Accepted_test

Rational design of human terminal deoxynucleotidyl transferase
by Ukladov Egor Olegovich | Tyugashev Timofey Evgenyevich | Kuznetsov Nikita Aleksandrovich | Institute of Chemical Biology and Fundamental Medicine SB RAS, Russia, Novosibirsk; Department of natural sciences NSU, Russia, Novosibirsk | Institute of Chemical Biology and Fundamental Medicine SB RAS, Russia, Novosibirsk | Institute of Chemical Biology and Fundamental Medicine SB RAS, Russia, Novosibirsk; Department of natural sciences NSU, Russia, Novosibirsk
Abstract ID: 399
Event: BGRS-abstracts
Sections: [Sym 3] Section “Structural biology of proteins nucleic acids and membranes”

The terminal deoxynucleotidyl transferase (TdT) possesses a unique capability for 5'-3' template-independent DNA synthesis facilitated by loop 1 structure, which has found widespread applications in molecular and cellular biology. However, a significant limitation in TdT utilization lies in its pronounced substrate selectivity: dGTP > dCTP ≈ dATP > dTTP. In this study, pre- and post-catalytic complexes of human TdT with DNA primer, dNTP, and Mg2+ ions were modeled based on crystallographic structures and homology modeling. Through in silico introduction and assessment of amino acid substitutions using molecular dynamics, the roles of several amino acid residues were elucidated: Asp345 participates in maintaining the geometry of the active site, Asp472 stabilizes the position of loop 1, Phe400 and Leu397 indirectly stabilize the position of the nitrogenous base (NB) of dNTP, Asp395 and Glu456 form hydrogen bonds with the NB of dNTP and are responsible for substrate selectivity. Amino acid substitutions D395N, E456N, D395N+E456N are proposed, potentially increasing selectivity towards dATP, dTTP, and dCTP.