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PDBsum entry 1dh3
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Transcription/DNA
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PDB id
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1dh3
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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The structure of a creb bzip.Somatostatin cre complex reveals the basis for selective dimerization and divalent cation-Enhanced DNA binding.
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Authors
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M.A.Schumacher,
R.H.Goodman,
R.G.Brennan.
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Ref.
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J Biol Chem, 2000,
275,
35242-35247.
[DOI no: ]
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PubMed id
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Abstract
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The cAMP responsive element-binding protein (CREB) is central to second
messenger regulated transcription. To elucidate the structural mechanisms of DNA
binding and selective dimerization of CREB, we determined to 3.0 A resolution,
the structure of the CREB bZIP (residues 283-341) bound to a 21-base pair
deoxynucleotide that encompasses the canonical 8-base pair somatostatin cAMP
response element (SSCRE). The CREB dimer is stabilized in part by ionic
interactions from Arg(314) to Glu(319') and Glu(328) to Lys(333') as well as a
hydrogen bond network that links the carboxamide side chains of
Gln(322')-Asn(321)-Asn(321')-Gln(322). Critical to family selective dimerization
are intersubunit hydrogen bonds between basic region residue Tyr(307) and
leucine zipper residue Glu(312), which are conserved in all CREB/CREM/ATF-1
family members. Strikingly, the structure reveals a hexahydrated Mg(2+) ion
bound in the cavity between the basic region and SSCRE that makes a
water-mediated DNA contact. DNA binding studies demonstrate that Mg(2+) ions
enhance CREB bZIP:SSCRE binding by more than 25-fold and suggest a possible
physiological role for this ion in somatostatin cAMP response element and
potentially other CRE-mediated gene expression.
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Figure 3.
Fig. 3. Electron density maps of the CREB bZIP divalent
cation-binding pocket. A, (2F[o]  F[c]) omit
electron density map (gray wire mesh) contoured at 1  and
calculated with phases from the final model in which residues
307 to 321 of each monomer, the hexahydrated magnesium ion and
the water molecules involved in DNA binding were deleted and
followed by xyzb refinement until convergence, which removes
phase bias. Labeled are the hexahydrated magnesium ion, residues
Tyr307 and Glu312', and Lys304 and Arg301 from each subunit. B,
(F[o] F[c]) omit
electron densities using coefficients F[o](BaCl[2])
F[o](MgCl[2]) (contoured at  6 and
displayed as red wire mesh) or F[o](Na[2]WO[4])
F[o]((NH[4])[2]SO[4]) (contoured at 4 and
displayed as blue wire mesh) confirming unequivocally the
identity of the density in A to be a divalent cation. The slight
off-centering of the Ba^2+ ion is very likely the result of its
preferred hepta aquo coordination versus the hexa aquo
coordination of a Mg2+ ion. A and B were generated with O (15).
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Figure 4.
Fig. 4. CREB-DNA affinity and its divalent cation
enhancement. A, representative fluorescence anisotropy-derived
DNA binding isotherm of the CREB bZIP-SSCRE measured in the
presence of 10 m M MgCl[2] (diamonds) or 10 mM BaCl[2] (boxes)
in buffer A or buffer A alone (circles). Plotted are the
normalized measured millipolarization units (mP) versus the CREB
bZIP concentration (nM). B, representative divalent cation
binding isotherm. Measurements were made by titrating CaCl[2]
into a 0.990-ml reaction buffer of 25 mM Tris, pH 7.5, 5%
glycerol, 50 mM NaCl, 6 µg of bovine serum albumin, 10
µg of poly d(I-C) containing 1 nM fluoresceinated DNA and
20 nM CREB bZIP. Plotted are the measured millipolarization
units (mP) versus the Ca^2+ ion concentration. Essentially
identical binding isotherms are generated by titration with
MgCl[2] or BaCl[2]. C, CREB bZIP:SSCRE phosphate contacts.
Residues making phosphate contacts are shown as light blue
sticks and labeled in black. The DNA is shown as sticks. The
carbon atoms are colored white, oxygen atoms are red, nitrogen
atoms are blue, and phosphate atoms are shown as yellow. Arg289,
which does not make any direct phosphate contacts, is also
shown. This residue is in position to make potential
water-mediated base contacts to bases outside the consensus
SSCRE or, alternatively, may contribute to binding through a
general electrostatic effect. The two identical DNA strands are
differentiated as strand 1 (S1) and strand 2 (S2) to distinguish
cross-strand phosphate contacts made by each CREB bZIP subunit.
D, specific interactions between CREB and the SSCRE DNA. Only
DNA base pairs 1 to 4 of the consensus SSCRE, are shown. DNA
atoms are colored as in C. CREB residues involved in base
specific contacts are shown as light blue sticks. Only residue
Arg301' from the second subunit is shown as the remaining
base-specific contacts are identical between subunits. Two water
molecules (W1 and W2) and the hexahydrated magnesium ion are
shown as ball-and-sticks. Oxygen atoms are colored red and the
magnesium atom is colored light blue. Hydrogen bonds are
indicated by black dashed lines and hydrophobic contacts by
light blue dashed lines.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2000,
275,
35242-35247)
copyright 2000.
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