Rational design and molecular characterization of a chimaeric response regulator protein.
BvgA and EvgA are closely related response regulators from Bordetella pertussis and Escherichia coli. To analyze the domain borders and linker sequences of these proteins, we used limited proteolysis and matrix-assisted laser desorption/ionization-mass spectrometry analysis of the in-gel-digested proteolytic fragments. The thermolysin-sensitive linker regions were found to extend from Leu130 to Thr144 for BvgA and from Leu127 to Ser133 for EvgA. These data provided the rationale for the construction of the chimaeric protein HA. HA carries the EvgA receiver and BvgA output domains, fused in the central part of the linker sequences of the parent proteins. Thermolysin-sensitive sites of HA were found at positions identical with those in the EvgA and BvgA linker sequences, indicating intact folding of its receiver and output domains. Consistent with this, the chimaera showed virtually unchanged phosphorylation and dimerization properties. However, BvgA and HA differed in the effect of phosphorylation on their DNA-binding activities. In the case of BvgA, phosphorylation resulted in an increased affinity and specificity in DNA binding, whereas the DNA-binding properties of HA were not affected by phosphorylation. The chimaera HA was unable to activate transcription of the BvgA-dependent fha promoter, either in vivo or in vitro. These results indicate that the phosphorylation-induced activation of BvgA requires specific interactions between the receiver and output domains that are disturbed in the chimaera.
Bock
A
A
Lehrstuhl für Mikrobiologie, Biozentrum, Universität Würzburg, Würzburg, D-97074, Germany.
Bantscheff
M
M
Perraud
A L
AL
Rippe
K
K
Weiss
V
V
Glocker
M O
MO
Gross
R
R
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001886:Bordetella pertussis; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial / Q000235:genetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism*; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D013820:Thermolysin / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation
D001426:Bacterial Proteins; C065601:BvgA protein, Bacteria; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C489314:EvgA protein, E coli; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D004247:DNA; D013820:Thermolysin