<br/>The emergence of multi-drug resistant strains of <i>Mycobacterium tuberculosis</i> has prioritized the need for new drug targets. β-clamp can serve as one such target as it interacts with many proteins involved in DNA replication and repair. The primary role of b-clamp in the development of antibiotic resistance is as an accessory factor in the DnaE2 mutasome. The mutasome is made up of an error-prone DNA polymerase (DnaE2), an inactive Y-family polymerase (ImuB), a homolog of the RecA protein (ImuA), and the β-clamp. During inducible mutagenesis, β-clamp forms a complex with ImuB, which in turn interacts with ImuA and DnaE2. Here, we explore the interaction between b-clamp and the b-clamp interaction motif of ImuB. We hypothesize that ImuB will have a higher affinity for b-clamp as compared to other DNA polymerases, so that upon DNA damage, the interaction between b-clamp and ImuB will be favored. We determined the thermodynamic binding parameters for complex formation between b-clamp and the predicted interaction motifs of ImuB, DinX (a Y-family polymerase), and DnaE1 (the replicative polymerase) using isothermal titration calorimetry. We found that the free energy of complex formation between b-clamp and the interaction motifs is largely driven by enthalpic forces and that b-clamp has a higher affinity for ImuB as compared to DnaE1 and DinX1. To better understand the differences in affinity between the three interaction motifs and b-clamp, we co-crystallized each peptide with the b-clamp protein and solved the structures of β-clamp with ImuB, DnaE1 and DinX1 peptides at resolutions of 2.5, 2.8 and 2.45 Å, respectively. The structures revealed that all peptides bound to the β-clamp with 1:1 stoichiometry at the same site near the C- terminus, in the domain III of the monomeric subunit. An increased number of hydrogen bonds were observed between the ImuB peptide and b-clamp compared to the DnaE1 and DinX1 peptides, consistent with the increased binding enthalpy determined by the ITC experiments. Together these findings confirm our hypothesis that β-clamp has a higher affinity for ImuB and therefore is likely to out-compete other β-clamp interacting proteins upon induction. Our findings here bring us a step closer in understanding the mechanism involved in mutagenic repair pathway and suggest that β-clamp-ImuB complex formation could serve as a potential drug target to prevent drug resistance.

Interaction of β-clamp with mycobacterial DNA polymerases

ACS Fall 2021

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