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References listed in PDB file
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Key reference
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Title
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Investigation by site-Directed mutagenesis of the role of cytochrome p450 2b4 non-Active-Site residues in protein-Ligand interactions based on crystal structures of the ligand-Bound enzyme.
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Authors
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P.R.Wilderman,
S.C.Gay,
H.H.Jang,
Q.Zhang,
C.D.Stout,
J.R.Halpert.
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Ref.
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Febs J, 2012,
279,
1607-1620.
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PubMed id
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Abstract
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Residues located outside the active site of cytochromes P450 2B have exhibited
importance in ligand binding, structural stability and drug metabolism. However,
contributions of non-active-site residues to the plasticity of these enzymes are
not known. Thus, a systematic investigation was undertaken of unique
residue-residue interactions found in crystal structures of P450 2B4 in complex
with 4-(4-chlorophenyl)imidazole (4-CPI), a closed conformation, or in complex
with bifonazole, an expanded conformation. Nineteen mutants distributed over 11
sites were constructed, expressed in Escherichia coli and purified. Most mutants
showed significantly decreased expression, especially in the case of
interactions found in the 4-CPI structure. Six mutants (H172A, H172F, H172Q,
L437A, E474D and E474Q) were chosen for detailed functional analysis. Among
these, the K(s) of H172F for bifonazole was ∼ 20 times higher than for
wild-type 2B4, and the K(s) of L437A for 4-CPI was ∼ 50 times higher than for
wild-type, leading to significantly altered inhibitor selectivity. Enzyme
function was tested with the substrates 7-ethoxy-4-(trifluoromethyl)coumarin,
7-methoxy-4-(trifluoromethyl)coumarin and 7-benzyloxyresorufin (7-BR). H172F was
inactive with all three substrates, and L437A did not turn over 7-BR.
Furthermore, H172A, H172Q, E474D and E474Q showed large changes in k(cat)/K(M)
for each of the three substrates, in some cases up to 50-fold. Concurrent
molecular dynamics simulations yielded distances between some of the residues in
these putative interaction pairs that are not consistent with contact. The
results indicate that small changes in the protein scaffold lead to large
differences in solution behavior and enzyme function.
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Secondary reference #1
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Title
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Structure of microsomal cytochrome p450 2b4 complexed with the antifungal drug bifonazole: insight into p450 conformational plasticity and membrane interaction.
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Authors
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Y.Zhao,
M.A.White,
B.K.Muralidhara,
L.Sun,
J.R.Halpert,
C.D.Stout.
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Ref.
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J Biol Chem, 2006,
281,
5973-5981.
[DOI no: ]
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PubMed id
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Figure 1.
A, structure of ligand-free 2B4. B, structure of 2B4-CPI. The
chemical formula of CPI is shown below the protein structure. C,
structure of 2B4-bifonazole. The chemical formula of bifonazole
is shown below the protein structure. The three structures are
shown in the same orientation by aligning the most conserved
tertiary structure (residues 61-98 and 306-465). Four regions
around the active site (residues 100-140, 203-262, 276-290, and
474-479) are colored green in A, orange in B, and yellow in C,
respectively, with the major helices labeled. Other parts of the
protein are colored gray in all three structures. Heme, CPI, and
bifonazole are shown as red, cyan, and forest green sticks,
respectively. Images were generated using PyMol (www.pymol.org)
unless otherwise credited.
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Figure 2.
A, divergent stereo view of the 2B4-bifonazole monomer.
Helices and strands are colored yellow and blue, respectively.
The termini and major helices are labeled. Heme, bifonazole, and
Cymal-5 molecules are shown as red, forest green, and cyan
sticks, respectively. B, the dimer interface of 2B4-bifonazole
is stabilized by interdigitating β-strands, and extra copies of
detergent and substrate that associate with exposed hydrophobic
regions. For clarity, only residues 98-117 and 204-230 are
shown. Molecules A and B are colored yellow and gray,
respectively. Heme is shown as red sticks. Bifonazole and
Cymal-5 of molecule A are shown as forest green and cyan sticks,
respectively. Bifonazole and Cymal-5 of molecule B are shown as
green and pale cyan sticks, respectively. Helix F′ and
β-strands 6-1, 7-1, 7-2, and 7-3 are labeled. The chemical
formula of Cymal-5 is shown.
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The above figures are
reproduced from the cited reference
with permission from the ASBMB
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Secondary reference #2
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Title
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Structure of mammalian cytochrome p450 2b4 complexed with 4-(4-Chlorophenyl)imidazole at 1.9-A resolution: insight into the range of p450 conformations and the coordination of redox partner binding.
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Authors
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E.E.Scott,
M.A.White,
Y.A.He,
E.F.Johnson,
C.D.Stout,
J.R.Halpert.
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Ref.
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J Biol Chem, 2004,
279,
27294-27301.
[DOI no: ]
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PubMed id
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Figure 2.
Divergent (walleye) stereo view of cytochrome P450
2B4dH(H226Y) with CPI bound. The sequence can be traced from the
blue N terminus to the red C terminus. Major helices and termini
are labeled. Heme and CPI are shown in red and cyan sticks,
respectively. Images generated using PyMOL (www.pymol.org) (41)
unless otherwise credited.
