Fig 1: Cryo-EM map at 7.9 Å resolution of POT1 bound by TPP1 (E266-L326) with fitted crystal structures.(A) Simulated 8 Å maps generated in Chimera for known crystal structures of POT1 domains were fitted into the 7.9 Å cryo-EM map to find structural complementarity. Simulated map for the crystal structure of POT1 (OB1-OB2) (PDB ID: 1XJV) (green) after removal of the ssDNA ligand fits the top half of the map, while simulated map for crystal structure of POT1 (OB3/HJR) complexed to the POT1-binding domain (PBD) of TPP1 (E266-L326) (PDB ID: 5UN7) (blue) fits the bottom half of the map. Placement of ssDNA ligand from PDB ID: 1XJV onto map depicts the location of the DNA-binding groove (red). (B) Simulated 8 Å maps for OB1 (magenta) and OB2 (green) demonstrate that improved complementarity of POT1 (OB1-OB2) can be achieved by fitting the OB1 and OB2 domains separately. (C) Cartoon representation of the structure of full-length POT1 (yellow) bound by the PBD of TPP1 (blue) which interacts with the OB3/HJR domain of POT1, fitted into the 7.9 Å cryo-EM map. (D) Fit of crystal structure of POT1 (OB3/HJR) complexed to the PBD of TPP1 shows that the a-helices of TPP1 (blue) overlap perfectly with four complimentary densities in the map (blue density).
Fig 2: EMSA demonstrate that binding of telomeric ssDNA to the POT1 OB1/OB2 domain is not limited to the minimal binding sequence, but to sequences separated by spacers as well.(A) Cartoon representation of the POT1 OB1/2 domains and its possible binding conformations that can allow binding to the minimal tight binding sequence TTAGGGTTAG (top), or the same sequence separated by either a single telomeric repeat (center) or a ~6-nt long non-telomeric spacer (bottom). (B) Horizontal scatter plot with median marking of the apparent binding affinity of various telomeric ligands for the POT1-TPP1-TIN2(1–354) subcomplex. Each individual Kd value was computed from the EMSA data shown in S6B–S6H Fig. The binding experiments confirm that telo64 is truly the minimal tight-binding sequence. Both shorter and longer oligos, with the notable exception of telo65 ligand, present reduced affinities for the subcomplex. (C) Horizontal scatter plot with median marking of the apparent binding affinity of various non-contiguous telomeric ligands for the POT1-TPP1-TIN2(1–354) subcomplex. Each individual Kd value was computed from the EMSA data shown in S6I–S6Q Fig. The binding experiments show that the minimal tight-binding sequence ligands that have been interrupted with various non-telomeric spacers retain binding affinity comparable to that of the contiguous telo64 ligand.
Fig 3: Cryo-EM map at 9.6 Å resolution of POT1 with fitted crystal structures indicates an alternative open conformation.(A) Simulated 8 Å maps generated with Chimera for POT1 OB1 (magenta) and OB2 (green) fitted into the 9.6 Å cryo-EM map suggest the two domains exist in an open/extended conformation. (B) Cartoon representation of full-length POT1 (yellow) bound by the PBD of TPP1 (blue) fitted into the 9.6 Å cryo-EM map. (C) Comparison between closed and open conformations of full-length POT1 complexed to the PBD of TPP1 observed through cryo-EM. The distance between the centres of OB1 and OB2 is increased by about 5 Å in the open conformation compared to the closed conformation.
Fig 4: Composition and architecture of the shelterin complex.(A) Cartoon representation of the full shelterin complex POT1-TPP1-TIN2-TRF2(2×)-RAP1(2×) bound to a telomeric double-stand/singe-stand junction. (B) Interaction map for the shelterin subcomplex POT1-TPP1-TIN2. The OB3/HJR domain of POT1 (orange) interacts with the PDB of TPP1 (blue), while the TBM of TPP1 (blue) interacts with TIN2 through the TRFH domain of TIN2 (green). TIN2 also has a TBM and a DC motif. (C) Published crystal structures of domains of the POT1-TPP1-TIN2 shelterin subcomplex: POT1 (OB1 & OB2) (yellow) bound to the minimal telomeric sequence TTAGGGTTAG (red) (PDB ID: 1XJV) (top left), POT1 (OB3 & HJRD) (yellow) bound by the PBD of TPP1 (E266-L326) (blue) (PDB ID: 5UN7, 5H65) (top right), TPP1 OB domain (blue) (PDB ID: 2I46) (bottom left), and TIN2(green)-TPP1(blue)-TRF2(grey) interface complex (PDB ID: 5XYF) (bottom right).
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