Crystal Structure of Human POT1 and TPP1 (PDB ID: 5H65) from Homo sapiens

Created by: Noah Vogler

          The DNA binding complex in Homo sapiens consisting of POT1 and TPP1 (PDB ID: 5H65) is critical in promoting and inhibiting telomere elongation (1). POT1 stands for Protection of Telomeres 1 and TPP1 stands for Tripeptidyl Peptidase 1. Missense mutations of the POT1-TPP1 complex have been found in human cancers that distorts the POT1-TPP1 interaction (2). Therefore, investigating how POT1-TPP1 works may lead to the discovery of drugs that combat cancer. The heterodimer POT1-TPP1 is part of a six-protein complex called a shelterin that has two functions (3,4). Firstly, the shelterin protects the natural chromosome ends from being misidentified as a broken end, which would lead to DNA repair. Secondly the shelterin negatively regulates telomerase by preventing telomerase from binding to the DNA (3). POT1-TPP1 consists of two macromolecules that allow it to work. POT1E, that protects telomeres, is 71442.05 Daltons and consists of 634 residues long. POT1E has an isoelectric point of 6.26. Adrenocortical dysplasia protein homolog (ACD), which is the part of TPP1 that binds to POT1, is 57733.41 Daltons and consitsts of 544 residues long. ACD also has an isoelectric point of 6.29 (5). The secondary structure for POT1E consists of 31% β-sheets and also 23% α-helices, while ACD has 0% β-sheets and 47% α-helices (1).

The POT1-TPP1 prefers to bind to the 3’ terminus of the chromosome. The binding is dictated primarily by POT1, as the subunit directly binds to the 3’ end. Alone, POT1 can still bind to the chromosome, however, the affinity for the chromosome is much lower (3). It has been shown that the N-terminal half of POT1 has two OB folds that allows POT1 to recognize telomeric ssDNA sequence. There is an increase in affinity of 900% when TPP1 is attached to POT1 compared to POT1 individually. When the POT1-binding recruitment domain (RD) of TPP1 was deleted there was no increase in the affinity as seen as in the POT1-TPP1 complex. This experiment suggests that the interaction between POT1 and TPP1 is necessary for the increase in telomerase activity. It is thought that the TPP1-RD domain binds to the POT1-PBR in order to target telomeres (4).

Overall, there is little effect on telomeric length when the entirety of TPP1 is expressed. However, a deletion of the first 86 amino acids of TPP1 results in the elongation of the telomere. This data suggests that the first 86 amino acids of TPP1 serve in a regulatory role. The TPP1-OB fold, consisting of residues 90 to 250, is particularly important to the heterodimer in recruiting telomeres. If the deletion of TPP1 is carried out further, and the OB fold is deleted, then the lengthening of the telomerase ceases, suggesting that the OB fold participates in telomerase recruitment (4).

The POT1-TPP1 protein complex functions as both a negative and positive regulator of telomerase (4). When POT1-TPP1 covers the 3’ terminus of the G-overhang, the heterodimer covers up the telomere and prevents telomerase from binding to it. This is an example of POT1-TPP1 negatively regulating telomerase and preventing elongation. If, however, the POT1-TPP1 protein is removed from the binding site then elongation will continue. The mechanism behind the removal of POT1-TPP1 is not yet known, but it is believed to involve the disruption of the 6-protein shelterin complex through post-translational modification. Once the length of the telomere reaches a certain threshold, POT1-TPP1 will again bind to the G-overhang and inhibit additional telomere elongation (3).

A new target for anti-cancer drugs is the TEL patch in TPP1. TEL patch stands for “TPP1 Glutamate Leucine patch” and consists of 7 critical residues, Glu-168, Glu-169, Glu-171, Arg-180, Leu-183, Leu-212, and Glu-215, clustered in a patch on the surface of the OB fold of TPP1 (6). The TEL patch promotes processivity and telomerase binding by recruiting telomerase to the telomeres (2,6).

The C-terminus of POT1, POT1C, contains two domains. In addition to the two OB folds in the N-terminus half of POT1 there is a third OB fold, OB3, in POT1C as well as a Holliday junction (2,4). Both the OB3 and Holliday junction domain are imperative in POT1C binding to TPP1. Crystal structure of the OB-fold reveals a curving 5-stranded β-barrel at one end of POT1C. From residues 393-538 of POT1C, POT1C adopts a compact, globular fold with a seven-stranded β-sheet surrounded by 4 α-helices. A Dali search indicated that this structure closely resembles archaeal Holliday junction resolvase Hjc, and will be called POT1_HJRL. POT1_HJRL will not bind to Holliday junctions since the outer regions of POT1_HJRL are different from Hjc (2).

