Catabolite Activator Protein

The Catabolite Activtor Protein (CAP)
David Marcey
©2003

I. Introduction
II. CAP-Cyclic AMP Structure
III. CAP-DNA Interaction
IV. CAP-DNA-alphaCTD Complex
V. References

I. Introduction

The catabolite activator protein (CAP), also known as the cyclic AMP (cAMP) receptor protein (CRP), is a positive transcriptional activator in E. coli. CAP activates transcription at a variety of promoters that drive operons involved in catabolite metabolism (e.g. P_lac, P_gal). CAP accomplishes this important regulatory function by binding cAMP, which increases CAP binding to a conserved DNA sequence located in or upstream of target core promoters. CAP-cAMP binds promoter DNA as a homodimer. CAP-cAMP assists RNA polymerase binding to the promoter by interacting with the carboxy-terminal domain of the alpha subunit of of RNA polymerase (alphaCTD), thereby enhnacing the frequency of transcription initiation of catabolic operons.

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 small 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 beta strands 1-8, a beta roll motif.

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 ~ 90^o in target DNA .

The C-terminal domain of each CAP monomer contains a helix-turn-helix DNA binding motif found in a variety of DNA binding proteins, including some eukaryotic transcription factors. This motif confers DNA binding specificity. The recognition helix (F) 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-alphaCTD 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 (arg²⁰⁹). Both AR1 and arg²⁰⁹ play key roles in CAP interaction with alpha-CTD. 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 alpha-CTD 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 and several bases in the DNA minor groove, into which this residue penetrates.

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V. 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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