Solution structure and tandem DNA recognition of the C-terminal effector domain of PmrA from Klebsiella pneumoniae

Klebsiella pneumoniae PmrA is a polymyxin-resistance-associated response regulator. The C-terminal effector/DNA-binding domain of PmrA (PmrA(C)) recognizes tandem imperfect repeat sequences on the promoters of genes to induce antimicrobial peptide resistance after phosphorylation and dimerization of its N-terminal receiver domain (PmrA(N)). However, structural information concerning how phosphorylation of the response regulator enhances DNA recognition remains elusive. To gain insights, we determined the nuclear magnetic resonance solution structure of PmrA(C) and characterized the interactions between PmrA(C) or BeF(3)(−)-activated full-length PmrA (PmrA(F)) and two DNA sequences from the pbgP promoter of K. pneumoniae. We showed that PmrA(C) binds to the PmrA box, which was verified to contain two half-sites, 5′-CTTAAT-3′ and 5′-CCTAAG-3′, in a head-to-tail fashion with much stronger affinity to the first than the second site without cooperativity. The structural basis for the PmrA(C)–DNA complex was investigated using HADDOCK docking and confirmed by paramagnetic relaxation enhancement. Unlike PmrA(C), PmrA(F) recognizes the two sites simultaneously and specifically. In the PmrA(F)–DNA complex, PmrA(N) may maintain an activated homodimeric conformation analogous to that in the free form and the interactions between two PmrA(C) molecules aid in bending and binding of the DNA duplex for transcription activation.