Recombinant AcnB, NrdR and RibD of Acinetobacter baumannii and their potential interaction with DNA adenine methyltransferase AamA.
Protein expression and purification(2022)
Abstract
In the last decades Acinetobacter baumannii developed into an increasingly challenging nosocomial pathogen. A. baumannii ATCC 17978 harbors a DNA-(adenine N6)-methyltransferase termed AamA. Previous studies revealed a low specific activity of AamA in vitro despite proven folding, which led us to speculate about possible interaction partners assisting AamA in targeting methylation sites. Here, applying a pulldown assay with subsequent mass spectrometry we identified aconitate hydratase 2 (AcnB) as possible interaction partner. In addition, we considered the putative transcriptional regulator gene nrdR (A1S_0220) and the pyrimidine deaminase/reductase gene ribD (A1S_0221) of A. baumannii strain ATCC 17978 to encode additional potential interaction partners due to their vicinity to the aamA gene (A1S_0222). Proteins were recombinantly produced in the milligram scale, purified to near homogeneity, and interactions with AamA were studied applying blue native gel electrophoreses, electrophoretic mobility shift assay, chemical cross-linking and co-immunoprecipitation. These analyses did not provide evidence of interaction between AamA and purified proteins. Solution structures of RibD, NrdR and AcnB were studied by small-angle X-ray scattering (SAXS) alone and in combination with AamA. While in the case of RibD and AcnB no evidence of an interaction with AamA was produced, addition of AamA to NrdR resulted in dissociation of long and rod-shaped polymeric NrdR structures, implying a specific but transient interaction. Moreover, we identified a molecular crowding effect possibly impeding the DNA methyltransferase activity in vivo and a sequence-independent DNA binding activity of AamA calling for continued efforts to identify the interaction network of AamA.
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Key words
AcnB,NrdR,RibD,AamA,Acinetobacter baumannii,DNA-Adenine-methyltransferase,Recombinant production,Small-angle X-ray scattering,SAXS,Solution structure
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