Structure-function study of deinococcal serine/threonine protein kinase implicates its kinase activity and DNA repair protein phosphorylation roles in radioresistance of Deinococcus radiodurans

The DR2518 (RqkA) a eukaryotic type serine/threonine protein kinase in Deinococcus radiodurans was characterized for its role in bacterial response to oxidative stress and DNA damage. The K42A, S162A, T169A and S171A mutation in RqkA differentially affected its kinase activity and functional complementation for γ radiation resistance in Δdr2518 mutant. For example, K42A mutant was completely inactive and showed no complementation while S171A, T169A and T169A/S171A mutants were less active and complemented proportionally to different levels as compared to wild type. Amongst, different DNA binding proteins that purified RqkA could phosphorylate, PprA a DNA repair protein, phosphorylation had improved its affinity to DNA by 4 fold and could enhance its supportive role in intermolecular ligation by T4 DNA ligase. RqkA phosphorylates PprA at threonine 72 (T72), serine 112 (S112) and threonine 144 (T144) in vitro with the majority of it goes to T72 site. Unlike wild type PprA and single mutants of T72, S112 and T144 residues, the T72AS112A double and T72AS112AT144A triple mutant derivatives of PprA did not phosphorylate in vivo and also failed to complement PprA loss in D. radiodurans. Deletion of rqkA in pprA::cat background enhanced radiosensitivity of pprA mutant, which became nearly similar to ΔrqkA resistance to γ radiation. These results suggested that K42 of RqkA is essential for catalytic functions and the kinase activity of RqkA as well as phosphorylation of PprA have roles in γ radiation resistance of D. radiodurans.