Structural and functional analysis of the human POT1-TPP1 telomeric complex

ARTICLE Received 30 Jun 2016 | Accepted 14 Feb 2017 | Published 10 Apr 2017 DOI: 10.1038/ncomms14928 OPEN Structural and functional analysis of the human POT1-TPP1 telomeric complex Cory Rice1,2, Prashanth Krishna Shastrula1, Andrew V. Kossenkov1, Robert Hills1, Duncan M. Baird3, Louise C. Showe1, Tzanko Doukov4, Susan Janicki1 & Emmanuel Skordalakes1,2 POT1 and TPP1 are part of the shelterin complex and are essential for telomere length regulation and maintenance. Naturally occurring mutations of the telomeric POT1–TPP1 complex are implicated in familial glioma, melanoma and chronic lymphocytic leukaemia. Here we report the atomic structure of the interacting portion of the human telomeric POT1– TPP1 complex and suggest how several of these mutations contribute to malignant cancer. The POT1 C-terminus (POT1C) forms a bilobal structure consisting of an OB-fold and a holiday junction resolvase domain. TPP1 consists of several loops and helices involved in extensive interactions with POT1C. Biochemical data shows that several of the cancerassociated mutations, partially disrupt the POT1–TPP1 complex, which affects its ability to bind telomeric DNA efficiently. A defective POT1–TPP1 complex leads to longer and fragile telomeres, which in turn promotes genomic instability and cancer. 1 The Wistar Institute, 3601 Spruce St, Philadelphia, Pennsylvania 19104, USA. 2 Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. 3 Division of Cancer and Genetics, School of Medicine, Cardiff University, Heath Park, Cardiff CF10 3AT, UK. 4 Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Stanford University, Menlo Park, California 94025, USA. Correspondence and requests for materials should be addressed to E.S. (email: ). NATURE COMMUNICATIONS | 8:14928 | DOI: 10.1038/ncomms14928 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14928 S helterin is a hexameric nucleoprotein complex responsible for maintaining the integrity of the ends of our chromosomes, known as telomeres1. Human shelterin consists of TRF1, TRF2, TIN2, RAP1, POT1 and TPP1 (refs 1–3), binds double and single-stranded telomeric DNA and is involved in a wide range of functions4,5. It suppresses DNA damage response by capping and protecting the ends of chromosomes from being recognized as DNA double-strand breaks6,7. It prevents exonuclease degradation by sequestering the telomeric overhang8. It also controls telomere length by regulating access of telomerase to the telomeric overhang9. POT1 has a wide range of functions at telomeres all of which are geared toward maintaining the integrity of the telomeric overhang. POT1 binds single-stranded, telomeric DNA with high affinity and specificity4,10,11. POT1-DNA binding sequesters the telomeric overhang, thus assisting in telomere capping, downregulation of telomere elongation and ATR dependent DNA damage response12–15. POT1 telomeric DNA binding is mediated by the two N-terminal OB-folds of the protein, while the C-terminal portion of the protein binds TPP1 (refs 10,16). Human and S. pombe TPP1 binding to POT1 enhances its DNA binding properties by 10-fold9,11,17,18. During the S-phase of the cell cycle, the human POT1–TPP1 complex recruits telomerase to telomeres9,16,19 via direct contacts of telomerase with the TEL patch located at the N-terminal OB-fold of TPP1 (ref. 20). Binding of POT1 to the telomeric overhang resolves G-quadruplexes and allows for telomerase loading to telomeres for telomere elongation21. It was recently discovered that POT1 is frequently mutated in chronic lymphocytic leukaemia, familial melanoma and glioma22–27. There are currently 137 naturally occurring mutations of POT1 reported to be associated with human disease (cBioPortal for Cancer Genomics). Many of these mutations localize at the N-terminal OB-folds and disrupt POT1-DNA binding, while others are located at the C-terminus of the protein and were TPP1 binds22–25. POT1 N-terminal mutations primarily disrupt DNA-binding and are associated with chromosomal abnormalities such as irregular telomere length, fragile telomeres and chromosome end-to-end fusions, phenotypes usually associated with telomere uncapping22–24,26,27. However, the precise role of human POT1 C-terminal (POT1C) mutations in human disease is currently unclear. Here, we investigate the mechanism of POT1–TPP1 assembly and how naturally occurring POT1 mutations contribute to cancer. Our data shows that POT1C consists of two domains, an OB-fold and a holiday junction resolvase (HJR) domain both of which make extensive interactions with TPP1 forming a tight heterodimer. Inspection of the structure reveals that several of these mutations either perturb the POT1C fold and/or disrupt POT1–TPP1 binding. Altering the natural state of the POT1–TPP1 complex affects the integrity of the telomeric overhang, leading to chromosomal abnormalities associated with a dysfunctional telomere capping complex leading to genomic instability and cancer. Results Structure of the human POT1–TPP1 complex. We generated the interacting domains of human POT1 and TPP1 by limited proteolysis and mass spec analysis of the full length POT1–TPP1 complex (Fig. 1a,b). POT1C consists of residues 330–634 and TPP1 255–337 (TPP1(PBD)) (Fig. 1a,b). We solved the structure by the single wavelength anomalous dispersion (SAD) method and a Hg derivative (Table 1). The map showed clear electron density for POT1C residues 332–633 and TPP1(PBD) residues 266–326 (Fig. 1c). 2 POT1C consists of a classic OB-fold and a holiday junction resolvase domain (HJR) (Fig. 1d). The POT1C(OB) is a canonical OB-fold and is structurally most similar to the Oxytricha Nova Telomere End-Binding Protein (TEBPa, PDB ID: 1OTC—RMSD ¼ 2.2 Å—Fig. 1e). It is worth noting that an overlay of the TEBP alpha and beta dimer (PDB ID: 1OTC (ref. 28)) with that of the POT1–TPP1 structure shows no similarities in the organization of the two heterodimers. The organization of the six beta strands of the OB-fold forms a deep and well defined indentation on the surface of the protein, which comprises the canonical binding pocket of an OB-fold (Fig. 1d). Interestingly, the HJR is an insertion of the OB-fold and comprises residues P392-L538. The HJR is structurally similar to the Archaeoglobus fulgidus resolvase domain (AfHJR, PDB:ID 2WIW) with an RMSD 2.7 Å (Fig. 1f). HJR consists of seven antiparallel beta-strands surrounded by five alpha helices (a3, 4, 5, 6, 7) (Fig. 1d,f). Structural comparison of POT1C (HJR) with the AfHJR domain highlighted two distinct differences between these two HJR domains. One difference lies with the organization of the helices present in both HJR domains with four out of the five helices not overlapping (Fig. 1f). Another striking difference between the two HJR domains lies with the DNA binding pocket of the HJR. In AfHJR do (...truncated)