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* Residue conservation analysis
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Enzyme class:
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Chains L, H:
E.C.3.4.21.5
- thrombin.
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Reaction:
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Preferential cleavage: Arg-|-Gly; activates fibrinogen to fibrin and releases fibrinopeptide A and B.
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DOI no:
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Protein Sci
3:2254-2271
(1994)
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PubMed id:
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The isomorphous structures of prethrombin2, hirugen-, and PPACK-thrombin: changes accompanying activation and exosite binding to thrombin.
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J.Vijayalakshmi,
K.P.Padmanabhan,
K.G.Mann,
A.Tulinsky.
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ABSTRACT
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The X-ray crystal structure of prethrombin2 (pre2), the immediate inactive
precursor of alpha-thrombin, has been determined at 2.0 A resolution complexed
with hirugen. The structure has been refined to a final R-value of 0.169 using
14,211 observed reflections in the resolution range 8.0-2.0 A. A total of 202
water molecules have also been located in the structure. Comparison with the
hirugen-thrombin complex showed that, apart from the flexible beginning and
terminal regions of the molecule, there are 4 polypeptide segments in pre2
differing in conformation from the active enzyme (Pro 186-Asp 194, Gly 216-Gly
223, Gly 142-Pro 152, and the Arg 15-Ile 16 cleavage region). The formation of
the Ile 16-Asp 194 ion pair and the specificity pocket are characteristic of
serine protease activation with the conformation of the catalytic triad being
conserved. With the determination of isomorphous structures of hirugen-thrombin
and D-Phe-Pro-Arg chloromethyl ketone (PPACK)-thrombin, the changes that occur
in the active site that affect the kinetics of chromogenic substrate hydrolysis
on binding to the fibrinogen recognition exosite have been determined. The
backbone of the Ala 190-Gly 197 segment in the active site has an average RMS
difference of 0.55 A between the 2 structures (about 3.7 sigma compared to the
bulk structure). This segment has 2 type II beta-bends, the first bend showing
the largest shift due to hirugen binding. Another important feature was the 2
different conformations of the side chain of Glu 192. The side chain extends to
solvent in hirugen-thrombin, which is compatible with the binding of substrates
having an acidic residue in the P3 position (protein-C, thrombin platelet
receptor). In PPACK-thrombin, the side chain of Asp 189 and the segment Arg
221A-Gly 223 move to provide space for the inhibitor, whereas in
hirugen-thrombin, the Ala 190-Gly 197 movement expands the active site region.
Although 8 water molecules are expelled from the active site with PPACK binding,
the inhibitor complex is resolvated with 5 other water molecules.
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Literature references that cite this PDB file's key reference
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PubMed id
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Reference
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H.N.Bradford,
J.A.Micucci,
and
S.Krishnaswamy
(2010).
Regulated cleavage of prothrombin by prothrombinase: repositioning a cleavage site reveals the unique kinetic behavior of the action of prothrombinase on its compound substrate.
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J Biol Chem,
285,
328-338.
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Z.Chen,
L.A.Pelc,
and
E.Di Cera
(2010).
Crystal structure of prethrombin-1.
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Proc Natl Acad Sci U S A,
107,
19278-19283.
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PDB code:
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J.A.Huntington
(2009).
Slow thrombin is zymogen-like.
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J Thromb Haemost,
7,
159-164.
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C.T.Veldkamp,
C.Seibert,
F.C.Peterson,
N.B.De la Cruz,
J.C.Haugner,
H.Basnet,
T.P.Sakmar,
and
B.F.Volkman
(2008).
Structural basis of CXCR4 sulfotyrosine recognition by the chemokine SDF-1/CXCL12.
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Sci Signal,
1,
ra4.
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PDB codes:
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E.Di Cera
(2008).
Thrombin.
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Mol Aspects Med,
29,
203-254.
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J.A.Huntington
(2008).
How Na+ activates thrombin--a review of the functional and structural data.
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Biol Chem,
389,
1025-1035.
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J.A.Huntington
(2008).
