Fig 1.
Structure and modeling of insert in TdT-long isoform.
a) Doughnut shaped structure of TdT-long modeled isoform and its structural sub-domains. Colour index: Index finger (cyan), fingers (green), palm (yellow), thumb (blue), Loop1 (orange), Loop3 (magenta), Loop2 (blue) b) Sequence composition of Loop3. Color coded according to their physiochemical properties c) Secondary structure prediction of TdT-long form by PSIPRED, d) Phylogenetic clustering of TdT and other DNA polymerases in mouse showed that TdT and Pol mu shared a sequence identity of 42% and clustered together.
Table 1.
List of simulation runs performed at different temperatures for TdT isoforms. Duration of simulation and root mean square deviation (RMSD) of simulations carried out.
Extended runs of TdT-long form at 311 K for 300 ns yielded RMSD for run1: 3.2± 0.21 Å and run2 (duplicate): 3.86 ±0.35 Å. RMSD of replicate run is shown in brackets.
Fig 2.
TdT-long form is more structurally dynamic than its shorter counterpart.
a) Root Mean Square Fluctuations (RMSF) of TdT-short form and TdT-long form in its ligand bound state at 311 K. Index: TdT-short form (run1: black, run2: red), TdT-long form (run1: green, run2:blue). b) RMSF of TdT-long form at all simulation temperatures. Index: 298 K (black), 300 K (red), 311 K (green) and 318 K (blue). Sub-domains and loops of TdT has been marked on X-axis in different colours as: index finger (blue), fingers (green), palm (yellow), thumb(violet), Loop1 (orange), Loop2 (blue) and Loop3 (magenta)
Fig 3.
Deviations within different subdomains (except within Loop1) observed in TdT-short and long isoform.
RMSD of subdomains of TdT-short and TdT-long for simulations runs at 311 K for 100 ns. Index: TdT-short (run1: black, run2: red), TdT-long form (run1: green, run2: blue).
Table 2.
RMSF of each sector of hand-like structure of TdT viz. thumb, finger, index finger and loop1 (Increased RMSF values are marked in bold).
Loop1 RMSF values reduce in TdT-long (wild and mutant). Simulations performed at 311 K for 100 ns except for the mutated TdT-long form.
Table 3.
RMSD (Root Mean Square Deviation) from the starting equilibrated structure of each part of hand-like structure of TdT at 311 K, 100 ns.
Fig 4.
Parameters to measure the interaction between Loop3 and Loop1 a) Distances between Loop3 & Loop1 of wild-type (red) and mutated (magenta) TdT-long form, b) & c) Number of hydrogen bonds formed during simulations of wild-type (red) and mutated (magenta) TdT-long form at 300 K, d) Salt bridges formed in wild-type TdT-long form but absent in mutated TdT-long form e) Left Panel: Distances between Loop1 (Asp396) and active site (Asp434) during simulations at 300 K in TdT-short form (black), wild-type TdT-long form (red) and mutated TdT-long form (magenta). Right Panel: Illustration of Loop1 projecting over active site.
Fig 5.
Inter-residue motions illustrated as cross-correlation matrices to depict rigid body motions within subdomains of TdT.
Correlation matrix for TdT-long heavy atoms when simulated at 311 K. Positive values (red spectrum) indicate correlated motion; negative values (blue spectrum) indicate anti-correlated motions between sub-domains. Correlated and anti-correlated motions observed between sub-domains of TdT-long form have been marked by arrows. Sub-domains and loops of TdT has been marked on X-axis and Y-axis in different colours as: index finger or “I_F” (blue), fingers or “F” (green), palm or “P” (yellow), thumb or “T” (violet), Loop1 or “L1” (orange), Loop2 or “L2” (blue) and Loop3 or “L3” (magenta). Similar colour coded TdT-long structure has been provided at top left corner for ease in understanding.
Table 4.
Percentage of occurrence of salt bridges between Loop3 and Loop1 throughout the simulations at 300 K and 311 K of TdT-long isoform.
Fig 6.
Analysis of global and inter-residue motions in both isoforms.
Essential dynamics studies in form of covariance matrices and their resultant PCA were performed at all simulation temperatures but shown here only for 300 K and 311 K. These analyses were performed to characterize the motion of Loop3 and its effect on other regions of TdT. The first two principal components have been analyzed in detail and mapped onto the TdT-short form and TdT-long form three-dimensional structures. Index: Lower frequency fluctuations (blue), higher frequency fluctuations (red), High amplitude -> covering larger space during motion.
Fig 7.
Protein residue network analysis to depict atomic level interactions.
a) Formation of clusters in TdT-short form and TdT-long form, b) Unique cliques detected in TdT-short form and TdT-long form. Absence of clique in TdT-long form has been pointed by a cyan coloured dotted circle; c) Distances measured between residues of thumb and index finger for both isoforms (run1 and run2) plotted as bar graph.
Fig 8.
Effect of insert on the catalytic triad and the neighbouring residues a) Superposition of active site triad in TdT-short (blue) and TdT-long form (green). Distances between active site triad (Asp343, Asp345 and Asp434) residues in pairwise manner at 300 K and 311 K temperature. Index: TdT-short form marked in complete lines, TdT-long form in dashed lines. b) Snapshots of the deviations in distances within catalytic triad across both isoforms. Asp343-Asp345 pair and Asp343-Asp434 pair show longer distances within them thus indicating distortion of active site triad. TdT-long form showed deviation in distances for two out of three pairs (D343-D434, D343-D345) at even physiological temperature (300 K). c) Orientation of His342 in TdT-short form (left) and TdT-long isoform (right) during simulations at 311 K temperature. His342 side chain is flipped out in TdT-long form for majority of the 100 ns of simulation time.
Fig 9.
Parameters to measure shifting of DNA primer in TdT-long isoform a) Superposed DNA primers at different instances during simulation (at 0, 20, 50 and 100 ns snapshots) for TdT-short form and TdT-long form at 311 K, b) Orientation of the bases and backbone of DNA primer at final snapshot of different temperatures for TdT-short form (green) and TdT-long form (cyan). c) Boxplot of distances measured between DNA phosphates 2 and 3 with active site Asp434 between TdT-short and TdT-long form at 311 K. The extent of variation in TdT-long form quantifies the drifting motion in the DNA primer.
Fig 10.
Schematic illustrating the major conformational changes observed during molecular dynamics mapped onto TdT-long isoform.
a) Events leading to increasing thermosensitivity: 1.Hydrogen and electrostatic interactions bind Loop1 and Loop3, 2. Weakening of interaction between thumb and index finger, 3) Increase in fluctuations in index finger and finger subdomains. b) Events leading to decrease in polymerization activity: 1. Deformation of active site triad, 2. Flipping away of His342 side chain, 3. Widening of active site cavity and 4. Shifting of DNA primer.