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Figure 7.
Changes in the position of the C helix and heme ligation upon
CPI binding shown in divergent (walleye) stereo. Residues in
helix C (ribbon) alter positions significantly between the 2B4
open (green) and the CPI closed (blue) conformations, resulting
in the loss of a hydrogen bond to the D ring propionate in the
open conformation. Hydrogen bonds between the D ring propionate
and helix C residues are indicated by dashed lines. Residues
Arg-98 (R98), Ser-430 (S430), Arg-434 (R434), and His-369 (H369)
have smaller changes in their positions and are labeled once
near the base of the side chain.
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The above figures are
reproduced from the cited reference
with permission from the ASBMB
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Secondary reference #3
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Title
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An open conformation of mammalian cytochrome p450 2b4 at 1.6-A resolution.
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Authors
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E.E.Scott,
Y.A.He,
M.R.Wester,
M.A.White,
C.C.Chin,
J.R.Halpert,
E.F.Johnson,
C.D.Stout.
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Ref.
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Proc Natl Acad Sci U S A, 2003,
100,
13196-13201.
[DOI no: ]
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PubMed id
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Figure 1.
Fig. 1. The 2B4 structure and comparison with 2C5. (A) P450
2B4 is oriented to view the large cleft from the protein surface
to the heme. The sequence can be traced starting at the blue N
terminus and ending at the red C terminus. (B and C) Comparison
of 2B4 (B) and 2C5/3LV (1N6B [PDB]
) (C) structures. Residues with the highest rms deviations
between the two structures include 2B4 residues 37-50 (helix A'
and adjacent residues, magenta), residues 92-140 (helix B C
terminus to helix D N terminus, blue), residues 206-250
(C-terminal turn of helix F through helix G, purple), residues
275-300 (loop between helices H and I encompassing a
three-residue insertion in 2B4 relative to 2C5 and N-terminal
half of helix I, orange), and 474-479 (  turn between L' and [3-2],
gray). Excluding these residues, the rms deviation of 324 C^
 atoms between 2B4 and
2C5 is 1.08 Å. The heme group is shown as a stick figure,
and the iron is shown as a red sphere. N and C termini are
labeled. Unless otherwise noted, molecular figures were
generated by using PYMOL (28).
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Figure 2.
Fig. 2. Comparison of structural elements composing the 2B4
cleft. (A) Clefts in the mammalian 2B4 (green) and the bacterial
154C1 (PDB ID code 1GWI [PDB]
, blue) P450s are composed of similar structural elements. (B)
In 2B4 (green) helices, F' and G' flex away from the B' helix,
whereas in 2C5 (yellow), they extend to form the roof of the
active site. For clarity, only the regions including helices B
through D, F through G, and I are shown. The heme is shown as a
stick figure.
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Secondary reference #4
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Title
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Structural and thermodynamic consequences of 1-(4-Chlorophenyl)imidazole binding to cytochrome p450 2b4.
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Authors
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Y.Zhao,
L.Sun,
B.K.Muralidhara,
S.Kumar,
M.A.White,
C.D.Stout,
J.R.Halpert.
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Ref.
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Biochemistry, 2007,
46,
11559-11567.
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PubMed id
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Secondary reference #5
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Title
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Crystal structures of cytochrome p450 2b4 in complex with the inhibitor 1-Biphenyl-4-Methyl-1h-Imidazole: ligand-Induced structural response through alpha-Helical repositioning.
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Authors
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S.C.Gay,
L.Sun,
K.Maekawa,
J.R.Halpert,
C.D.Stout.
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Ref.
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Biochemistry, 2009,
48,
4762-4771.
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PubMed id
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Secondary reference #6
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Title
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Structures of cytochrome p450 2b4 complexed with the antiplatelet drugs ticlopidine and clopidogrel .
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Authors
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S.C.Gay,
A.G.Roberts,
K.Maekawa,
J.C.Talakad,
W.X.Hong,
Q.Zhang,
C.D.Stout,
J.R.Halpert.
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Ref.
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Biochemistry, 2010,
49,
8709-8720.
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PubMed id
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Secondary reference #7
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Title
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Plasticity of cytochrome p450 2b4 as investigated by hydrogen-Deuterium exchange mass spectrometry and x-Ray crystallography.
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Authors
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P.R.Wilderman,
M.B.Shah,
T.Liu,
S.Li,
S.Hsu,
A.G.Roberts,
D.R.Goodlett,
Q.Zhang,
V.L.Woods,
C.D.Stout,
J.R.Halpert.
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Ref.
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J Biol Chem, 2010,
285,
38602-38611.
[DOI no: ]
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PubMed id
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Secondary reference #8
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Title
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Structural analysis of mammalian cytochrome p450 2b4 covalently bound to the mechanism-Based inactivator tert-Butylphenylacetylene: insight into partial enzymatic activity.
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Authors
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S.C.Gay,
H.Zhang,
P.R.Wilderman,
A.G.Roberts,
T.Liu,
S.Li,
H.L.Lin,
Q.Zhang,
V.L.Woods,
C.D.Stout,
P.F.Hollenberg,
J.R.Halpert.
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Ref.
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Biochemistry, 2011,
50,
4903-4911.
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PubMed id
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