How Na(+) activates thrombin - a review of the functional and structural data.
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Biol Chem,
(),
0.
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P.S.Gandhi,
Z.Chen,
F.S.Mathews,
and
E.Di Cera
(2008).
Structural identification of the pathway of long-range communication in an allosteric enzyme.
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Proc Natl Acad Sci U S A,
105,
1832-1837.
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PDB codes:
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S.Lancellotti,
S.Rutella,
V.De Filippis,
N.Pozzi,
B.Rocca,
and
R.De Cristofaro
(2008).
Fibrinogen-elongated gamma chain inhibits thrombin-induced platelet response, hindering the interaction with different receptors.
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J Biol Chem,
283,
30193-30204.
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A.Bah,
Z.Chen,
L.A.Bush-Pelc,
F.S.Mathews,
and
E.Di Cera
(2007).
Crystal structures of murine thrombin in complex with the extracellular fragments of murine protease-activated receptors PAR3 and PAR4.
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Proc Natl Acad Sci U S A,
104,
11603-11608.
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PDB codes:
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E.Di Cera,
M.J.Page,
A.Bah,
L.A.Bush-Pelc,
and
L.C.Garvey
(2007).
Thrombin allostery.
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Phys Chem Chem Phys,
9,
1291-1306.
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H.K.Kroh,
G.Tans,
G.A.Nicolaes,
J.Rosing,
and
P.E.Bock
(2007).
Expression of allosteric linkage between the sodium ion binding site and exosite I of thrombin during prothrombin activation.
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J Biol Chem,
282,
16095-16104.
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P.E.Bock,
P.Panizzi,
and
I.M.Verhamme
(2007).
Exosites in the substrate specificity of blood coagulation reactions.
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J Thromb Haemost,
5,
81-94.
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D.Zhang,
and
I.M.Kovach
(2006).
Deuterium solvent isotope effect and proton-inventory studies of factor Xa-catalyzed reactions.
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Biochemistry,
45,
14175-14182.
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V.De Filippis,
R.Frasson,
and
A.Fontana
(2006).
3-Nitrotyrosine as a spectroscopic probe for investigating protein protein interactions.
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Protein Sci,
15,
976-986.
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E.P.Bianchini,
S.J.Orcutt,
P.Panizzi,
P.E.Bock,
and
S.Krishnaswamy
(2005).
Ratcheting of the substrate from the zymogen to proteinase conformations directs the sequential cleavage of prothrombin by prothrombinase.
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Proc Natl Acad Sci U S A,
102,
10099-10104.
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R.Murali,
X.Cheng,
A.Berezov,
X.Du,
A.Schön,
E.Freire,
X.Xu,
Y.H.Chen,
and
M.I.Greene
(2005).
Disabling TNF receptor signaling by induced conformational perturbation of tryptophan-107.
|
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Proc Natl Acad Sci U S A,
102,
10970-10975.
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S.Akhavan,
M.A.Miteva,
B.O.Villoutreix,
L.Venisse,
F.Peyvandi,
P.M.Mannucci,
M.C.Guillin,
and
A.Bezeaud
(2005).
A critical role for Gly25 in the B chain of human thrombin.
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J Thromb Haemost,
3,
139-145.
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S.Krishnaswamy
(2005).
Exosite-driven substrate specificity and function in coagulation.
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J Thromb Haemost,
3,
54-67.
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V.De Filippis,
S.De Boni,
E.De Dea,
D.Dalzoppo,
C.Grandi,
and
A.Fontana
(2004).
Incorporation of the fluorescent amino acid 7-azatryptophan into the core domain 1-47 of hirudin as a probe of hirudin folding and thrombin recognition.
|
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Protein Sci,
13,
1489-1502.
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R.Friedrich,
P.Panizzi,
P.Fuentes-Prior,
K.Richter,
I.Verhamme,
P.J.Anderson,
S.Kawabata,
R.Huber,
W.Bode,
and
P.E.Bock
(2003).
