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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 solution structure of human thioredoxin complexed with its target from ref-1 reveals peptide chain reversal.
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Authors
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J.Qin,
G.M.Clore,
W.P.Kennedy,
J.Kuszewski,
A.M.Gronenborn.
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Ref.
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Structure, 1996,
4,
613-620.
[DOI no: ]
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PubMed id
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Abstract
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BACKGROUND: Human thioredoxin (hTRX) is a 12 kDa cellular redox protein that has
been shown to play an important role in the activation of a number of
transcriptional and translational regulators via a thiol-redox mechanism. This
activity may be direct or indirect via another redox protein known as Ref-1. The
structure of a complex of hTRX with a peptide comprising its target from the
transcription factor NF kappa B has previously been solved. To further extend
our knowledge of the recognition by and interaction of hTRX with its various
targets, we have studied a complex between hTRX and a Ref-1 peptide. This
complex represents a kinetically stable mixed disulfide intermediate along the
reaction pathway. RESULTS: Using multidimensional heteronuclear edited and
filtered NMR spectroscopy, we have solved the solution structure of a complex
between hTRX and a 13-residue peptide comprising residues 59-71 of Ref-1. The
Ref-1 peptide is located in a crescent-shaped groove on the surface of hTRX, the
groove being formed by residues in the active-site loop (residues 32-36), helix
3, beta strands 3 and 5, and the loop between beta strands 3 and 4. The complex
is stabilized by numerous hydrogen-bonding and hydrophobic interactions that
involve residues 61-69 of the peptide and confer substrate specificity.
CONCLUSIONS: The orientation of the Ref-1 peptide in the hTRX-Ref-1 complex is
opposite to that found in the previously solved complex of hTRX with the target
peptide from the transcription factor NF kappa B. Orientation is determined by
three discriminating interactions involving the nature of the residues at the
P-2' P-4 and P-5 binding positions. (P0 defines the active cysteine of the
peptide, Cys65 for Ref-1 and Cys62 for NF kappa B. Positive and negative numbers
indicate residues N-terminal and C-terminal to this residue, respectively, and
vice versa for NF kappa B as it binds in the opposite orientation.) The
environment surrounding the reactive Cys32 of hTRX, as well as the packing of
the P+3 to P-4 residues are essentially the same in the two complexes, despite
the opposing orientation of the peptide chains. This versatility in substrate
recognition permits hTRX to act as a wide-ranging redox regulator for the cell.
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Figure 3.
Figure 3. Stereoview showing the interactions between the Ref-1
peptide and hTRX. The backbones (N, Cα, C) of hTRX and the
Ref-1 peptide are shown in blue and red, respectively; the side
chains of hTRX and the Ref-1 peptide at the interface of the
complex are shown in magenta and green, respectively; and the
disulfide bond between Cys32 of hTRX and cys62 of the Ref-1
peptide is shown in yellow. Figure 3. Stereoview showing the
interactions between the Ref-1 peptide and hTRX. The backbones
(N, Cα, C) of hTRX and the Ref-1 peptide are shown in blue and
red, respectively; the side chains of hTRX and the Ref-1 peptide
at the interface of the complex are shown in magenta and green,
respectively; and the disulfide bond between Cys32 of hTRX and
cys62 of the Ref-1 peptide is shown in yellow. (The figure was
generated with the program VISP [3][44].)
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Figure 5.
Figure 5. Schematic summary of the interactions observed in the
hTRX–Ref-1 and hTRX–NFκB complexes. (a) All interactions
with the exception of those involving backbone–backbone
hydrogen bonds: hydrophobic, hydrogen-bonding and salt bridge
interactions are represented by continuous (—), long dashed
(– – –) and short dashed (- - -) lines, respectively.
(b) Backbone–backbone hydrogen bonds. Figure 5. Schematic
summary of the interactions observed in the hTRX–Ref-1 and
hTRX–NFκB complexes. (a) All interactions with the exception
of those involving backbone–backbone hydrogen bonds:
hydrophobic, hydrogen-bonding and salt bridge interactions are
represented by continuous (—), long dashed (– – –) and
short dashed (- - -) lines, respectively. (b)
Backbone–backbone hydrogen bonds.
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The above figures are
reprinted
by permission from Cell Press:
Structure
(1996,
4,
613-620)
copyright 1996.
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Secondary reference #1
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Title
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Solution structure of human thioredoxin in a mixed disulfide intermediate complex with its target peptide from the transcription factor nf kappa b.
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Authors
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J.Qin,
G.M.Clore,
W.M.Kennedy,
J.R.Huth,
A.M.Gronenborn.
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Ref.
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Structure, 1995,
3,
289-297.
[DOI no: ]
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PubMed id
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Figure 3.
