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PDBsum entry 1kli
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Contents |
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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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Crystal structures of uninhibited factor viia link its cofactor and substrate-Assisted activation to specific interactions.
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Authors
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K.Sichler,
D.W.Banner,
A.D'Arcy,
K.P.Hopfner,
R.Huber,
W.Bode,
G.B.Kresse,
E.Kopetzki,
H.Brandstetter.
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Ref.
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J Mol Biol, 2002,
322,
591-603.
[DOI no: ]
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PubMed id
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Abstract
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Factor VIIa initiates the extrinsic coagulation cascade; this event requires a
delicately balanced regulation that is implemented on different levels,
including a sophisticated multi-step activation mechanism of factor VII. Its
central role in hemostasis and thrombosis makes factor VIIa a key target of
pharmaceutical research. We succeeded, for the first time, in recombinantly
producing N-terminally truncated factor VII (rf7) in an Escherichia coli
expression system by employing an oxidative, in vitro, folding protocol, which
depends critically on the presence of ethylene glycol. Activated recombinant
factor VIIa (rf7a) was crystallised in the presence of the reversible S1-site
inhibitor benzamidine. Comparison of this 1.69A crystal structure with that of
an inhibitor-free and sulphate-free, but isomorphous crystal form identified
structural details of factor VIIa stimulation. The stabilisation of
Asp189-Ser190 by benzamidine and the capping of the intermediate helix by a
sulphate ion appear to be sufficient to mimic the disorder-order transition
conferred by the cofactor tissue factor (TF) and the substrate factor X. Factor
VIIa shares with the homologous factor IXa, but not factor Xa, a bell-shaped
activity modulation dependent on ethylene glycol. The ethylene glycol-binding
site of rf7a was identified in the vicinity of the 60 loop. Ethylene glycol
binding induces a significant conformational rearrangement of the 60 loop. This
region serves as a recognition site of the physiologic substrate, factor X,
which is common to both factor VIIa and factor IXa. These results provide a
mechanistic framework of substrate-assisted catalysis of both factor VIIa and
factor IXa.
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Figure 5.
Figure 5. Overview of rf7a^BA. The protein is shown in
ribbon representation (blue, catalytic domain, grey,
EGF2-domain), the residues of the catalytic triad, the inhibitor
benzamidine, the four sulphate groups and the glycerol moiety
are shown in stick representation, and the calcium ion is shown
as a green ball.
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Figure 7.
Figure 7. A stereo view of the 170 loop and the
intermediate helix. Orange, arrow pointing to the intermediate
helix (Met164-Ser170B) and Ser170H-Cys182; pink, insertion loop
(Arg170C-Asp170G); green, Trp215-Arg230; purple, sulphate; light
blue, disulfide bond Cys168-Cys182; white ball, water. Residues
Met164, Cys168, Gln170A-Asn175, Cys182, Trp215 and Arg230 are
shown including their side-chains, for the other residues, only
the backbone atoms are shown. The factor VIIa specific insertion
loop Arg170C-Asp170G and the sulphate groups are shown with
their 2F[o] -F[c] electron density maps. The hydrogen bonds
stabilising the sulphate groups are indicated by white dotted
lines.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2002,
322,
591-603)
copyright 2002.
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