The Catabolite Activator Protein (CAP)
David Marcey and Jennifer Lovick
© 2021

I. Introduction
II. CAP-cAMP Structure
III. CAP-DNA Interaction

IV. CAP-DNA-alpha CTD Complex
V. References


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I. Introduction

The protein shown at left is the catabolite activator protein (CAP), also known as the cyclic AMP (cAMP) receptor protein (CRP), a transcriptional activator in E. coli. CAP activates transcription of a variety of genes including many involved in the metabolism of sugars (e.g. genes encoding proteins involved in metabolism of lactose, galactose and also arabinose). CAP binds as a homodimer to specific DNA sequences upstream of these genes, but only when the protein is in complex with cAMP. CAP activates transcription by contacting RNA polymerase. Thus, for example, at the lac operon, it recruits RNA polymerase to the promoter by interacting with the carboxy-terminal domain of the alpha subunit of RNA polymerase (alphaCTD). This enhances the frequency of transcription initiation.

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II. CAP-cAMP Structure

Each monomer consists of an amino-terminal domain responsible for dimerization as well as cAMP binding and a carboxy-terminal domain that binds to DNA and also interacts with alpha-CTD (see below). These domains are connected by a short hinge sequence.

Dimerization is largely due to hydrophobic interactions between amino acid sidechains of the long, central alpha helix in the N-terminal domain of each monomer, the C helix.

cAMP is bound in a pocket of the N-terminal domain of each CAP monomer. This pocket is formed between the C helix and a beta roll motif that includes beta strands 1-8.

Numerous electrostatic interactions are involved in cAMP binding, including:

  • a salt bridge between the sidechain of arginine82 and a phosphate oxygen of cAMP
  • hydrogen bonds between cAMP atoms and side chain atoms of glutamate72, serine83, and threonine127
  • hydrogen bonds between main chain atoms (serine83) and cAMP
  • a hydrogen bond between cAMP and a serine128, on the C helix from the opposite monomer.


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III. CAP-DNA Interaction

The CAP homodimer (with bound cAMP) binds a 22-basepair DNA consensus sequence with a two-fold axis of symmetry:

CAP can be seen to induce a sharp bend of ~ 90o in target DNA .

The C-terminal domain of each CAP monomer contains a helix-turn-helix (H-T-H) DNA binding motif found in most bacterial transcription factors. This motif is found, in a modified form (the homeodomain), in some eukaryotic transcription factors as well. The H-T-H motif confers DNA binding specificity. The recognition helix of the motif is inserted into the DNA major groove, where base sequence specific contacts are available.

Examining one monomer and its DNA half site, numerous protein-DNA contacts can be identified, including:

  • hydrogen bonds between recognition helix residues (arg180, glu181, and arg185) and bases lining the DNA major groove
  • hydrogen bonds between recognition helix residues (ser179, thr182), and phosphate oxygens on the backbone of DNA
  • interaction of residues not in the recognition helix (e.g. val139, lys26) with the DNA backbone

Many of the CAP-DNA interactions are facilitated by the bending of DNA in response to CAP binding.


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IV. CAP-DNA-alpha CTD Complex

Shown at left is a CAP monomer (with bound cAMP) complexed with a DNA sequence representing one half of the consensus CAP binding sequence plus the carboxy-terminal domain of the alpha subunit of of RNA polymerase (alphaCTD). The C-terminal and N-terminal domains of CAP are indicated.

The activation of transcription by CAP requires an activating region (AR1) in the C-terminal domain. AR1 is a loop of nine residues (156-164). CAP transcriptional activation also requires the C-terminal residue of CAP (arg209). Both AR1 and arg209 play key roles in CAP interaction with polymerase (alphaCTD). For example:

  • The sidechain of AR1 residue thr158 forms two hydrogen bonds with alphaCTD residues, one with thr285, the second with glu286. The backbone carbonyl of thr158 also makes two hydrogen bonds, one with thr285 and one with val287.
  • van der Waals interactions between AR1and alphaCTD contribute to CAP-alphaCTD binding.
  • The backbone carboxylate of the C-terminal arg209 of CAP forms a salt bridge with arg317 of alphaCTD. The side chain of arg209 participates in a hydrogen bond with gly315 of alphaCTD.

alphaCTD binds to a DNA sequence centered 19 base pairs from the center of the CAP binding site: 5'- A A A A A G - 3'. Binding is achieved through extensive contact of the DNA backbone by alphaCTD residues, and by water-mediated H-bonds between protein and DNA bases. For example:

  • Asn268, gly296, lys298, and ser299 form H-bonds with several DNA phosphate oxygens.
  • Although no direct contact is made between alphaCTD and DNA bases, water-mediated H-bonds connect arg265 to several bases in the DNA minor groove, into which this residue penetrates (water hydrogens not shown).


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IV. References

Benoff, B., Yang, H., Lawson, C. L., Parkinson, G., Lui, J., Blatter, E., Ebright, Y. W., Berman, H. M., Ebright, R. H.: Structural Basis of Transcription Activation: The Structure of CAP-Alphactd-DNA Complex. Science 297: 1562-1566 (2002).

Parkinson, G., Gunasekera, A., Vojtechovsky, J., Zhang, X., Kunkel, T. A., Berman, H., Ebright, R. H.: Aromatic hydrogen bond in sequence-specific protein DNA recognition. Nat Struct Biol 3: 837-841 (1996).

Passner, J. M., Schultz, S. C., Steitz, T. A.: Modeling the Camp Induced Allosteric Transition Using the Crystal Structure of CAP-Camp at 2.1 A Resolution. J.Mol.Biol. 304: 847-859 (2000).

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