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References listed in PDB file
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Key reference
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Title
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A specificity switch in selected cre recombinase variants is mediated by macromolecular plasticity and water.
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Authors
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E.P.Baldwin,
S.S.Martin,
J.Abel,
K.A.Gelato,
H.Kim,
P.G.Schultz,
S.W.Santoro.
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Ref.
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Chem Biol, 2003,
10,
1085-1094.
[DOI no: ]
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PubMed id
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Abstract
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The basis for the altered DNA specificities of two Cre recombinase variants,
obtained by mutation and selection, was revealed by their cocrystal structures.
The proteins share similar substitutions but differ in their preferences for the
natural LoxP substrate and an engineered substrate that is inactive with
wild-type Cre, LoxM7. One variant preferentially recombines LoxM7 and contacts
the substituted bases through a hydrated network of novel interlocking
protein-DNA contacts. The other variant recognizes both LoxP and LoxM7 utilizing
the same DNA backbone contact but different base contacts, facilitated by an
unexpected DNA shift. Assisted by water, novel interaction networks can arise
from few protein substitutions, suggesting how new DNA binding specificities
might evolve. The contributions of macromolecular plasticity and water networks
in specific DNA recognition observed here present a challenge for predictive
schemes.
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Figure 3.
Figure 3. Details of ALSHG/LoxM7 ComplexCre/LoxP (green
sticks) was superimposed on ALSHG/LoxM7 (atom-colored balls and
sticks) as described in Figure 2. The dashed lines represent
potential hydrogen bonds in ALSHG/LoxM7 (black) and Cre/LoxP
(yellow).(A) Specific contacts to bases C7 and T8. Residues
258–266 of helix J are rolled 7° and shifted 0.6 Å
toward the DNA as a consequence of steric interactions between
Leu258 and Ala175. This repositioning facilitates hydrogen
bonding between Ser259 O^γ and C7 O^4 atoms. In addition, a
network involving water molecules Sol67, Sol179, and Sol503 (B
factors of 45, 52, and 50 Å^2, respectively) and the
Ser257 O^γ atom, the Leu258 N atom, and the Ser259 N and O^γ
atoms couples recognition of bases C7 and T8 and replaces the
water bridge between Thr258 O^γ1 atom and the N^4 atom of base
C8 in Cre/LoxP.(B) Coupled recognition of nucleotide T26, base
A27, and the phosphate backbone via a tripartite hydrogen bond
bridge. Base A27 is contacted by a hydrogen bond bridge mediated
by Sol501 and Sol502 with the Ser259 carbonyl. His262 is rotated
from the position of Glu262 in Cre/LoxP, which avoids a steric
clash and forms a tight Van der Waals contact with the 5-methyl
group of base T26. In addition, His262 forms a hydrogen bond
bridge between Sol501 and the phosphate of nucleotide 26,
connecting the T26 and A27 contacts.(C) Atomic level details of
ALSHG/LoxM7 interactions. Symbols and distances are as described
in Figure 1D.
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Figure 4.
Figure 4. Structure of the Substituted Region of the
LNSGG/LoxM7 ComplexFor comparison, Cre/LoxP (green sticks) or
ALSHG/LoxM7 (purple sticks) are superimposed on LNSGG/LoxM7
(atom-colored balls and sticks), as described in Figure 2.
Potential hydrogen bonds are denoted by dashed lines.(A)
LNSGG/LoxM7 has contacts between the DNA backbone and base C7
but not bases A27 and T26. Helix J maintains a position similar
to that in Cre/LoxP-G5. Ser259 forms a hydrogen bond with C7,
and Asn258 is positioned to form hydrogen bond with the
phosphate backbone at residue 24 (orange dashes). In addition,
Sol49 and new solvents Sol501 and Sol505 (B factors of 61, 52,
and 51 Å^2, respectively), form a hydrogen bond network
that interconnects the Ser259 carbonyl with the phosphates of
nucleotides 25 and 26. Sol49 and Sol84 occupy similar positions
in Cre/LoxP-G5. Although Sol502 is still bound by A27, the
increased length of the bridging contact with Sol501 (3.6
Å) indicates a weaker protein-DNA interaction.(B) Since
helix J is not rotated as in ALSHG/LoxM7 (purple) and Sol501 is
shifted toward Gly262, water molecules Sol501 and Sol502 are 1.2
Å farther apart (gray dashed lines) than in ALSHG/LoxM7
(cyan dashed lines), perhaps diminishing the strength of the
contact. Note the correspondences of Sol49 and Sol505 in LNSGG
and His262 in ALSHG. Sol84 is conserved in the Cre/LoxP-G5 and
1CRX structures.(C) Atomic level details of LNSGG/LoxM7
interactions. Symbols and distances are as described in Figure
1D. The gray stippled line indicates a weakened hydrogen bond
with a contact distance that is greater than 3.5 Å.
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The above figures are
reprinted
by permission from Cell Press:
Chem Biol
(2003,
10,
1085-1094)
copyright 2003.
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Secondary reference #1
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Title
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Directed evolution of the site specificity of cre recombinase.
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Authors
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S.W.Santoro,
P.G.Schultz.
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Ref.
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Proc Natl Acad Sci U S A, 2002,
99,
4185-4190.
[DOI no: ]
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PubMed id
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Figure 1.
Fig. 1. LoxP, the natural recombination site of Cre. The
outermost 13-bp regions on each side (black) are inverted
repeats. The middle region (gray) is asymmetric and confers
directionality to the site. The base pair identities most
important for Cre binding (12) are boxed. Arrows indicate sites
of cleavage during recombination.
