Structural studies of thymidine kinases from Bacillus anthracis and Bacillus cereus provide insights into quaternary structure and conformational changes upon substrate binding

Abstract

Thymidine kinase (TK) is the key enzyme in salvaging thymidine to produce thymidine monophosphate. Owing to its ability to phosphorylate nucleoside analogue prodrugs, TK has gained attention as a rate‐limiting drug activator. We describe the structures of two bacterial TKs, one from the pathogen Bacillus anthracis in complex with the substrate dT, and the second from the food‐poison‐associated Bacillus cereus in complex with the feedback inhibitor dTTP. Interestingly, in contrast with previous structures of TK in complex with dTTP, in this study dTTP occupies the phosphate donor site and not the phosphate acceptor site. This results in several conformational changes compared with TK structures described previously. One of the differences is the way tetramers are formed. Unlike B. anthracis TK, B. cereus TK shows a loose tetramer. Moreover, the lasso‐domain is in open conformation in B. cereus TK without any substrate in the active site, whereas in B. anthracis TK the loop conformation is closed and thymidine occupies the active site. Another conformational difference lies within a region of 20 residues that we refer to as phosphate‐binding β‐hairpin. The phosphate‐binding β‐hairpin seems to be a flexible region of the enzyme which becomes ordered upon formation of hydrogen bonds to the α‐phosphate of the phosphate donor, dTTP. In addition to descriptions of the different conformations that TK may adopt during the course of reaction, the oligomeric state of the enzyme is investigated.

Synthesis and evaluation of thymidine kinase 1-targeting carboranyl pyrimidine nucleoside analogs for boron neutron capture therapy of cancer

“…The enzymes undergo significant conformational changes upon binding of 1 and ATP [25, 26]. In the apo state, the lasso loop is folded away from the substrate-binding site whereas in the holo state, the lasso loop covers the substrate-binding site tightly by forming hydrogen bonds with 1 , primarily via main chain atoms.…”

Multistep Virtual Screening for Rapid and Efficient Identification of Non‐Nucleoside Bacterial Thymidine Kinase Inhibitors

“…[17,18] In contrast, inhibitors of bacterial thymidine kinases have not attracted much attention in antibacterial research. [10,15,19,20] In most bacteria intracellular dTMP can be synthesized by two different pathways, which suggests combinations of bioactive agents inhibiting both pathways simultaneously. [10] Thymidine kinase inhibitors impair the salvage pathway for dTMP, which is initiated by thymidine kinase catalyzing the transfer of a gamma-phosphate group from adenosine-5'-triphosphate (ATP) to thymidine.…”

Cell cycle regulation and novel structural features of thymidine kinase, an essential enzyme in Trypanosoma brucei

“…Unfortunately, only heavily truncated versions of Tb TK are expressed in soluble and stable form, so structural work was limited to the core protein closely similar to those of other species ( Hs TK1, (Welin et al ., ; Birringer et al ., ); Uu TK, (Kosinska et al ., ); Ba TK and Bc TK, (Kosinska et al ., ); Tm TK, (Segura‐Pena et al ., 2007a,b); Ca TK. All these Tb TK structures form closed tetramers with the blocked phosphate donor site.…”

“…Type II TKs (TKIIs) are present in many organisms, for example protozoan parasites like T. brucei and Leishmania spp., plants, mammals (cytosolic), bacteria and certain viruses (Lee and Cheng, 1976;Ranjbarian et al, 2012;Timm et al, 2015). The crystal structures of many TKIIs have been solved, including those from human (HsTK1, (Welin et al, 2004;Birringer et al, 2005)), Ureaplasma urealyticum (UuTK, (Kosinska et al, 2005)), Bacillus anthracis and B. cereus (BaTK and BcTK, (Kosinska et al, 2007)), Thermotoga maritima (TmTK, (Segura-Pena et al, 2007a,b)), Clostridium acetobutylicum and L. major (LmTK, (Timm et al, 2015)). All these structures have the enzyme packed as a tetramer in the crystal with similar quaternary interactions: most forming a 'closed' tetramer with no ligand in the phosphate donor site, and a smaller number with an 'open' tetramer with the site occupied by a nucleobase.…”

“…10A). A detailed description of the active site was given previously for HsTK1 (Welin et al, 2004) and BaTK and BcTK (Kosinska et al, 2007). In brief for TbTK, the dThd molecule is coordinated by hydrogen bonds to the main chain of residues, R361, L363 and G365, located in the lasso domain, and Y316 from the a/b-domain.…”