Structure-function studies with the cAMP receptor protein of Escherichia coli



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Texas Tech University


Cyclic AMP Receptor protein (CRP) regulates the transcription of more than 100 genes. In the absence of cAMP, CRP is inactive. Cyclic AMP binding induces a structure change in CRP that promotes its interaction with RNA polymerase and DNA. CRP is a dimer of identical subunits; each consisting of 209 amino acids. A CRP subunit is composed of two domains. The larger N-terminal domain binds the allosteric effector, cAMP. This domain consists of eight j8-sheets that provide a hydrophobic pocket for cAMP binding. One cAMP is bound to each subunit contacting amino acid residues from both subunits. The C-terminal domain contains a helix-tum-helix motif that binds specific DNA sequences.

The structure of CRP in the absence of cAMP is unknown, therefore the details of the allosteric mechanism mediated by cAMP remain obscure. The allosteric conformational change in CRP upon binding cAMP can be understood by comparing CRP and the CRP-cAMP complex by similar biophysical characterization. Several groups have used Raman spectroscopy, and circular dichroism techniques to compare these two different states of CRP. These methods used high concentrations of salt to improve CRP solubility. Our laboratory has used Fourier Transform Infrared Spectroscopy (FTIR) along with STIR cards to overcome the problems of protein solubility and high salt concentration. Analysis of the Amide I region indicated a secondary structure distribution of 35% a-helix, 31% jS-sheet, 21% turn, and 13% unordered for both states of WT and its E72D, E72Q, and R82Q mutants. This result is consistent with X-ray analysis of CRP-CAMP2 (37% a-helix, 36% ^-sheet).

Fluorimetric binding studies showed that cAMP binding exhibits negative cooperativity in cAMP binding to the second subunit and amino acid substitution at positions 72 and 82 reduced binding affinities for cAMP by factors of 2 to 25 fold. DNA binding studies indicated that the equilibrium constants of the mutant CRP: cAMP complexes measured for lac? were reduced compared to that of WT CRP: cAMP complex. In addition, the mutant complexes failed to footprint in the presence of RNA polymerase.

The level of j8-galactosidase expression in the mutants varied depending on this negative allostery. Since, under the conditions utilizied in this study, cAMP makes no contact with the DNA-binding domains, it cannot induce a conformational change in them by direct interaction. This suggests that cAMP induces a change in the relative orientation of the two subunits because it binds close to the subunit interaction area. This change could be relayed to the DNA binding domain and could change the relative position and orientation of the recognition helices and the activity. Thus these results can explain the allosteric transition mediated by the binding of cyclic AMP that converts CRP from a protein having low DNA activity to one that exhibits high, sequence-specific, affinity for DNA