DNA-induced conformational changes in cyclic AMP receptor protein: detection and mapping by a protein footprinting technique using multiple chemical proteases.
Cyclic AMP receptor protein (CRP) is a regulator of transcription in Escherichia coli which mediates its activity by binding specific DNA sequences in a cyclic AMP-dependent manner. The interaction of CRP with specific DNA was probed by a protein footprinting technique using chemical proteases of different charge, size, and hydrophobicity. The experimental data were compared with known crystal structures of cAMP-CRP and cAMP-CRP-DNA complexes to determine a correlation between the structure of the complexes, the nature of the chemical protease and protein cleavage patterns. In addition, such comparison allowed us to determine if DNA binding in solution induced conformational changes in the protein not apparent in the crystal structure. In the cAMP-CRP-DNA complex, both the protections and the enhancements of proteolytic cleavage were observed outside of the known CRP-DNA interface, suggesting that CRP undergoes a conformational change upon binding DNA. Among the observed changes, the most interesting were those around the B alpha-helix and beta-strand 8, since this region overlaps with the activation region 2 which CRP uses for protein-protein interactions with RNA polymerase. DNA-induced changes were observed also in the region involved in CRP-CytR interaction and in CRP intersubunit contact regions. These data suggest that binding of DNA in solution induces conformational changes in CRP which can be transmitted via intersubunit contacts to regions of the protein involved in interactions with other members of transcriptional machinery.
Baichoo
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Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University Medical School, 1402 S. Grand Blvd., St. Louis, MO, 63104, USA.
Heyduk
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D002614:Chelating Agents / Q000737:chemistry; D002373:Cyclic AMP Receptor Protein / Q000737:chemistry* / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D008670:Metals / Q000737:chemistry; D008958:Models, Molecular; D011487:Protein Conformation
D002614:Chelating Agents; D002373:Cyclic AMP Receptor Protein; D004268:DNA-Binding Proteins; D008670:Metals; D004247:DNA