Sequence-based Identification of Functional Residues in Mycobacterium Tuberculosis DnaE1 and DnaE2 using Two-Entropy Analysis
R.C.M Kuin1, T.H.W Bäck2, M.H. Lamers3 and G.J.P. van Westen1
1Leiden Academic Centre for Drug Research (LACDR), Einsteinweg 55, 2333 CC Leiden, The Netherlands
2Leiden Institute of Advanced Computer Science (LIACS), Niels Bohrweg 1, 2333 CA Leiden, The Netherlands
3Department of Cell & Chemical Biology, Leiden University Medical Center (LUMC), Einthovenweg 20, 2333 ZC Leiden, The Netherlands
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) is a leading cause of death from an infectious disease [1]. The emergence of drug-resistant TB increases the need for new antibiotics. Here, we explore Mtb DNA polymerase as novel drug target. Mtb contains two copies of DNA polymerases involved in replication, the replicative DNA polymerase DnaE1 and DNA polymerase DnaE2 which is expressed in adverse conditions and induces drug-resistance mutations [2]. In this project we aim to find differences between DnaE1 and DnaE2 to explain their different enzymatic properties using sequence analysis.
In this project, we focused on sequences of the Mycobacterium genus and constructed a MSA of 358 DnaE1 and DnaE2 sequences. Using a Two-Entropy Analysis (TEA) approach, for every position in the MSA the Shannon Entropy and similarity were calculated for both proteins together and individually [3]. This way we have identified positions that are be important for the enzymatic properties of the protein, or for interaction with the ligands that bind to it.
From the TEA analysis a group of residues was found that is conserved between the two proteins, suggesting an essential role for polymerase activity, such as the polymerase active site residues. Next, a set of residues was found that is conserved, but different in DnaE1 and DnaE2. These residues could play a role in the difference in the enzymatic properties. Finally, a group of residues that is conserved in DnaE1, but not in DnaE2 and vice versa was identified, suggesting a possible role for these residues in polymerase fidelity.
To experimentally validate the impact of these residues on polymerase fidelity, a set of residues was selected based on visual inspection in the E1 structure and E2 AlphaFold structure. Several mutations in the palm and fingers domain were selected to assert the polymerase activity, mutations in the PHP were made to assert exonuclease activity for which the PHP in DnaE1 is responsible. Moreover, mutations were selected to validate binding to other proteins of the replisome upon mutation.
DnaE1 residues will be mutated to their DnaE2 equivalent and the effect on mutation rates upon mutation will be measured. Interestingly, for many of these positions the residue is smaller in DnaE2 compared to DnaE1 and might affect the flexibility of the protein. Molecular Dynamics (MD) simulations will be applied to rationalize this. We anticipate that these results will enable an understanding of DnaE2 and will allow for the development of drugs targeting DnaE1 and DnaE2.
References
[1] Natarajan A, “A systemic review on tuberculosis”. Indian J Tuberc. 2020 Jul;67(3):295-311. doi: 10.1016/j.ijtb.2020.02.005. Epub 2020 Feb 28. PMID: 32825856.
[2] Ditse Z, “DNA Replication in Mycobacterium tuberculosis” Microbiol Spectr. 2017 Mar;5(2):10.1128/microbiolspec.TBTB2-0027-2016. doi: 10.1128/microbiolspec.TBTB2-0027-2016. PMID: 28361736; PMCID: PMC5507077.
[3] Kai Ye, “Tracing evolutionary pressure” Bioinformatics, Volume 24, Issue 7, 1 April 2008, Pages 908–915, https://doi.org/10.1093/bioinformatics/btn057
