BASIL2022GV3HDT
Characterizing Putative Kinase 3HDT and its Potential Substrates
Putative kinase (3HDT) shows limited activity as a cytidylate kinase, utilizing ATP and dCMP as ligands. ContentsIntroductionKinases (or phosphotransferases) facilitate the transfer of a phosphate group from one molecule to another and are involved in cell growth and signaling. The goal of this research is to study a protein (PDB ID 3HDT) with unknown function and elucidate its potential substrates. This work uses in silico techniques to characterize the protein before biochemical assays were used to test for kinase activity. 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 anaylsisWe used a variety of in silico tools to find similarities between 3HDT and 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 function was hypothesized for 3HDT and potential substrates such as dCMP were selected for kinase assays.
BLASTpBeginning with BLASTp[1], we queried the FASTA sequence for our protein, PDB ID 3HDT. Our query sequence aligned with the NK superfamily and showed a high degree of overlap indicating that 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 hypothesis that 3HDT is a cytidylate kinase. Additionally, the query hits showed several cytidylate kinase-like family proteins almost completely aligning with our protein, 3HDT.  
PfamWe used 3HDT's FASTA sequence to search in Pfam[2] for protein families that 3HDT may belong to. Pfam predicted that 3HDT is part of the cytidylate kinase-like family which concurs with the BLASTp results shown previously.  
DALITwo 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 structural similarity between the proteins (structure of the protein = function) indicating a similarity in function between these kinases and our protein.
DockingWe 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 binding pocket (in purple) is a potential pocket the substrate dCMP may bind to in the protein, 3HDT. This area was also where dCMP bound with the highest affinity in PyRx. The amino acids (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, Lys146 (purple) and Leu 202 (yellow) 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 due to an issue in the docking process where we were unsuccessful in getting PyRx to recognize where the ATP cofactor was bound before docking our substrate into the protein.  Some of the predicted binding pockets were areas where ATP (red arrow) and dCMP (yellow arrow) bound with the highest affinity in PyRx.  
Laboratory ExperimentsPlasmid AnalysisBL21-Gold E.Coli were transformed with a plasmid containing the gene for 3HDT to produce our protein of interest. A pMCSG19 vector with ampicillin resistance was inserted into the cells via heat shock and incubated on a plate containing carbenicillin. To ensure we inserted the gene of interest the plasmid was digested with Xho1 and Nde1 before gel electrophoresis was run to verify its identity. The vector was 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 and could move forward with protein over-expression and purification.  
Coupled kinase assayTwo rounds of coupled kinase assays were run using 3HDT with ATP and dCMP as substrates. The stock concentration of dCMP was measured to be 109mM, and various concentrations between 5-10mM were used in the assays. The first round of assays (3 assays) used 45.6ng of 3HDT and increasing concentrations of 5.0, 7.5, and 10.0mM dCMP in a well with 100μL total. A concentration of 7.5mM dCMP resulted in the highest specific activity (0.377 U/mg) while increasing the substrate concentration to 10mM had a slightly lower specific activity of 0.348 U/mg. 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 improper mixing, bubbles being present in the well, or too much time passing between adding substrate to the well and measuring absorbance. Additionally, the protein in the second round of assays was older (original protein but approximately a week had passed from time of over-expression) which could have affected the specific activity in those trials due to the protein starting to expire/decrease in function.
 
SDS-PAGESDS-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. 
Conclusion/Future ExperimentsOur 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 was likely not dCMP due to the low specific activity values. 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, in the future we would like to repeat the protein purification process to try and get a higher protein concentration than what we achieved as the low concentration may have affected our coupled kinase assay results.
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