1. Abstract

In this part, we predicted the ZaTdT protein structure, performed molecular docking and molecular dynamics using AlphaFold, AutoDock and GROMACS software, respectively. These models and methods provide directions for conducting experiments and help us to excavate more commercially valuable TdTs.

2. Protein structure prediction of ZaTdT

At present, the protein 3D structure of ZaTdT remains unreported. We started with the original amino acid sequence of ZaTdT and predicted its structure by AlphaFold software.

3. Molecular Docking

To make the active center more compatible with 3’-O-(2-nitrobenzyl)-modified nucleotides, we attempted to reshape the catalytic cavity using mutagenesis on the residues within 6 Å around 3’-O-(2-nitrobenzyl) and the base moiety. We predicted the 3D structure of ZaTdT by homology modelling and molecular docking it with 3’-O-(2-nitrobenzyl)-dATP. The results of molecular docking indicate that residues Arg335 and Lys337 are closely related to the catalytic activity of ZaTdT.

4. Molecular Dynamics

Analysis of the molecular dynamics trajectories showed that R335 and its spatially neighboring residue K337 in ZaTdT contacted the triphosphate group through hydrogen bonds. We anticipate that eliminating the hydrogen bonds binding force between the above residues and the triphosphate group will release the greater freedom of the modified nucleoside within the active pocket, thereby increasing catalytic activity. Consequently, We performed molecular dynamics simulations of the 3’-O-(2-nitrobenzyl)-dATP docked into ZaTdT, ZaTdT-R335P, ZaTdT-R335W, ZaTdT-R335I, ZaTdT-R335F, ZaTdT-R335M, ZaTdT-K337A and K337L. The predicted results show that above substitution can abolish the hydrogen-bond connection at position 335 or reduce the frequency of hydrogen-bond formation between the triphosphate group and Lys337.