Biosynthesis of a site-specific DNA cleaving protein

Article abstract of DOI:10.1021/ja804653f

An E. coli catabolite activator protein (CAP) has been converted into a sequence-specific DNA cleaving protein by genetically introducing (2,2′-bipyridin-5-yl)alanine (Bpy-Ala) into the protein. The mutant CAP (CAP-K26Bpy-Ala) showed comparable binding affinity to CAP-WT for the consensus operator sequence. In the presence of Cu(II) and 3-mercaptopropionic acid, CAP-K26Bpy-Ala cleaves double-stranded DNA with high sequence specificity. This method should provide a useful tool for mapping the molecular details of protein-nucleic acid interactions. Copyright

Full text of DOI:10.1021/ja804653f

Published on Web 09/13/2008
Biosynthesis of a Site-Specific DNA Cleaving Protein
Hyun Soo Lee and Peter G. Schultz*
Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, California 92037
Received June 18, 2008; E-mail: schultz@scripps.edu
A number of chemical tools have been developed to map
biomolecular interactions, including chemical and photocrosslinking
reagents,1 DNA footprinting methods,2 and fluorescent resonance
energy transfer pairs.3 These reagents have helped to elucidate the
molecular details of protein-protein and protein-nucleic acid
interactions, as well as the higher level architecture of cellular
regulatory and signaling networks. A major challenge with all of
these methods involves placement of the chemical or spectroscopic
probe at a defined site in the protein. This is generally accomplished
by exploiting the unique reactivity of cysteine or lysine side chains
but can be complicated by the presence of multiple such residues
in a protein and cannot easily be carried out within cells. Recently,
we developed methods that allow one to genetically encode
additional amino acids, beyond the common 20, directly in
Figure 1. (a) Structure of Bpy-Ala. (b) Crystal structure of the CAP-DNA
prokaryotic and eukaryotic organisms. This approach has been used
to site-specifically introduce chemically reactive and photocrosslink-
ing amino acids4 as well as spectroscopic probes5 into proteins.
Here we demonstrate that redox active metal ions can be selectively
introduced at defined sites in a DNA binding protein to directly
map its binding site on duplex DNA.
complex with K26 highlighted.10,11
It has been shown that attachment of EDTA-Fe(II) or 1,10-
phenanthroline-Cu(II) derivatives to a DNA binding agent near the
DNA interface leads to oxidative cleavage of the phosphosugar
moiety of the nucleic acid backbone.6 Subsequent high resolution
gel electrophoresis allows precise determination of the binding site
size, sequence, orientation, and groove location of the ligand on
DNA. Recently, the metal-chelating amino acid, (2,2′-bipyridin-5-
yl)alanine (Bpy-Ala, Figure 1a), was genetically encoded in E. coli
with an orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pair
from Methanococcus jannashii.7 The evolved tRNA/aaRS pair is
able to incorporate Bpy-Ala into proteins with high efficiency and
specificity in response to the amber nonsense codon. Since 2,2′-
bipyridyl strongly chelates many transition-metal ions such as Cu2+
and Fe2+, the genetic incorporation of Bpy-Ala into DNA-binding
proteins should similarly make possible oxidative cleavage of the
DNA backbone adjacent to the Bpy-Ala residue.
Figure 2. Gel-retardation assay of CAP-WT and CAP-K26Bpy with 50-
bp 5′-32P-end-labeled DNA fragment. Conditions: 2 nM DNA, CAP, 50
µg/mL BSA, 0.2 mM cAMP, 10 mM MOPS (pH 7.3), 200 mM NaCl, 10
µL total volume, 37 °C, 10 min.
To incorporate Bpy-Ala, an amber codon was substituted for
Lys26 in a CAP derivative containing a C-terminal His6 tag. Bpy-
Ala was prepared according to a literature procedure with minor
modifications.7 Expression of CAP was carried out in the presence
of the evolved tRNA/aaRS pair and 1 mM of Bpy-Ala in E. coli
strain DH10B grown in glycerol minimal medium.13 Mutant and
wildtype (WT) proteins were purified by Ni-NTA, and the yield of
the mutant CAP was 4-8 mg/L (CAP-WT yield was 10-15 mg/
L). SDS-PAGE analysis showed that a small amount of full-length
protein was produced in the absence of Bpy-Ala. However, ESI-
mass spectrometric analysis indicated that the full-length mutant
protein contained Bpy-Ala with no detectable common amino acid
incorporation (Figures S1 and S2).
