Structural Basis of a Physical Blockage Mechanism for the Interaction of Response Regulator PmrA with Connector Protein PmrD from Klebsiella pneumoniae

Abstract

Background: PmrD binds to phospho-PmrA and sustains its phosphorylation state. Results: Phospho-PmrA interacts with PmrD via several specific intermolecular interactions. Conclusion: A steric inhibition mechanism was proposed for protecting phospho-PmrA against dephosphorylation. Significance: This work provides novel data revealing how a connector protein protects an activated response regulator.

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

“…5a). Two activated RECs form a symmetric dimer mediated by the a4-b5-a5 interface, which is consistent with our previously determined crystal structure of the stand-alone REC dimer 19 with an r.m.s.d. value of 0.55 Å for C a atoms ( Supplementary Fig.…”

“…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.…”

“…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.…”

“…Precursors (a-keto-3-methyl-d 3 -butyric acid-4-13 C and 2-ketobutyric acid-4-13 C) were added to cells once A 600 reached 0.8. Recombinant protein was purified by use of nickelnitrilotriacetic acid affinity resin 19 (Qiagen, Hilden, Germany). The purity of samples was checked with use of Coomassie-blue-stained SDS-polyacrylamide gel and was 495%.…”

“…The protein samples were activated by adding 5.3 mM BeCl 2 , 35 mM NaF and 7 mM MgCl 2 (ref. 19). Double-stranded DNA was prepared by mixing an equal amount of two complementary oligonucleotides in 20 mM sodium phosphate and 30 mM NaCl at pH 6.0, heating to 95°C for 30 min and cooling slowly to room temperature, then further purified on a Mono-Q 5/50 GL column (Amersham Biosciences) with elution by NaCl concentration gradient from 0.1 to 1 M. The final concentration of DNA was calculated by ultraviolet absorbance at 260 nm.…”