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PDBsum entry 1jk1
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Transcription/DNA
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PDB id
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1jk1
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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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Rearrangement of side-Chains in a zif268 mutant highlights the complexities of zinc finger-Dna recognition.
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Authors
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J.C.Miller,
C.O.Pabo.
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Ref.
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J Mol Biol, 2001,
313,
309-315.
[DOI no: ]
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PubMed id
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Abstract
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Structural and biochemical studies of Cys(2)His(2) zinc finger proteins
initially led several groups to propose a "recognition code" involving
a simple set of rules relating key amino acid residues in the zinc finger
protein to bases in its DNA site. One recent study from our group, involving
geometric analysis of protein-DNA interactions, has discussed limitations of
this idea and has shown how the spatial relationship between the polypeptide
backbone and the DNA helps to determine what contacts are possible at any given
position in a protein-DNA complex. Here we report a study of a zinc finger
variant that highlights yet another source of complexity inherent in protein-DNA
recognition. In particular, we find that mutations can cause key side-chains to
rearrange at the protein-DNA interface without fundamental changes in the
spatial relationship between the polypeptide backbone and the DNA. This is clear
from a simple analysis of the binding site preferences and co-crystal structures
for the Asp20-->Ala point mutant of Zif268. This point mutation in finger one
changes the specificity of the protein from GCG TGG GCG to GCG TGG GC(G/T), and
we have solved crystal structures of the D20A mutant bound to both types of
sites. The structure of the D20A mutant bound to the GCG site reveals that
contacts from key residues in the recognition helix are coupled in complex ways.
The structure of the complex with the GCT site also shows an important new water
molecule at the protein-DNA interface. These side-chain/side-chain interactions,
and resultant changes in hydration at the interface, affect binding specificity
in ways that cannot be predicted either from a simple recognition code or from
analysis of spatial relationships at the protein-DNA interface. Accurate
computer modeling of protein-DNA interfaces remains a challenging problem and
will require systematic strategies for modeling side-chain rearrangements and
change in hydration.
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Figure 4.
Figure 4. Stereo representation of simulated-annealing F[o]
-F[c] electron density of the D20A- Image structure with
residues 18 and 21 omitted from the calculation. The map is
contoured at 3 s, and was generated using the program O and
rendered using the SwissPDBViewer and POV Ray.
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Figure 5.
Figure 5. Comparison of the D20A mutant bound to different
DNA sites and comparison with wt Zif268 structure. (a) Region of
the D20A-GCG structure focusing on the interacting residues.
Broken lines indicate hydrogen bonds. Simple modeling indicates
that Glu21 could not obtain this new conformation in the
wild-type complex without making electrostatically unfavorable
contacts with Asp20. Modeling also suggests that this new Glu21
conformation would collide with the thymine methyl group if the
mutant was bound to a Image site and this observation fits
nicely with the binding data for the two proteins at the Image
and Image sites. (b) Same region of the wild-type Zif268
structure[16] shown in previous panel. (c) Comparison of the
interaction between Glu21 and Arg18 in the D20A- Image structure
with the interaction between Asp20 and Arg18 in the wild-type
structure. Broken lines indicate hydrogen bonds and distances
are given in Å. (d) Corresponding region of the D20A-GCT
structure. Only the most relevant contact made by the secondary
Arg18 conformation is shown for clarity. We explored the
possibility that the electron density modeled as an ordered
water was actually due to an alternate conformation of Arg18,
but we were not able to fit the Arg18 guanidinium group into
this density without severely distorting the side-chain geometry
and generating several unacceptable steric clashes.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2001,
313,
309-315)
copyright 2001.
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Secondary reference #1
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Title
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Zif268 protein-Dna complex refined at 1.6 a: a model system for understanding zinc finger-Dna interactions.
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Authors
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M.Elrod-Erickson,
M.A.Rould,
L.Nekludova,
C.O.Pabo.
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Ref.
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Structure, 1996,
4,
1171-1180.
[DOI no: ]
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PubMed id
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Figure 2.
Figure 2. Overview of the Zif268-DNA complex, showing the
side chains that make direct base contacts. The peptide is
color-coded by finger: finger one is red, finger two is yellow,
and finger three is purple. The DNA is shown in dark blue, and
the zinc ions in pale blue.
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The above figure is
reproduced from the cited reference
with permission from Cell Press
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Secondary reference #2
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Title
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Binding studies with mutants of zif268. Contribution of individual side chains to binding affinity and specificity in the zif268 zinc finger-Dna complex.
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Authors
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M.Elrod-Erickson,
C.O.Pabo.
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Ref.
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J Biol Chem, 1999,
274,
19281-19285.
[DOI no: ]
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PubMed id
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Figure 1.
Fig. 1. a, overview of the Zif268 zinc finger-DNA
complex. Only those side chains that make direct base contacts
are shown. Finger one is red, fingers two and three are yellow,
the DNA is blue, and the zinc ions are gray (adapted from Ref.
12). b, schematic diagram of the base contacts made by Zif268.
Arrows indicate hydrogen bonds; dotted arrows represent hydrogen
bonds with marginal geometry. Lines ending in filled circles
represent van der Waals' interactions. The numbering scheme is
the same as that used in papers describing the structure of the
complex (11, 12) (adapted from Ref. 11).
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Figure 2.
Fig. 2. Sequences of the zinc finger peptides and of the
oligonucleotide binding sites. a, sequence of the wild type
Zif268 zinc finger peptide. The residues at positions  1, 2, 3,
and 6 of the  helix of
finger one, which have been the focus of this study, are
circled. The three fingers are aligned to highlight conserved
residues and conserved secondary structure elements. The helix is
indicated by a cylinder, and the  strands are
indicated by arrows. The cysteine and histidine residues that
are ligands for the zinc ions are highlighted in bold (adapted
from Ref. 12). b, sequences of the wild type and mutant
oligonucleotide binding sites used in the gel shift assays. The
Zif268 binding site is highlighted in bold; the numbering scheme
is the same as that used in papers describing the structure of
the complex (11, 12). Boxes indicate bases that are altered in
the mutant binding sites.
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The above figures are
reproduced from the cited reference
with permission from the ASBMB
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