Structural analysis of the DNA-binding domain of the Erwinia amylovora RcsB protein and its interaction with the RcsAB box.
The transcriptional regulator RcsB interacts with other coactivators to control the expression of biosynthetic operons in enterobacteria. While in a heterodimer complex with the regulator RcsA the RcsAB box consensus is recognized, DNA binding sites for RcsB without RcsA have also been identified. The conformation of RcsB might therefore be modulated upon interaction with various coactivators, resulting in the recognition of different DNA targets. We report the solution structure of the C-terminal DNA-binding domain of the RcsB protein from Erwinia amylovora spanning amino acid residues 129-215 solved by heteronuclear magnetic resonance (NMR) spectroscopy. The C-terminal domain is composed of four alpha-helices where two central helices form a helix-turn-helix motif similar to the structures of the regulatory proteins GerE, NarL, and TraR. Amino acid residues involved in the RcsA independent DNA binding of RcsB were identified by titration studies with a RcsAB box consensus fragment. Data obtained from NMR spectroscopy together with surface plasmon resonance measurements demonstrate that the RcsAB box is specifically recognized by the RcsAB heterodimer as well as by RcsB alone. However, the binding constant of RcsB alone at target promoters from Escherichia coli, E. amylovora, and Pantoea stewartii was approximately 1 order of magnitude higher compared with that of the RcsAB heterodimer. We present evidence that the obvious role of RcsA is not to alter the DNA binding specificity of RcsB but to stabilize RcsB-DNA complexes.
Pristovsek
Primoz
P
Kemijski Institute, National Institute of Chemistry, Hajdrihova 19, P. O. Box 660, SI-1001 Ljubljana, Slovenia.
Sengupta
Kaushik
K
Löhr
Frank
F
Schäfer
Birgit
B
von Trebra
Markus Wehland
MW
Rüterjans
Heinz
H
Bernhard
Frank
F
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D019992:Databases as Topic; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004885:Erwinia / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D005838:Genotype; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020349:Surface Plasmon Resonance; D013997:Time Factors; D014157:Transcription Factors
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D009841:Oligonucleotides; C489568:RcsB protein, E coli; D014157:Transcription Factors; C074889:RcsB protein, Bacteria; D004247:DNA