The binding affinity of the CAP-K26Bpy-Ala to a 50-bp double-
stranded DNA fragment containing the 22-bp DNA recognition site
for CAP was determined by a gel-retardation assay (Figure 2). The
dissociation constants for CAP-WT (Kd ) 0.85 nM) and CAP-
K26Bpy-Ala (Kd ) 1.9 nM) determined from the gel shift data
show that CAP-K26Bpy-Ala has comparable binding affinity to
that for CAP-WT and the Bpy-Ala group does not significantly
perturb the interaction of CAP with its operator sequence. In
contrast, previous chemical labeling experiments of the endogenous
Cys178 of CAP12a with an iodoacetamide phenanthroline derivative
led to a 400-fold reduction in affinity to the operator sequence,
possibly due to nonspecific modification of the protein or a
To demonstrate the feasibility of this approach, Bpy-Ala was
introduced into the E. coli catabolite activator protein (CAP). CAP
is a structurally characterized transcriptional activator which regu-
lates a number of catabolite-sensitive operons in E. coli.8 CAP
functions by binding a 2-fold symmetric 22-bp consensus binding
site upstream of CAP-dependent promoters in the presence of the
allosteric effector cAMP.8,9 Upon binding to DNA as a homodimer,
CAP induces an approximate 90° bend in the double helix.10 Based
on the crystal structure of the CAP-DNA complex,10,11 Lys26 was
substituted by Bpy-Ala. Lys26 lies at the protein-DNA interface
near bp 1 and 22 of the DNA recognition site (Figure 1b). Indeed,
earlier studies showed that chemical derivatization of a Lys26Cys
CAP mutant with an electrophilic phenanthroline derivative afforded
a selective affinity cleaving agent.12b
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10.1021/ja804653f CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
restriction fragment (3.3kb) was also selective (Figure S3), indicat-
ing that the genetic incorporation of the Cu2+-bipyridyl side chain
into a DNA binding protein effectively localizes its oxidative
chemistry at a defined site on DNA.
In conclusion, we have demonstrated that CAP can be converted
into a site-specific DNA cleaving protein by genetically introducing
Bpy-Ala into the protein. The mutant DNA binding protein, CAP-
K26Bpy-Ala, was able to cleave double-stranded DNA at its
consensus sequence with high specificity. Theoretically, any DNA
binding protein can be converted into a DNA cleaving protein by
this method. Because Bpy-Ala can be placed at almost any surface
site on a protein, this method should provide a high resolution
picture of interactions between specific regions of the protein and
nearby sites on DNA. Moreover, because introduction of Bpy-Ala
only requires simple mutagenesis, it should be possible to rapidly
generate a series of Bpy-Ala mutants to define an optimal site of
incorporation even in the absence of structural information. Finally,
it may also be possible to use a similar approach to map
protein-RNA14 or protein-protein interfaces, as it has been
previously shown that selective delivery of Cu2+-phenanthroline
to these biopolymers also results in selective backbone cleavage.15
Acknowledgment. We thank Dr. Mary Sever, Dan Groff, and
Dr. Youngha Ryu for experimental assistance and helpful discus-
sions. We are grateful to the U.S. Department of Energy, Division
of Materials Sciences, under Award No. DE-FG03-00ER46051, and
the Skaggs Institute for Chemical Biology for support of this work.
Figure 3. Autoradiogram of a 15% TBE-Urea gel of CAP-K26Bpy-Ala
cleavage of a 5′-32P-end-labeled 50-bp DNA fragment. Conditions: 2 nM
DNA (50 bp), 100 nM CAP, 200 nM CuSO4, 2.5 mM 3-mercaptopropionic
acid, 50 µg/mL BSA, 0.2 mM cAMP, 2.2% EtOH, 10 mM MOPS (pH
7.3), 200 mM NaCl, 30 µL total volume, 37 °C, 12 h. (a) Data for the
bottom strand. Lane 1, DNA only; lane 2, CAP-WT with all components;
lane 3, all components without CAP; lane 4, CAP-K26Bpy-Ala without
reducing agent; lane 5, CAP-K26Bpy-Ala with all components; lane 6, DNA
marker. (b) Data for the top strand. Lane 1, DNA only; lane 2, CAP-
K26Bpy-Ala with all components; lane 3, DNA marker. (c) Arrows indicate
the cleavage sites and the lengths of arrows indicate the relative extents of
DNA cleavage.
Supporting Information Available: Materials and Methods. This
material is available free of charge via the Internet at http://pubs.acs.org.
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