Catabolite Activator Protein (CAP)
and Jennifer Lovick
II. CAP-cAMP Structure
III. CAP-DNA Interaction
IV. CAP-DNA-alpha CTD Complex
This exhibit displays molecules in the left part of the screen,
and text that addresses structure-function relationships of
the molecules in the right part (below). Use the scrollbar to
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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.
Each monomer consists of an amino-terminal
domain responsible for dimerization 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
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.
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
electrostatic interactions are involved in cAMP binding, including:
- a salt bridge
between the sidechain of arginine82
and a phosphate
oxygen of cAMP
bonds between cAMP atoms and side chain atoms of glutamate72,
- hydrogen bonds
between main chain atoms (serine83)
- a hydrogen
bond between cAMP and a serine128,
on the C helix from the opposite monomer.
The CAP homodimer
(with bound cAMP)
binds a 22-basepair DNA consensus sequence with a two-fold axis of
CAP can be seen
to induce a sharp bend of ~ 90o in target DNA .
domain of each CAP monomer contains a helix-turn-helix
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
binding specificity. The
recognition helix of the
motif is inserted into the DNA major groove, where base
sequence specific contacts are available.
one monomer and its DNA half site, numerous protein-DNA contacts can
be identified, including:
bonds between recognition helix residues
(arg180, glu181, and arg185) and
bases lining the DNA major groove
recognition helix residues (ser179,
thr182), and phosphate oxygens on the backbone of DNA
of residues not in the recognition helix
(e.g. val139, lys26) with the DNA backbone
of the CAP-DNA interactions are facilitated by the bending of DNA
in response to CAP binding.
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
domains of CAP are indicated.
activation of transcription by CAP requires an activating region (AR1)
in the C-terminal
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).
sidechain of AR1 residue
thr158 forms two hydrogen bonds with alphaCTD
residues, one with thr285,
the second with glu286.
carbonyl of thr158
also makes two
hydrogen bonds, one with thr285
and one with
der Waals interactions between AR1and
contribute to CAP-alphaCTD
backbone carboxylate of the C-terminal arg209
of CAP forms
a salt bridge with arg317
of alphaCTD. The side chain of arg209
in a hydrogen bond with gly315
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
residues, and by water-mediated H-bonds between protein and DNA bases.
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).
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).
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).