Klebsiella pneumoniae PmrA is a polymyxin-resistance-associated response regulator. The C-terminal effector/DNA-binding domain of PmrA (PmrAC), a member of OmpR-PhoB superfamily, recognizes tandem imperfect repeat DNA sequences on the promoters of several genes to mediate lipopolysaccharide modification and induce antimicrobial peptide resistance after the phosphorylation and dimerization of its N-terminal receiver domain. Here, we identified the PmrA box on the pbgP promoter of K. pneumoniae, which contains 2 hexanucleotide half sites and found that PmrAC binds to the first site with much stronger affinity than to the second site. However, full-length PmrA recognizes two sites specifically and simultaneously when it is activated with the phosphoryl analog beryllofluoride (BeF3-). The solution structure of PmrAC was determined and the structural basis for the PmrAC/DNA interaction was investigated using HADDOCK docking base on NMR and mutational studies. The complex model shows that Thr187, Asn188, and His193 play key roles in specific recognition and two PmrAC molecules bind to two half sites with a head to tail fashion, which was further verified by NMR paramagnetic relaxation enhancement. The structural basis for the interactions between activated full-length PmrA and DNA (MW ~ 70 kDa) is still on going. The NMR strategies to investigate this high-molecular-weight system will also be discussed in this poster.