Putative kinase (3HDT) shows limited activity as a cytidylate kinase, 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. 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 families, and 3-dimensional structures of 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 such as dCMP were selected.
BLASTp
Beginning with BLASTp[1], we queried the FASTA sequence for our protein, PDB ID 3hdt. The alignment of our query sequence with the NK superfamily shows a high degree of overlap indicating this protein is likely a member. The importance of this is that the cytidylate kinase family is a member of the NK superfamily, supporting our claim that 3hdt is a cytidylate kinase. Also, the query hits show several cytidylate kinase-like family proteins almost completely aligning with our protein 3HDT.
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 FASTA sequence from 3HDT to do a comparative search in Pfam[2] to find similar protein families that our protein may belong to. Pfam predicted that our protein is part of the cytidylate kinase-like family which was also shown in the BLASTp results.
Pfam query aligned with a cytidylate kinase-like family.
Top domain results from Pfam.
DALI
Two of the top results from our DALI[3] query (both cytidylate kinases) show a high structural resemblance with 3hdt when overlaid in DALI’s viewer. It is also worth noting that the majority of the results from our DALI query consisted of cytidylate kinases. This information helped us hypothesize the function cytidylate kinase because of the similar alignment between the proteins (structure of the protein = function) indicating a similarity in function between these kinases and our protein.
Spatial alignment of putative kinase 3HDT (green) with two cytidylate kinases: 7L4A (dark brown) and 1KDO (light brown).
Docking
We used POCASA [4] to determine potential binding pockets within our protein and PyRx[5] to dock dCMP into 3HDT. PyMOL[6] was then used to visualize the binding pockets and dCMP docked in the protein. This (in purple) is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. This area was also where dCMP binded with the highest affinity in PyRx. The (Gly21, Ser22, Gly23, Val27, Thr 142, Gln149, Arg150, Thr197, Leu200, Thr201) interact within that area to help hold the substrate in place with hydrophobic interactions. Also, there are two residues, within the active site containing dCMP and cofactor ATP that are important in substrate binding. They interact with the substrate forming hydrogen bonds with the oxygens on the phosphate groups and the 6-membered ring. However, these results may not be as accurate because during the docking process on PyRx, we were unsuccessful in getting the program to recognize where the ATP was bound in the protein first before docking our substrate with ATP already in the protein.
Predicted binding pockets for 3HDT represented by the white stippling.
Some of the predicted binding pockets were areas where ATP (red arrow) and dCMP (yellow arrow) bound with the highest affinity in PyRx.
dCMP (purple) docked with 3HDT and cofactor ATP (pink). Visualized in PyMOL.
Binding affinity was increased when hydroxyl group removed from ribose ring on CMP to make dCMP.
Binding affinities for compounds docked using PyRx.
Laboratory Experiments
Plasmid Analysis
BL21-Gold E.Coli were transformed with a plasmid containing the gene for 3HDT. We used a pMCSG19 vector with ampicillin resistance. The plasmid was digested with Xho1 and Nde1 before gel electrophoresis was run to verify its identity. The vector had a length of 6,441 bp with the insert being an additional 660 bp for a total of 7,101 bp. This is approximately the size of the combined bands in lane five demonstrating that we had the correct plasmid in order to move forward with protein over-expression.
Lane one contains ladder. Lane two contains pDNA without enzyme. Lane three contains pDNA + Xho1. Lane four contains pDNA + Nde1. Lane 5 contains pDNA + Xho1 + Nde1. Image has been modified to only show lanes of interest.
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 in a well with 100μL total. 6.88μL of dCMP (7.5mM) resulted in the highest specific activity (0.377 U/mg) and increasing the substrate concentration to 10mM had a similar but slightly less specific activity of 0.348 U/mg. However, when we repeated the first three kinase assays (5.0, 7.5, 10.0mM dCMP), there were discrepancies in the results indicating a potential experimental error such as not pipetting up and down to mix, bubbles, or taking too long between mixing the substrate in and reading the plate. In addition, the protein in the second round of assays was older (original protein but about a week old from when it was over-expressed) which could have affected the specific activity in those trials due to the protein starting to expire/decrease in function.
representation of the reaction of the coupled assay with 3HDT.
Table of data showing all eight coupled kinase assays with 3HDT.
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. Additionally, if we had more time, we would repeat the protein purification process to try and get a higher protein concentration than what we achieved.
