Role of glutamic acid-181 in DNA-sequence recognition by the catabolite gene activator protein (CAP) of Escherichia coli: altered DNA-sequence-recognition properties of [Val181]CAP and [Leu181]CAP.
It has been proposed that Glu-181 of the catabolite gene activator protein (CAP) makes direct contact with certain base pairs of the specific DNA site. We have purified wild-type CAP and two substituted CAP variants, [Val181]CAP and [Leu181]CAP, and have assessed the DNA-sequence-recognition properties in vitro with respect to positions 5, 6, 7, 8, and 16 of the DNA site. The data indicate that [Val181]CAP and [Leu181]CAP fail to discriminate between the consensus DNA base pair and the three non-consensus-DNA base pairs at 2-fold-related positions 7 and 16 of the DNA site. In contrast, [Val181]CAP and [Leu181]CAP retain the ability to discriminate between different base pairs at positions 5 and 8 of the DNA site. We conclude that Glu-181 of CAP makes a direct contact with 2-fold-related positions 7 and 16 of the DNA site, as proposed previously based on in vivo results. We propose that upon replacement of Glu-181 by valine or leucine, this contact is eliminated and is replaced by no other functional contact. We estimate that the contact by Glu-181 with each position contributes -0.7 kcal/mol to the total CAP-DNA binding free energy.
D000595:Amino Acid Sequence; D001665:Binding Sites; D004269:DNA, Bacterial / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D005971:Glutamates; D018698:Glutamic Acid; D007930:Leucine; D009154:Mutation; D011953:Receptors, Cyclic AMP / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics; D014633:Valine
D004269:DNA, Bacterial; D005971:Glutamates; D011953:Receptors, Cyclic AMP; D018698:Glutamic Acid; D007930:Leucine; D014633:Valine