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Figure 3.
Fig. 3. Cre recombination of loxP and loxP variant sites.
Each of the eight loxP variants shown (M1-M8) were inserted into
pS, replacing both of the loxP sites, and tested for
recombination in vivo. All of the sites, except lox M3, M5, and
M7, supported detectable levels of recombination by wild-type
Cre.
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Secondary reference #2
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Title
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Modulation of the active complex assembly and turnover rate by protein-Dna interactions in cre-Loxp recombination.
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Authors
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S.S.Martin,
V.C.Chu,
E.Baldwin.
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Ref.
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Biochemistry, 2003,
42,
6814-6826.
[DOI no: ]
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PubMed id
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Secondary reference #3
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Title
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The order of strand exchanges in cre-Loxp recombination and its basis suggested by the crystal structure of a cre-Loxp holliday junction complex.
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Authors
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S.S.Martin,
E.Pulido,
V.C.Chu,
T.S.Lechner,
E.P.Baldwin.
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Ref.
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J Mol Biol, 2002,
319,
107-127.
[DOI no: ]
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PubMed id
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Figure 2.
Figure 2. Cre-Lox complex structure, represented by
Cre^*/LoxP-G5. (a) Overall architecture of the synapse. The
non-cleaving A (purple) and cleaving B (green) Cre subunits are
shown in ribbon representation. The active-site tyrosine
residues for both subunits are shown for the upper dimer in gray
CPK spheres. The crossover C (yellow) and non-crossover D (blue)
DNA strands are shown as ball-and-sticks. The 3CRX-like
alternative conformation for the HJ crossover strand is also
shown (white). The tetramer is generated by crystallographic
2-fold symmetry from the unique Cre[2]-Lox dimer. The position
of the 2-fold axis is indicated by the black oval at the center
of the complex. (b) Main-chain structural differences between
residues 138-341 of the cleaving (green) and non-cleaving
subunits (purple) after superposition. The broken green line
depicts the connection made by residues 329-332, which are
disordered in the cleaving subunit of Cre^*/LoxP-G5. The largest
difference between subunits is in the 198-208 loop, shown in
thick cable representation. In the cleaving subunit loop
(black), Lys201 contacts the scissile base (201B, S1) while in
the non-cleaving subunit (red), the loop positions this residue
(201A) far from the scissile base.
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Figure 6.
Figure 6. Stereo views of positional differences in the
198-208 loop and scissile base regions of Cre^*/LoxP-G5 and
other structures. (a) The 198-208 loop in Cre^*/LoxP-G5 (atom
colored ball-and-stick) is displaced 3.4 Å (main-chain
rmsd for residues 198-208) compared to 3CRX, after superposition
of residues 20-197 and 209-341 (green sticks). Note the large
shift in the Lys201 position (arrow). The density in the
omit-refine F[o] -F[c] map (magenta, +2.5s) corroborates the
placement of this segment. Relative to Cre^*/LoxP-G5, the
R[work] and R[free] values after 50 refinement cycles changed
<±0.1% and +0.2%. (b) Comparison of interactions between
Lys86 and Lys201 in Cre^*/LoxP-G5 and 1CRX, superimposed and
colored as in (a). In 1CRX, Lys86 NZ makes a hydrogen bond with
the scissile guanine O6 atom (black dotted line), while in
Cre^*/LoxP-G5 it forms hydrogen bonds with N7 (red dotted line).
Lys201 NZ atom forms a hydrogen bond with the Ade14 N3 in
Cre^*/LoxP-G5 (red dotted line), while in 1CRX atoms CE and NZ
make only van der Waals contacts with the Gua14. The omit-refine
F[o] -F[c] omit map density (magenta, +2.3s) corroborates the
Cre^*/LoxP-G5 atom placements. Relative to Cre^*/LoxP-G5, the
R[work] and R[free] values after 50 refinement cycles changed
-0.3% and +0.1%.
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The above figures are
reproduced from the cited reference
with permission from Elsevier
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Author's comment:
Crystallizing wildtype Cre (containing a minimal N-terminal His-6 extension) with the intact LoxP substrate results in the HJ(L) intermediate, that is, with Cre poised to cleave the upper strand (left arm) of the HJ intermediate. We have utilized this outcome to characterize variant Cre/Lox complexes (1MA7, 1PVP, 1PVQ, and 1PVR). Interactions with Lys201 and the scissile Ade14 base suggest a mechanism for strand selection in the HJ intermediate, biasing the reaction pathway towards the products. It is generally accepted that Cre initiates recombination by cleaving the lower LoxP strand (right arm) and thus, 1KBU represents the HJ resolution complex. In Martin et al. ((2002) JMB 319, 107), we suggested that Cre initiates recombination via upper strand cleavage. Indeed, wildtype Cre prefers to cleave the upper strand first in all substrates except intact LoxP, and a Cre mutant His289->Ala accumulates HJs that arise from left-arm initiation. However, we have since demonstrated that the mutation inverts Cre's cleavage preferences (Gelato et al. (2005) JMB 354, 233). This data as well as that of many others (summarized in Lee and Sadowski, (2005) Prog Nucleic Acid Res Mol Biol 80, 1) suggests that conformational strain or asymmetric DNA bending in the initiation complex modify Cre's cleavage specicifity from the default left-arm preference to that for the right arm. Such a mechanism would serve to drive the reaction towards products in the recombination complex.
Enoch Baldwin
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