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PDBsum entry 2pgq
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Hydrolase/hydrolase inhibitor
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PDB id
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2pgq
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References listed in PDB file
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Key reference
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Title
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Important role of the cys-191 cys-220 disulfide bond in thrombin function and allostery.
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Authors
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L.A.Bush-Pelc,
F.Marino,
Z.Chen,
A.O.Pineda,
F.S.Mathews,
E.Di cera.
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Ref.
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J Biol Chem, 2007,
282,
27165-27170.
[DOI no: ]
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PubMed id
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Abstract
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Little is known on the role of disulfide bonds in the catalytic domain of serine
proteases. The Cys-191-Cys-220 disulfide bond is located between the 190 strand
leading to the oxyanion hole and the 220-loop that contributes to the
architecture of the primary specificity pocket and the Na+ binding site in
allosteric proteases. Removal of this bond in thrombin produces an approximately
100-fold loss of activity toward several chromogenic and natural substrates
carrying Arg or Lys at P1. Na+ activation is compromised, and no fluorescence
change can be detected in response to Na+ binding. A 1.54-A resolution structure
of the C191A/C220A mutant in the free form reveals a conformation similar to the
Na+-free slow form of wild type. The lack of disulfide bond exposes the side
chain of Asp-189 to solvent, flips the backbone O atom of Gly-219, and generates
disorder in portions of the 186 and 220 loops defining the Na+ site. This
conformation, featuring perturbation of the Na+ site but with the active site
accessible to substrate, offers a possible representation of the recently
identified E* form of thrombin. Disorder in the 186 and 220 loops and the flip
of Gly-219 are corrected by the active site inhibitor H-D-Phe-Pro-Arg-CH(2)Cl,
as revealed by the 1.8-A resolution structure of the complex. We conclude that
the Cys-191-Cys-220 disulfide bond confers stability to the primary specificity
pocket by shielding Asp-189 from the solvent and orients the backbone O atom of
Gly-219 for optimal substrate binding. In addition, the disulfide bond
stabilizes the 186 and 220 loops that are critical for Na+ binding and
activation.
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Figure 1.
FIGURE 1. Overlay of the ribbon plots of the structures of
the thrombin mutant C191A/C220A in the free (wheat) and
PPACK-inhibited (cyan) forms. The r.m.s.d. between the two
structures is 0.42 Å. Structures are displayed in the
standard Bode orientation (3) with the active site in the
middle. The catalytic residues His-57, Asp-102, and Ser-195 are
rendered as sticks as is Asp-189 in the primary specificity
pocket and the inhibitor PPACK (green). Relevant regions of the
enzyme are noted. The C  atoms of Ala-191 and
Ala-220 at the sites of mutation are indicated by arrows (black
for Ala-220, red for Ala-191). Note the autolysis loop that is
completely ordered only in the PPACK-bound form.
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Figure 2.
FIGURE 2. Electron density maps 2F[0] - F[C] contoured at
0. 7  for the thrombin mutant
C191A/C220A in its free CCF (A) and PPACK-bound CCB (B) forms.
Shown is the region around the mutations (arrows) with the
adjacent 186 loop, the 217–220 strand, the primary specificity
pocket up to the catalytic Ser-195 and His-57. Removal of he
Cys-191–Cys-220 disulfide bond increases exposure of Asp-189
to solvent. Note the flip of the backbone O atom of Gly-219 in
the CCF structure. The O  atom of Ser-195 is
oriented away from His-57 in CCF, as seen in the slow form of
wild type (22). Disorder in the side chains of residues in the
186-loop and around Glu-217 and Gly-219 in the CCF structure (A)
is corrected by the presence of PPACK (stick model in green) in
the CCB structure (B). Disorder in the Na^+ binding site (186
and 220 loops) suggests that the conformation of CCF is unable
to bind Na^+, in agreement with functional data on the mutant.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2007,
282,
27165-27170)
copyright 2007.
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Secondary reference #1
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Title
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Molecular dissection of na+ binding to thrombin.
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Authors
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A.O.Pineda,
C.J.Carrell,
L.A.Bush,
S.Prasad,
S.Caccia,
Z.W.Chen,
F.S.Mathews,
E.Di cera.
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Ref.
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J Biol Chem, 2004,
279,
31842-31853.
[DOI no: ]
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PubMed id
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Figure 7.
FIG. 7. Stereo view of the Na^+ binding environment in the
structures of F (free fast form, gold), S (free slow form, red),
FL (PPACK-bound fast form, blue), and SL (PPACK-bound slow form,
green). Shown are all atoms within 3 Å of the bound Na^+
in the F structure, in addition to the side chains of Asp-189
and Asp-221. Note the similarity of the Na^+ coordination shell
between F and FL; the bound Na^+ is coordinated octahedrally by
the backbone O atoms of Lys-224 and Arg-221a and by four buried
water molecules that H-bond to (clockwise) Asp-189, Asp-221,
Gly-223, and Tyr-184a. Only some of these water molecules are
replaced in the absence of Na^+ (S and SL). Note the
rearrangement of the side chain of Asp-189 in the S structure
and the significant shift in the backbone O atom of Arg-221a
that assumes a position incompatible with Na^+ coordination.
H-bonds are shown by broken lines and refer to the F structure.
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Figure 8.
FIG. 8. Stereo view of the electron density maps of the S
(A), F (B), SL (C), and FL (D) intermediates of thrombin in the
regions bearing the most significant structural transitions.
Residues are rendered in CPK. The bound Na^+ is rendered as a
cyan ball. Shown are the 221–224 loop region and the 187–195
domain. Note how Asp-222 and Arg-187 have joined densities in
the F form, indicative of ion pair interaction, but not in the S
form. Also notable are the reorientation of Asp-189 and Glu-192
in the S form, as well as the shift in the position of Ser-195.
Other changes observed in the slow  fast transition involve
the network of water molecules (red balls) embedding the Na^+
site, the S1 pocket, and the active site region. In the fast
form, this network is well organized and contains 11 water
molecules. In the slow form, the water molecules are reduced to
seven, and the long range connectivity of the network is lost
(see also Fig. 9). The 2F[o] - F[c] electron density maps are
contoured at 0.7 for S and F and at 1.0
for
SL and FL.
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The above figures are
reproduced from the cited reference
with permission from the ASBMB
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