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

Abstract

Klebsiella pneumoniae PmrA is a polymyxin-resistance-associated response regulator. The C-terminal effector/DNA-binding domain of PmrA (PmrAC) 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 (PmrAN). 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 PmrAC and characterized the interactions between PmrAC or BeF3−-activated full-length PmrA (PmrAF) and two DNA sequences from the pbgP promoter of K. pneumoniae. We showed that PmrAC 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 PmrAC–DNA complex was investigated using HADDOCK docking and confirmed by paramagnetic relaxation enhancement. Unlike PmrAC, PmrAF recognizes the two sites simultaneously and specifically. In the PmrAF–DNA complex, PmrAN may maintain an activated homodimeric conformation analogous to that in the free form and the interactions between two PmrAC molecules aid in bending and binding of the DNA duplex for transcription activation.

Structural dynamics of the two-component response regulator RstA in recognition of promoter DNA element

“…On the other hand, the DNA-bound structure of PhoB DBD has for a long time been the only source of information for structural changes of the DBD upon binding to DNA (21). Recently, the structures of K. pneumoniae PmrA DBD and E. coli KdpE bound to their respective cognate DNA sequences have been solved (22,56). Although PhoB, PmrA and KdpE belong to the same OmpR/PhoB subfamily, their DBDs have shown very different structural and functional characteristics when bound to DNA.…”

“…For example, the two DBDs of the OmpR dimer can bind to different DNA sequences in either head-to-tail or head-to-head orientations (18–20), whereas the DBDs of PhoB and PmrA bind to DNA exclusively in a head-to-tail orientation (21,22). Moreover, binding of PhoB to DNA induces bending in the DNA, which is not observed for PmrA.…”

Structure and dynamics of the polymyxin-resistance-associated response regulator PmrA in complex with the promoter DNA

“…1). However, our previous NMR study showed that REC amide resonances from BeF 3 À -activated PmrA in complex with DNA are similar to those from the stand-alone BeF 3 À -activated REC dimer 20 , which suggests that the inclusion of DBD and addition of DNA do not greatly change the chemical environment of the PmrA REC dimer. Therefore, the preliminary NMR findings do not agree with the extensive REC-DBD interactions identified in PmrA-1 in the crystal structure.…”

“…PmrA was activated by the phosphoryl analogue BeF 3 À , which has been used to activate the REC domain to determine its activated structure 19 . The pmra box on the pbgP promoter of Klebsiella pneumoniae was verified previously 20 , and a series of various-length DNAs covering the half-1 and half-2 sites were mixed with an equal amount of BeF 3 À -activated PmrA for co-crystallization (Supplementary Table 1) to obtain the crystals of complexes with 25-and 26-bp DNA (Supplementary Fig. 3).…”

“…We screened different pH values, buffer types, salt concentrations and additives systematically but found no significant increase in solubility. We then calculated solvent-accessible surface areas from the X-ray structure of the REC domain 19 and NMR structure of the DBD domain 20 and identified two highly exposed hydrophobic residues, Trp 181 and Ile 220 . The double-substitution W181G/I220D PmrA exhibited the best solubility and highest thermal stability ( Supplementary Fig.…”