 |
PDBsum entry 2gp9
|
|
|
|
References listed in PDB file
|
 |
|
Key reference
|
|
|
Title
|
|
Crystal structure of thrombin in a self-Inhibited conformation.
|
|
|
Authors
|
|
A.O.Pineda,
Z.W.Chen,
A.Bah,
L.C.Garvey,
F.S.Mathews,
E.Di cera.
|
|
|
Ref.
|
|
J Biol Chem, 2006,
281,
32922-32928.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
|
|
|
Abstract
|
|
|
The activating effect of Na(+) on thrombin is allosteric and depends on the
conformational transition from a low activity Na(+)-free (slow) form to a high
activity Na(+)-bound (fast) form. The structures of these active forms have been
solved. Recent structures of thrombin obtained in the absence of Na(+) have also
documented inactive conformations that presumably exist in equilibrium with the
active slow form. The validity of these inactive slow form structures, however,
is called into question by the presence of packing interactions involving the
Na(+) site and the active site regions. Here, we report a 1.87A resolution
structure of thrombin in the absence of inhibitors and salts with a single
molecule in the asymmetric unit and devoid of significant packing interactions
in regions involved in the allosteric slow --> fast transition. The structure
shows an unprecedented self-inhibited conformation where Trp-215 and Arg-221a
relocate >10A to occlude the active site and the primary specificity pocket, and
the guanidinium group of Arg-187 penetrates the protein core to fill the empty
Na(+)-binding site. The extreme mobility of Trp-215 was investigated further
with the W215P mutation. Remarkably, the mutation significantly compromises
cleavage of the anticoagulant protein C but has no effect on the hydrolysis of
fibrinogen and PAR1. These findings demonstrate that thrombin may assume an
inactive conformation in the absence of Na(+) and that its procoagulant and
anticoagulant activities are closely linked to the mobility of residue 215.
|
|
|
|
|
|
|
|
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).
|
|
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.
|
|
|
|
|
|
The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2006,
281,
32922-32928)
copyright 2006.
|
|
|
Secondary reference #1
|
|
|
Title
|
|
Molecular dissection of na+ binding to thrombin.
|
|
|
Authors
|
|
A.O.Pineda,
C.J.Carrell,
L.A.Bush,
S.Prasad,
S.Caccia,
Z.W.Chen,
F.S.Mathews,
E.Di cera.
|
|
|
Ref.
|
|
J Biol Chem, 2004,
279,
31842-31853.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
|
|
|
|
|
|
|
|
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.
|
|
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.
|
|
|
|
|
|
The above figures are
reproduced from the cited reference
with permission from the ASBMB
|
|
|
|
|
|
|
