Inactive-to-Active Transition of Human Thymidine Kinase 1 Revealed by Molecular Dynamics Simulations

Abstract

Despite its importance in the nucleoside (and nucleoside prodrug) metabolism, the structure of the active conformation of human thymidine kinase 1 (hTK1) remains elusive. We perform microsecond molecular dynamics simulations of the inactive enzyme form bound to a bisubstrate inhibitor that was shown experimentally to activate another TK1-like kinase, Thermotoga maritima TK (TmTK). Our results are in excellent agreement with the experimental findings for the TmTK closed-to-open state transition. We show that the inhibitor induces an increase of the enzyme radius of gyration due to the expansion on one of the dimer interfaces; the structural changes observed, including the active site pocket volume increase and the decrease in the monomer-monomer buried surface area and of the number of hydrogen bonds (as compared to the inactive enzyme control simulation), indicate that the catalytically competent (open) conformation of hTK1 can be assumed in the presence of an activating ligand.

Figure 1

hTK1 (PDB code: 2ORV, violet) overlapped with TmTK (PDB code: 2ORW, orange). Only one monomer is shown for each protein. hTK1 is cocrystallized with TTP and TmTK with the bisubstrate inhibitor, 4TA. The phosphoryl transfer reaction catalyzed by hTK1 that leads to thymidine monophosphate (TMP) formation is shown in the frame. Glu98 serves as a base accepting the proton of the 5′-OH-group of T.

Figure 2

Overlap of hTK1-4TA (pink) and hTK1-TTP (green) most populated cluster representatives (first replica). In the top view of the tetramer, B and D monomers in the hTK1-4TA structure are visibly shifted down because of the expansion on the weak dimer interface. Additionally, the strong and weak dimer interfaces’ front views are shown. The corresponding distances between the monomers (as measured between chosen atom-pairs distances averaged over the last 900 ns of each replica) and hydrogen bonds (averaged over the last 900 ns of each replica) and the buried surface area (BSA) (averaged over all replicas most populated representatives) between monomers are shown in respective pink and green colors for hTK1-4TA and hTK1-TTP.

Figure 3

First (A) and second (B) PCs of hTK1 simulations are opening and twisting the tetrameric structure, as shown by the arrows.

Figure 4

PC projection of the replica simulations shows a great overlay for hTK1-TTP and hTK1-apo structures and the expected closed state for hTK1-TTP and the open state for hTK1-4TA simulations.

Figure 5

(A,B) Representative structure (first replica and chain A only) binding site overlaps with the 2ORW crystal structure (chain B, cyan): hTK1 structures (A, pink: hTK1-4TA and green: hTK1-TTP) and TmTK control systems (B, gray: TmTK-DIM and blue: TmTK-TET). (C) Stereo view of the active center of the hTK1-4TA representative (chain A) with the flexible loops discussed in the text marked orange (amino acids no. 56–61) and green (166–180).