Figure 3. View of the molecular surface of hTRX illustrating
the cleft in which the NFκB peptide is located. The degree of
curvature of the molecular surface is color coded from white
(convex) to dark gray (concave). Hence the cleft is visualized
as the contiguous boot-shaped gray region on the surface of
hTRX. The backbone of the peptide is shown in green, and side
chains are colored as follows: Phe, Tyr, Val, Pro and Cys in
yellow; Arg and His in blue; Glu in red and Ser in magenta. Note
that the side chains of Phe56 and His67 of the bound NFκB
peptide are disordered in solution. Figure 3. View of the
molecular surface of hTRX illustrating the cleft in which the
NFκB peptide is located. The degree of curvature of the
molecular surface is color coded from white (convex) to dark
gray (concave). Hence the cleft is visualized as the contiguous
boot-shaped gray region on the surface of hTRX. The backbone of
the peptide is shown in green, and side chains are colored as
follows: Phe, Tyr, Val, Pro and Cys in yellow; Arg and His in
blue; Glu in red and Ser in magenta. Note that the side chains
of Phe56 and His67 of the bound NFκB peptide are disordered in
solution. (Figure generated with the program GRASP [[3]83].)
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Figure 4.
Figure 4. Interactions between the NFκB peptide and hTRX. Only
residues 57–65 of the NFκB peptide, which are in contact
with hTRX, are shown, and the residues of the peptide and hTRX
are depicted by lower-case and upper-case letters, respectively.
(a) Stereoview of the backbone (N, Cα, C) of hTRX (blue) and
the NFκB peptide (red). The side chains of hTRX and the NFκB
peptide at the interface of the complex are shown in pink and
green, respectively, and the disulfide bond between Cys32 of
hTRX and Cys62 of the NFκB peptide is shown in yellow. (Figure
generated with the program VISP [84].) (b) Schematic
representation of the hTRX–NFκB peptide complex. Residues of
hTRX involved in hydrophobic interactions with the peptide are
shown circled, and the dashed lines indicate hydrogen bonds,
salt bridges or electrostatic interactions. Figure 4.
Interactions between the NFκB peptide and hTRX. Only residues
57–65 of the NFκB peptide, which are in contact with hTRX,
are shown, and the residues of the peptide and hTRX are depicted
by lower-case and upper-case letters, respectively. (a)
Stereoview of the backbone (N, Cα, C) of hTRX (blue) and the
NFκB peptide (red). The side chains of hTRX and the NFκB
peptide at the interface of the complex are shown in pink and
green, respectively, and the disulfide bond between Cys32 of
hTRX and Cys62 of the NFκB peptide is shown in yellow. (Figure
generated with the program VISP [[4]84].) (b) Schematic
representation of the hTRX–NFκB peptide complex. Residues of
hTRX involved in hydrophobic interactions with the peptide are
shown circled, and the dashed lines indicate hydrogen bonds,
salt bridges or electrostatic interactions. (The NFκB peptide
chain was generated by the program MOLSCRIPT [[5]85].)
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The above figures are
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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The high-Resolution three-Dimensional solution structures of the oxidized and reduced states of human thioredoxin.
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Authors
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J.Qin,
G.M.Clore,
A.M.Gronenborn.
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Ref.
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Structure, 1994,
2,
503-522.
[DOI no: ]
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PubMed id
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Figure 2.
Figure 2. Differences in (a), (b) measured ^3J [HNα]coupling
constants, (c), (d) number of NOE restraints per residue, and
(e) differences in number of NOE restraints between the reduced
and oxidized states of the (C62A, C69A, C73A) mutant of human
thioredoxin. A schematic illustration of the secondary structure
is shown below the figure with α-helices depicted as coils and
β-strands as arrows. Figure 2. Differences in (a), (b)
measured ^3J [HNα]coupling constants, (c), (d) number of NOE
restraints per residue, and (e) differences in number of NOE
restraints between the reduced and oxidized states of the (C62A,
C69A, C73A) mutant of human thioredoxin. A schematic
illustration of the secondary structure is shown below the
figure with α-helices depicted as coils and β-strands as
arrows.
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Figure 8.
Figure 8. Best fit superposition of (a) the active site and (b)
a portion of the hydrophobic core of the restrained minimized
mean structures of the reduced and oxidized states of the
(C62A, C69A, C73A) mutant of human thioredoxin. The backbone
and side chains are shown in dark and light blue, respectively
for the reduced form, and in red and pink, respectively, for the
oxidized form. The models were generated with the program VISP
[87]. Figure 8. Best fit superposition of (a) the active site
and (b) a portion of the hydrophobic core of the restrained
minimized mean structures of the reduced and oxidized states of
the (C62A, C69A, C73A) mutant of human thioredoxin. The backbone
and side chains are shown in dark and light blue, respectively
for the reduced form, and in red and pink, respectively, for the
oxidized form. The models were generated with the program VISP
[[4]87].
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The above figures are
reproduced from the cited reference
with permission from Cell Press
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Secondary reference #3
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Title
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High-Resolution three-Dimensional structure of reduced recombinant human thioredoxin in solution.
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Authors
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J.D.Forman-Kay,
G.M.Clore,
P.T.Wingfield,
A.M.Gronenborn.
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Ref.
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Biochemistry, 1991,
30,
2685-2698.
[DOI no: ]
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PubMed id
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