Introduction
Thymidylate kinase or TMK is found in methicillin-resistant Staphylococcus aureus or MRSA and has been targeted as a possible target for antibacterial drugs. TMK is essential for DNA synthesis as it phosphorylates deoxythymidine monophosphate to deoxythymidine diphosphate [1]. One reason that TMK has been targeted for antibacterial drugs are the structural differences between human and bacterial TMK's. These structural differences could possibly minimize long term resistance to the drug from the bacteria. One of the conformational differences between human and bacterial TMK's has to do with the difference in the TMP-binding site of SaTMK at the base of the TMP-binding cavity [1]. The of TMK can be seen here. In order to establish enzyme affinity hydrogen bonds were formed between Arginine48 and TMK is MRSA thus allowing for possible target sites for drugs to be identified [1]. This is shown in this where the black sticks are the sites that have identified as being positively charged, and the blue ribbons are where Arginine has been identified and thus a possible source of attachment for a drug or enzyme.
Overall Structure
Thymidylate Kinase is a protein dimer with clearly defined secondary and tertiary structures. The consists of 5 beta sheets and 8 alpha helices. The beta sheets and alpha helices are connected by turns, or chains of amino acids, shown in black. Additionally, it is good to note that for Thymidylate Kinase, beta sheets are arranged in an parallel fashion using short turns and alpha helices to connect.
Coloring from red to blue from the , it is clear the Thymidylate Kinase consists of two clear mirrored units with each unit binding a Sulfonylpiperidine ligand. Additionally, the C terminus constitutes the catalytic region of the protein, binding with the ligand. The polarity of amino acid units in the beta sheets and alpha helices is a large factor which determined the tertiary structure of this protein. residues are shown in purple, whereas nonpolar are shown in gray. The parts of the alpha helices and beta sheets that make up the surface contact are polar, whereas the hydrophobic core of the protein contains the nonpolar regions of the helices and sheets.
Binding Interactions
Green scene gives a rough outline of the TMK backbone and a look at its active site. Outlined, in ball and stick model, are the active site residues as well as the bound sulfonylpiperidine ligand. The key residues are Arg 48, Phe 66, Ser 97, and Gln 101. In green scene you are able to see that Arg 48, Ser 97, and Gln 101 form hydrogen bonds with the sulfonylpiperidine ligand. The hydrogen bond locations are shown with distances between the atoms which hydrogen bond with each other in one of the subunits. Arg 48 in particular forms one hydrogen bond with the phenolic group on the ligand. The ligand's ability to hydrogen bond with this particular residue was crucial because Arg 48 is a highly conserved residue. Moreover the hydrogen bond with Arg 48 is necessary in order for the ligand to have high enzyme affinity. If this sulfonylpiperidine is able to hydrogen bond with Arg 48, it will also be able to have relatively high binding affinity to the TMK of other organisms. The highly conserved residues, that bind with the ligand, in the active site of TMK can be seen in [1]
Additional Features
Substrate Binding:
Thymidylate kinase is associated with pyrimidine metabolism and deoxythymidine trisphosphate (dTTP) biosynthesis. Its catalytic activity is responsible for the conversion of deoxythymidine monophosphate (dTMP) and ATP into deoxythymidine diphosphate (dTDP) and ADP through phosphoryl transfer. Deoxythymidine trisphosphate is a key component in the synthesis of DNA. By inhibiting the formation of dTDP, dTTP cannot be synthesized and thus DNA synthesis is halted. A key difference in the substrate-binding site of MRSA-TMK compared to other species is the base of the TMP binding cavity, where the cis-proline that forms the base for other species is turned to instead provide a space for to link to Glu 37 of the main chain and form a new TMP binding site base.
Certain TMK binding residues are conserved throughout all species:
- P-loop carboxylic residue Glu 11 with 3'-OH of TMP
- aromatic ring stacking interactions (pyrimidine ring with phenylalanine (Phe 66) side chain
- Tyr 100 positioning (deoxynucleotide specific)
- h-bond between O4 of pyrimidine moeity with the side chain of Arg 70 (T specific)
Conformational Changes (:
For substrate-binding, there's a conformation change to a region a residues covering the phosphate donor site through TMP binding, rotating this region by 31 degrees. The folding points occur between residues 43 and 75 (specifically the α2 and α3 helics) and adopting a closed conformation which brings Arg 48 into position for binding interactions.
Quiz Question 1
A mutation occurs involving the function of magnesium permeable membrane proteins, resulting in a decreased concentration of Mg+2 in the cell. How would this effect the activity of Thymidylate Kinase? How might the cell counteract this/these effects?
Quiz Question 2
What properties would a substrate binding to the active site of Thymidylate Kinase probably possess and why? Consider size, polarity, and similarity to known substrates.
See Also
Credits
Introduction - Michael Grunwald
Overall Structure - Jeremy Gilbride
Drug Binding Site - Ross Furash
Additional Features - Randy Phan/Laura Ornes
Quiz Question 1 - Laura Ornes
Quiz Question 2 - Lauren Okamoto
References
- ↑ Martinez-Botella G, Loch JT, Green OM, Kawatkar SP, Olivier NB, Boriack-Sjodin PA, Keating TA. Sulfonylpiperidines as novel, antibacterial inhibitors of Gram-positive thymidylate kinase (TMK). Bioorg Med Chem Lett. 2013 Jan 1;23(1):169-73. doi: 10.1016/j.bmcl.2012.10.128., Epub 2012 Nov 5. PMID:23206863 doi:http://dx.doi.org/10.1016/j.bmcl.2012.10.128
[1] Kotaka M, Dhaliwal B, Ren J, et al. Structures of S. aureus thymidylate kinase reveal an atypical active site configuration and an intermediate conformational state upon substrate binding. Protein Science : A Publication of the Protein Society. 2006;15(4):774-784. doi:10.1110/ps.052002406.
