POT1-TPP1 binding stabilizes POT1, promoting efficient telomere maintenance

Abstract

Telomeric POT1-TPP1 binding is critical to telomere maintenance and disruption of this complex may lead to cancer. Current data suggests a reduction of intracellular POT1 levels in the absence of TPP1. Here we provide evidence of POT1 plasticity that contributes to its lack of stability in the absence of TPP1 binding. Structural data reveals inter- and intramolecular POT1C domain flexibility in the absence of TPP1. Thermostability and proteolytic resistance assays show that POT1C and the mutant complex POT1C(Q623H)-TPP1(PBD) are less stable than the wild type POT1C-TPP1(PBD), suggesting that TPP1 binding to POT1 stabilizes POT1C and makes it less accessible to proteasomal degradation in the cell. Disruption of the POT1-TPP1 complex such as through cancer-associated mutations leads to a reduction of intracellular POT1, telomere uncapping, and telomere associated DNA damage response (DDR). DDR in turn leads to senescence or genomic instability and oncogenesis.

Keywords: Cancer; POT1; TPP1; Telomeres.

Graphical abstract

Fig. 1

Structures of POT1C and POT1C-TPP1(PBD). A & B. Primary structures of POT1 and TPP1. Domains and binding partners are indicated. C. POT1C monomer from CF1/2. The four-cysteine cluster (C382, C385, C503, C506) which coordinates a Zn²⁺ ion and stabilizes the two POT1 domains promoting the formation of an elongated bilobal structure, which provides an extensive surface area for TPP1 binding. D. Structure of POT1C-TPP1(PBD) from CF3, CF4 and CF5.

Fig. 2

Structural and DDMP comparison of the POT1C from two different crystal forms (CF1 and CF2). A. Overlay of CF1A (purple), CF1B (violet), CF2A (green) and CF2B (lime). Monomers are aligned to the HJRD. A hinge-like motion is observed between the OB fold and the HJRD of CF1/2A/B. B. 90° rotation of panel A shows a side view of the OB fold and illustrates the rigid domain differences between CF1A/B and CF2A/B. C. 90° rotation of panel B. Intradomain differences between the HJRD domains of the four monomers are shown. These include the loop connecting β5 to β6 and β6 to β7 and form part of the TPP1 binding pocket of POT1C. D. 90° rotation of panel C. E. Alignment of the OB fold shows that there are almost no intradomain motions, other than minor shifts along the solvent accessible loop connecting β14 and β15. F-K. DDMP comparison of the CF1A/B and CF2A/B POT1C monomers further supports the conformational flexibility of the POT1C structure. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Structural comparison of the five POT1C-TPP1(PBD) heterodimers from three distinct crystals forms (CF3, CF4 and CF5). A. Overlay of CF3A/B/C/D, CF4 and CF5 structures (POT1C – blue/yellow; TPP1 – green/red). B. 90° upward rotation of panel A. C. 90° downward rotation of panel A shows the canonical OB binding pocket. D. 90° downward rotation of panel C. E-M. DDMP comparison of the five independent POT1C monomers present in the CF3 and CF4 crystal forms shows the rigidity of POT1C when in complex with TPP1. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Structural comparison of CF1/CF2 (TPP1 free) and CF3/CF4/CF5 (TPP1 bound). A. Structural comparison of CF1, CF2 (both colored grey), and CF4 (POT1 - red; TPP1 - green). Structures are aligned to the HJRD to illustrate the hinge-like motion between the two domains. B. 90° rotation of panel A. C. 90° of panel B. and D. 90° rotation of panel C. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Structural stability of POT1C vs POT1C-TPP1(PBD). A, B & C. B factor analysis of the POT1C and POT1C-TPP1(PBD) structures. The analysis clearly shows that POT1C becomes rigid upon TPP1 complex formation. D. DSF assays of POT1C, POT1C(Q623H)-TPP1(PBD) cancer associated mutant and the wild type POT1C-TPP1(PBD) complex. The results indicate that the wild type POT1C-TPP1(PBD) complex has a much higher melting temperature than POT1C alone and the cancer mutant. E. Proteasomal degradation assays of POT1C, POT1C(Q623H)-TPP1(PBD) cancer associated mutant and the wild type POT1C-TPP1(PBD) complex. The data shows that POT1C and POT1C(Q623H)-TPP1(PBD) are more susceptible to proteasomal degradation compared to wild type POT1C-TPP1(PBD). F. ImageJ intensity plot of the POT1C bands from panel E.