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PDBsum entry 3bef
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References listed in PDB file
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Key reference
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Title
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Structural identification of the pathway of long-Range communication in an allosteric enzyme.
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Authors
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P.S.Gandhi,
Z.Chen,
F.S.Mathews,
E.Di cera.
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Ref.
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Proc Natl Acad Sci U S A, 2008,
105,
1832-1837.
[DOI no: ]
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PubMed id
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Abstract
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Allostery is a common mechanism of regulation of enzyme activity and
specificity, and its signatures are readily identified from functional studies.
For many allosteric systems, structural evidence exists of long-range
communication among protein domains, but rarely has this communication been
traced to a detailed pathway. The thrombin mutant D102N is stabilized in a
self-inhibited conformation where access to the active site is occluded by a
collapse of the entire 215-219 beta-strand. Binding of a fragment of the
protease activated receptor PAR1 to exosite I, 30-A away from the active site
region, causes a large conformational change that corrects the position of the
215-219 beta-strand and restores access to the active site. The crystal
structure of the thrombin-PAR1 complex, solved at 2.2-A resolution, reveals the
details of this long-range allosteric communication in terms of a network of
polar interactions.
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Figure 1.
Structure of the human thrombin mutant D102N in complex with
the extracellular fragment of human PAR1. (A) Thrombin is
rendered in surface representation (wheat) with residues <4
Å from the bound fragment of PAR1 (stick model) colored in
light blue. The orientation is centered on the 30-loop that
separates exosite I on the right from the active site cleft on
the left. The 60-loop occupies the upper rim of the active site.
The electron density 2F[o] − F[c] map (green mesh) is
contoured at 1.0σ. (B) Details of the molecular contacts at the
thrombin–PAR1 interface, with hydrophobic regions of the
thrombin epitope colored in orange and polar regions colored in
light blue. H bonds are depicted as broken lines. Residues
involved in contacts <4 Å are listed in Table 1 and are
labeled in black for thrombin and red for PAR1. The
extracellular fragment of PAR1 engages exosite I through polar
and hydrophobic interactions.
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Figure 2.
Allosteric effect induced by binding of the extracellular
fragment of PAR1 (stick model in gold) to exosite I of thrombin
(ribbon model in light green) on the conformation of the
215–219 β-strand and the 220-loop (blue). The position of
Trp-215 and Arg-221a is indicated as a stick model. Thrombin is
shown in the standard Bode orientation (29) with the active site
cleft in the middle and exosite I to the right. Comparison with
the free structure of thrombin (ribbon model in wheat, with the
215–219 β-strand and the 220-loop, Trp-215, and Arg-221a
in red) shows a drastic rearrangement that pushes the 215–219
β-strand back >6 Å. Trp-215 and Arg-221a relocate >9
Å to restore access to the active site and primary
specificity pocket that was obliterated in the free form. The
allosteric communication between exosite I and the 215–219
β-strand and 220-loop spans almost 30 Å across the
thrombin molecule (see also Fig. 3) and reveals a possible
mechanism for the conversion of thrombin from its inactive form
E* into the active form E.
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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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Secondary reference #2
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Title
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Crystal structure of thrombin in a self-Inhibited conformation.
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Authors
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A.O.Pineda,
Z.W.Chen,
A.Bah,
L.C.Garvey,
F.S.Mathews,
E.Di cera.
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Ref.
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J Biol Chem, 2006,
281,
32922-32928.
[DOI no: ]
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PubMed id
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Figure 1.
FIGURE 1. Surface rendering of the structures of inactive
thrombin in the absence of Na^+, labeled with their
corresponding Protein Data Bank accession codes. Except for the
structure of the W215A/E217A mutant (14) (middle right, 1TQ0),
all of the molecules are in the standard Bode orientation (27),
with the active site at the center and the Na^+ site in the
southwest quadrant. The structure of D102N (top left, 2GP9) is
used as reference, with key residues labeled. Also shown for
reference is the structure of the active slow form (9) (top
right, 1SGI). The areas in cyan correspond to the intermolecular
contacts <4Å of the two molecules in the asymmetric unit,
related by noncrystallographic 2-fold symmetry (see also Table
2). Only one representative monomer in the asymmetric unit is
shown for clarity. The structure of the W215A/E217A mutant
(middle right, 1TQ0) is rotated 120° about the y axis
relative to the standard orientation to show the contact areas.
The other structures refer to the E217K mutant (13) (middle
left, 1RD3), wild type in the presence of Li^+ (17) (bottom
left, 2AFQ), and molecule 2 of the R77aA mutant in the presence
of K^+ (15, 16) (bottom right, 2A0Q).
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Figure 3.
FIGURE 3. Stereo view of the overlay of the structures of
D102N (CPK, with C in yellow) and the PPACK-inhibited Na^+-bound
form (CPK, with C in cyan) (9) reveals the molecular basis of
self-inhibition in the D102N structure. Trp-215 and Arg-221a of
D102N produce a self-inhibited conformation of the enzyme by
occupying positions analogous to Pro and Arg of PPACK (stick
model, green) in the fast form. Also shown is the bound Na^+,
with the coordinating water molecules and the H-bonding network
(dashed lines). Note the significant shift of the 220 loop with
disruption of the ionic interactions with the 186 loop, causing
Arg-187 to position its guanidinium group within 1 Å from
where Na^+ binds in the fast form. The arrows point to the
position of residue Asp-189 and the flip of the nitrogen atom of
Gly-193 in the oxyanion hole. The structure is a remarkable
example of molecular mimicry of bound substrate/inhibitor
(Trp-215 and Arg-221a) and Na^+ (Arg-187) made possible by the
flexibility of the thrombin fold in the free form.
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The above figures are
reproduced from the cited reference
with permission from the ASBMB
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