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PDBsum entry 3ela
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Hydrolase/hydrolase inhibitor
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PDB id
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3ela
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Contents |
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95 a.a.
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254 a.a.
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191 a.a.
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References listed in PDB file
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Key reference
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Title
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Mechanism of the ca2+-Induced enhancement of the intrinsic factor viia activity.
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Authors
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J.R.Bjelke,
O.H.Olsen,
M.Fodje,
L.A.Svensson,
S.Bang,
G.Bolt,
B.B.Kragelund,
E.Persson.
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Ref.
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J Biol Chem, 2008,
283,
25863-25870.
[DOI no: ]
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PubMed id
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Abstract
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The intrinsic activity of coagulation factor VIIa (FVIIa) is dependent on Ca(2+)
binding to a loop (residues 210-220) in the protease domain. Structural analysis
revealed that Ca(2+) may enhance the activity by attenuating electrostatic
repulsion of Glu(296) and/or by facilitating interactions between the loop and
Lys(161) in the N-terminal tail. In support of the first mechanism, the
mutations E296V and D212N resulted in similar, about 2-fold, enhancements of the
amidolytic activity. Moreover, mutation of the Lys(161)-interactive residue
Asp(217) or Asp(219) to Ala reduced the amidolytic activity by 40-50%, whereas
the K161A mutation resulted in 80% reduction. Hence one of these Asp residues in
the Ca(2+)-binding loop appears to suffice for some residual interaction with
Lys(161), whereas the more severe effect upon replacement of Lys(161) is due to
abrogation of the interaction with the N-terminal tail. However, Ca(2+)
attenuation of the repulsion between Asp(212) and Glu(296) keeps the activity
above that of apoFVIIa. Altogether, our data suggest that repulsion involving
Asp(212) in the Ca(2+)-binding loop suppresses FVIIa activity and that optimal
activity requires a favorable interaction between the Ca(2+)-binding loop and
the N-terminal tail. Crystal structures of tissue factor-bound FVIIa(D212N) and
FVIIa(V158D/E296V/M298Q) revealed altered hydrogen bond networks, resembling
those in factor Xa and thrombin, after introduction of the D212N and E296V
mutations plausibly responsible for tethering the N-terminal tail to the
activation domain. The charge repulsion between the Ca(2+)-binding loop and the
activation domain appeared to be either relieved by charge removal and new
hydrogen bonds (D212N) or abolished (E296V). We propose that Ca(2+) stimulates
the intrinsic FVIIa activity by a combination of charge neutralization and loop
stabilization.
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Figure 2.
Carbamylation inhibition assay using KOCN. At the indicated
time points, aliquots of wild-type FVIIa (♦), FVIIa[D212N]
(▴), and FVIIa[DVQ] (▪) were withdrawn and the residual
activity measured. The curves show the result of a
representative experiment. Similar results were obtained in two
additional experiments in which samples were withdrawn at time
points different from the experiment presented (data not shown).
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Figure 4.
Highlight of residues in the Ca^2+-binding loop of the
protease domain in the structures of FVIIa[D212N] (A and B; 2.7
Å) and wild-type FVIIa (C; 2.0 Å; PDB code 1DAN).
The N terminus is indicated with an arrow and hydrogen bonds
shown by dotted lines.2F[o] – F[c] electron density maps are
shown at 1σ (blue) and 2σ (red). The mutant structure shows
changes of hydrogen bond networks in the Ca^2+-binding loop
(Ca^2+ shown as a green sphere), for example, surrounding
mutated residue Asn^212 and Ser^214 and Glu^296. A hydrogen bond
is abolished between Asn^212 and Ser^214 because of a side chain
movement of Ser^214 in the mutant structure. A hydrogen bond
network is introduced between Asn^212, Glu^296, and two strongly
defined water molecules. In the wild-type structure, Asp^212 and
Glu^296 are not in an electrostatically optimal configuration
because of charge repulsion and the conformations of the two
residues are slightly changed in the mutant structure. A
distinct side chain movement of Asp^217 can be observed as well.
In turn, a hydrogen bond between Lys^161 and Asp^217 is lost,
whereas bonding to Asp^219 is strengthened: Asp^219 to Lys^161
is 2.6 Å in the mutant structure (see B) versus 3.9
Å in the wild-type structure (see C). Root mean square
displacements (Cα) of the Ca^2+-binding loop of the mutant
structure versus the wild structure were 0.74 Å compared
with an overall of 0.65 Å for the heavy chains.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2008,
283,
25863-25870)
copyright 2008.
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