This protein (3HDT) is a putative kinase with possibly cytidylate kinase function utilizing ATP and dCMP as ligands.
Introduction
Kinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another, and are involved in cell growth and signaling. This work characterizes a protein (PDB ID 3HDT) with unknown function, and tests for kinase activity. In silico tools including BLASTp, Pfam, SPRITE, DALI, DeepFRI, and PyRx were used to hypothesize the function and taxonomy of 3HDT. These results indicated the protein may be a cytidylate kinase (EC: 2.7.4.X) that transfers a phosphate from ATP to dCMP. E. coli. were transformed with a plasmid containing the gene for 3HDT. The protein was then overexpressed, purified and quantified. SDS-PAGE was used to verify the purity of the protein. Kinetics assays were carried out using dCMP as a substrate, showing only limited activity. Future research should focus on other nucleotide substrates such as TMP or GMP, which also showed high affinities in docking simulations.
OR
The goal of this research is to characterize the protein, 3HDT, that has an unknown function. This research is part of the BASIL project that involved performing in silico and in vitro modules to make predictions and study the function of this protein.
In silico anaylsis
We used a variety of in silico tools with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP.
BLASTp
BLASTp Alignment showing a hit with cytidylate kinase-like family, part of the NK superfamily.
Query hits 1-2 show proteins with unknown function while 3-6 match a cytidylate kinase-like family protein.
Pfam
We used the FASA sequence from 3HDT to do a comparative search in Pfam to find similar protein families our protein may belong. Pfam predicted that our protein is part of the cytidylate kinase which was also shown in the BLASTp results.
Pfam results showing cytidylate kinase-like family result.
DALI
Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).
PANZ
Docking
Laboratory Experiments
Coupled kinase assay
Two rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The concentration of dCMP was 109mM. The first round of assay (3 total assays) used 5μL of 3HDT and various amounts of 109mM of dCMP. 6.88μL of dCMP resulted in the highest specific activity (0.37669 U/mg) and increasing the substrate amount ~2μL had a similar but slightly less specific activity of 0.3268 U/mg. However, when we repeated the first kinase assays we did (using 5μL of 3HDT), the results did not turn out the same or even similar to the first round indicating a potential error could have happened. In addition, the protein in the second round was older (original protein but about a week old from when it was made) which could have affected the specific activity with the substrate because the protein was starting to expire/decrease function.
Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right).
SDS-PAGE
SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present.
Results from running an SDS-PAGE with the purified 3HDT
Conclusion/Future Experiments
Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT.
