Allosteric changes in the cAMP receptor protein of Escherichia coli: hinge reorientation.
The cAMP receptor protein (CRP) of Escherichia coli is a dimer of a two-domain subunit. It requires binding of cAMP for a conformational change in order to function as a site-specific DNA-binding protein that regulates gene activity. The hinge region connecting the cAMP-binding domain to the DNA-binding domain is involved in the cAMP-induced allosteric change. We studied the structural changes in CRP that are required for gene regulation by making a large number of single and double amino acid substitutions at four different positions in or near the hinge. To achieve cAMP-independent transcription by CRP, amino acid residues 138 (located within the hinge region) and 141 (located in the D alpha-helix adjacent to the hinge) must be polar. This need for polar residues at positions 138 and 141 suggests an interaction that causes the C and D alpha-helices to come together. As a consequence, the F alpha-helix is released from the D alpha-helix and can interact with DNA. At position 144 in the D alpha-helix and within interacting distances of the F alpha-helix, replacement of alanine by an amino acid with a larger side chain, regardless of its nature, allows cAMP independence. This result indicates that pushing against the F alpha-helix may be a way of making the helix available for DNA binding. We believe that the cAMP-induced allosteric change involves similar hinge reorientation to adjust the C and D alpha-helices, allowing outward movement of the F alpha-helix.
Kim
J
J
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Adhya
S
S
Garges
S
S
D000494:Allosteric Regulation; D001426:Bacterial Proteins / Q000648:ultrastructure*; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000648:ultrastructure; D004926:Escherichia coli; D007763:Lac Operon; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011953:Receptors, Cyclic AMP / Q000648:ultrastructure*; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D011953:Receptors, Cyclic AMP