for the TRF proteins. Together, the previously determined X-ray structure and our MD provide a detailed view of the TRF-peptide binding mode and the structure of TRF1/2 binding pockets. Particular TRF-peptide interactions are very specific for the formation of each protein-peptide complex, identifying TRF proteins as potential targets for the design of inhibitors/drugs modulating telomere machinery for anticancer therapy.

Keywords: Apollo; Molecular dynamics; Shelterin complex; TIN2; TRF1; TRF2.