The POT1C is important because it prevents the genome from becoming unstable and allowing tumorigenesis. The short fragment consisting of residues 266-320 in TPP1 is all that is needed for binding to occur with POT1C. This POT1 binding motif TPP1_PBM contains two α-helices, H1 and H2, and one 3₁₀-helix. The H1 helix is from Glu-266 to Cys-278. The surface of the complementary portion of POT1 is hydrophobic and contains a shallow groove formed by a curved β-sheet and helix αB of the HJRL domain. The Leu-271, Ala-275, Leu-279, and Leu-281 are four hydrophobic residues of H1 that interact with the POT1 groove. Additionally, the main chain carbonyl and amino groups of TPP1 Thr-280 work closely with the side chains of Arg-432 and Glu-461 of POT1, respectively, to act as tethers to stabilize the position of the H1 helix onto POT1. The H2 helix fits into a depression between the POT1_OB3 and POT1_HJRL domains, opposite the zinc ion. There are 4 cysteines that work together to stabilize the zinc ion. At the beginning of the H2 is a Trp-293, which fits into a hydrophobic pocket formed by the helix-α2 and 3₁₀-helix η1 of POT1_OB3. Hydrogen bonding among TPP1^His-292, TPP1^Trp-293, POT1^Asp-577, and POT1^Asp-584 stabilizes the configuration even more. There is a side chain, TPP1^Arg-297 that goes into the pocket between POT1_OB3 and POT1_HJRL, making a hydrogen bond with the carbonyl group of the main chain of POT1^Val-391. The final interaction in TPP1_PBM to recognize and bind to POT1 is the 3₁₀-helix η1. The 3₁₀-helix fits into a hydrophobic groove on the concave side of POT1_OB3. The groove acts as the ssDNA binding site for the OB folds. Residues in TPP1 from Val-305 – Ser-316 go through the groove perpendicular to β2 and β3 strands of POT1_OB3. TPP1 has both a hydrophobic core and hydrogen bonding interactions at the edges that enable it to stabilize the TPP1_PBM into the groove of POT1_OB3 (2).

Scientists discovered that the crystal structure of TPP1 was very similar to the structure of the β-subunit of TEBP (telomere end-binding protein) from Oxytricha nova, a protozoan. Upon further research POT1 was found to be the human homologue to the α-subunit of TEBP. This finding shows that the capping of the telomeres by a dimer, such as TEBP α-β dimer (PDB ID: 1PA6), is much more evolutionary conserved than previously expected (1,3). Both TPP1 and TEBP-β use a central region to interact with the C-terminus of their binding partners. Like in TPP1, the C-terminus of TEBP-β is not involved in the interaction with POT1 or TEBP-α. The structure of the OB-fold in TPP1 is most similar to the OB fold in the TEBP-β subunit from O. nova. Like POT1, TEBP-α contains three OB folds, with the first two folds serving the purpose of ssDNA recognition and the third OB fold interacting with TEBP-β. The TEBP-αβ subunits form a heart-shaped structure, with the telomeric ssDNA in the middle (2). This is different from the structure of the POT1-TPP1 heterodimer, which has a V-shaped conformation with POT1_OB1-OB2 and TPP1_OB on opposite sides. In H. sapiens, the chromosomes have the 3’ overhang extend much further than in O. nova, explaining the difference in structure. The V-shape is useful for binding both the 3’ overhang as well as the internal single stranded telomeric regions. Another different between POT1-TPP1 and TEBP-αβ is that while both TEBP-α and POT1C have a third OB-fold that is used to interact with TEBP-β and TPP1, respectively, the structure of POT1C is not similar to TEBP-α (2). The similarity in the tertiary structure of the POT1-TPP1 complex and TEBP-αβ can be seen quantitatively with Dali Server. Dali Server uses sum-of-pairs method to find proteins with a similar tertiary structure to the protein in question. Proteins with significant similarities have a Z-score greater than 2. The Z-score for TEBP-αβ compared to POT1-TPP1 is 14.3, which shows that the two structures are similar (7). PSI-BLAST is a way to find proteins that have a similar primary structure as the protein query. Each protein will get an E value, which is calculated by looking at the total sequence of the protein and assigning gaps (8). The closer the E value is to zero, the more similar the primary structure of the two proteins are. An E value less than 0.05 means the protein’s primary structure is significantly similar to the protein query. An E value for TEBP-αβ was not obtained, however, given the previous findings, the primary structure is most likely similar to that of POT1-TPP1. Because the POT1-TPP1 complex regulates the length of telomeres, studying how the complex works can lead to breakthroughs in cancer research.