 |
PDBsum entry 2od3
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Hydrolase/hydrolase inhibitor
|
PDB id
|
|
|
|
2od3
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
References listed in PDB file
|
|
|
Key reference
|
|
|
Title
|
|
Structural basis of na+ activation mimicry in murine thrombin.
|
|
|
Authors
|
|
F.Marino,
Z.W.Chen,
C.E.Ergenekan,
L.A.Bush-Pelc,
F.S.Mathews,
E.Di cera.
|
|
|
Ref.
|
|
J Biol Chem, 2007,
282,
16355-16361.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
Abstract
|
|
|
Unlike human thrombin, murine thrombin lacks Na(+) activation due to the charge
reversal substitution D222K in the Na(+) binding loop. However, the enzyme is
functionally stabilized in a Na(+)-bound form and is highly active toward
physiologic substrates. The structural basis of this peculiar property is
unknown. Here, we present the 2.2 A resolution x-ray crystal structure of murine
thrombin in the absence of inhibitors and salts. The enzyme assumes an active
conformation, with Ser-195, Glu-192, and Asp-189 oriented as in the Na(+)-bound
fast form of human thrombin. Lys-222 completely occludes the pore of entry to
the Na(+) binding site and positions its side chain inside the pore, with the
Nzeta atom H-bonded to the backbone oxygen atoms of Lys-185, Asp-186b, and
Lys-186d. The same architecture is observed in the 1.75 A resolution structure
of a thrombin chimera in which the human enzyme carries all residues defining
the Na(+) pore in the murine enzyme. These findings demonstrate that Na(+)
activation in thrombin is linked to the architecture of the Na(+) pore. The
molecular strategy of Na(+) activation mimicry unraveled for murine thrombin is
relevant to serine proteases and enzymes activated by monovalent cations in
general.
|
|
|
|
|
|
|
|
Figure 1.
FIGURE 1. A-C, surface rendering of the pore of entry to
the Na^+ binding site of human thrombin in the structure 1SG8
(15) (A) when compared with the same region in murine thrombin
(B) and the thrombin chimera (C). Residues lining the pore are
color-coded according to their physical properties (red =
positively charged, blue = negatively charged, orange =
hydrophobic, white = all others). In the human enzyme, the pore
is defined by residue Asp-222 in the 220-loop and the sequence
PDEGKR from Pro-186 to Arg-187 in the 186-loop (Table 1) (A). In
murine thrombin (B), residue 222 is Lys, and the corresponding
sequence in the 186-loop is VNDTKR (Table 1). The side chain of
Lys-222 completely occludes the pore. The side chain of Asn-186a
is glycosylated (NAG). Occlusionofthe pore is also seen in the
thrombin chimera (C), in which the human enzyme carries all
residues around the pore as in murine thrombin. There is no
glycosylation of Asn-186a in the chimera. D-F, architecture of
the pore of entry to the Na^+ binding site in the same
orientation as shown in the surface rendering (A-C), with
relevant residues rendered in Corey-Pauling-Koltun model (carbon
in yellow) and the 2F[o] - F[c] electron density maps contoured
at the 0.7  level for the
structures presented in this study (E and F). The human enzyme
(D) shows the pore wide open, whereas Lys-222 in murine thrombin
(E) occludes the pore and positions the N  atom within H-bonding
distance from Lys-185, Asp-186b, and Lys-186d. The backbone
oxygen atom of residue 186b is flipped relative to the position
assumed in the fast form of the human enzyme. Also shown is the
indole side chain of Trp-20, which is Ser in human thrombin, as
a structural signature of the murine enzyme. Lys-222 in the
thrombin chimera (F) is positioned as in the murine thrombin
structure.
|
|
Figure 2.
FIGURE 2. Overlay of key residues in murine thrombin
(Corey-Pauling-Koltun, with carbon in yellow) and in the fast
form (Corey-Pauling-Koltun, with carbon in green) of human
thrombin (15). H-bonds (broken lines) refer to the murine
thrombin structure. The presence of Lys-222 in murine thrombin
stabilizes the conformation in a fast-like form. The O  atom of
the catalytic Ser-195 is within H-bonding distance (3.05
Å) from His-57. This H-bond is present in the fast form of
the human enzyme (3.09 Å) but is broken (3.70 Å) in
the Na^+-free slow form (15). The side chain of Asp-189 in the
primary specificity pocket is oriented optimally for
coordination of Arg of substrate, as seen in the fast form. The
conformations of Asp-189 and Ser-195 are maintained by H-bonding
interactions mediated by water molecules, as in the fast form of
the human enzyme. However, only seven water molecules (red
balls) are present in this region of the murine thrombin
structure, as opposed to Na^+ (green ball) and 11 water
molecules (cyan balls) present in the fast form of the human
enzyme (15). The presence of Lys-222 in murine thrombin pushes
Arg-187 away and closer (2.55 Å) to Asp-221. The N atom of
Lys-222 and the O  1 atom of Asp-221
H-bond to water w153, which in turn stabilizes water w51 in a
position equivalent (<1 Å away) to the bound Na^+ in the
fast form (green ball) and in contact with the backbone oxygen
atoms of Arg-221a (2.77 Å) and Lys-224 (2.61 Å). The
H-bonding network around water w51 mimics that seen around the
bound Na^+ in the fast form of the human enzyme (15) and
establishes a connection to the O 2 atom of Asp-189 via
water w97. The O 1 atom of Asp-189 is
held in place by an H-bond with water w55 (2.74 Å).
Ser-195 is fixed in its orientation by a water-mediated contact
with the O  1 atom of Glu-192, with
water w63 positioned 3.19 Å away from the O atom of
Ser-195 and 2.82 Å away from the O 1 atom of Glu-192. The
only two water molecules, w141 and w142, between Asp-189 and
Ser-195 are too far away from either residue. Thus, murine
thrombin lacks the connectivity between the primary specificity
pocket and the catalytic triad seen in the fast form of the
human enzyme.
|
|
|
|
|
|
The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2007,
282,
16355-16361)
copyright 2007.
|
|
|
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
|
|
|
|
|
|
|