Staphylocoagulase is a prototype for the mechanism of cofactor-induced zymogen activation.
|
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Nature,
425,
535-539.
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PDB codes:
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M.Budayova-Spano,
M.Lacroix,
N.M.Thielens,
G.J.Arlaud,
J.C.Fontecilla-Camps,
and
C.Gaboriaud
(2002).
The crystal structure of the zymogen catalytic domain of complement protease C1r reveals that a disruptive mechanical stress is required to trigger activation of the C1 complex.
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EMBO J,
21,
231-239.
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PDB code:
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C.Eigenbrot,
D.Kirchhofer,
M.S.Dennis,
L.Santell,
R.A.Lazarus,
J.Stamos,
and
M.H.Ultsch
(2001).
The factor VII zymogen structure reveals reregistration of beta strands during activation.
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Structure,
9,
627-636.
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PDB code:
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E.Persson,
L.S.Nielsen,
and
O.H.Olsen
(2001).
Substitution of aspartic acid for methionine-306 in factor VIIa abolishes the allosteric linkage between the active site and the binding interface with tissue factor.
|
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Biochemistry,
40,
3251-3256.
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R.Egelund,
T.E.Petersen,
and
P.A.Andreasen
(2001).
A serpin-induced extensive proteolytic susceptibility of urokinase-type plasminogen activator implicates distortion of the proteinase substrate-binding pocket and oxyanion hole in the serpin inhibitory mechanism.
|
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Eur J Biochem,
268,
673-685.
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Y.M.Ayala,
A.M.Cantwell,
T.Rose,
L.A.Bush,
D.Arosio,
and
E.Di Cera
(2001).
Molecular mapping of thrombin-receptor interactions.
|
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Proteins,
45,
107-116.
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L.Perera,
C.Foley,
T.A.Darden,
D.Stafford,
T.Mather,
C.T.Esmon,
and
L.G.Pedersen
(2000).
Modeling zymogen protein C.
|
| |
Biophys J,
79,
2925-2943.
|
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R.Krishnan,
I.Mochalkin,
R.Arni,
and
A.Tulinsky
(2000).
Structure of thrombin complexed with selective non-electrophilic inhibitors having cyclohexyl moieties at P1.
|
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Acta Crystallogr D Biol Crystallogr,
56,
294-303.
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PDB codes:
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R.Krishnan,
J.E.Sadler,
and
A.Tulinsky
(2000).
Structure of the Ser195Ala mutant of human alpha--thrombin complexed with fibrinopeptide A(7--16): evidence for residual catalytic activity.
|
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Acta Crystallogr D Biol Crystallogr,
56,
406-410.
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PDB code:
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R.Recacha,
M.J.Costanzo,
B.E.Maryanoff,
M.Carson,
L.DeLucas,
and
D.Chattopadhyay
(2000).
Structure of human alpha-thrombin complexed with RWJ-51438 at 1.7 A: unusual perturbation of the 60A-60I insertion loop.
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Acta Crystallogr D Biol Crystallogr,
56,
1395-1400.
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PDB code:
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H.Jing,
K.J.Macon,
D.Moore,
L.J.DeLucas,
J.E.Volanakis,
and
S.V.Narayana
(1999).
Structural basis of profactor D activation: from a highly flexible zymogen to a novel self-inhibited serine protease, complement factor D.
|
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EMBO J,
18,
804-814.
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PDB code:
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I.Mochalkin,
and
A.Tulinsky
(1999).
Structures of thrombin retro-inhibited with SEL2711 and SEL2770 as they relate to factor Xa binding.
|
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Acta Crystallogr D Biol Crystallogr,
55,
785-793.
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PDB codes:
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J.Shobe,
C.D.Dickinson,
and
W.Ruf
(1999).
Regulation of the catalytic function of coagulation factor VIIa by a conformational linkage of surface residue Glu 154 to the active site.
|
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Biochemistry,
38,
2745-2751.
|
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B.A.Katz,
B.Liu,
M.Barnes,
and
E.B.Springman
(1998).
Crystal structure of recombinant human tissue kallikrein at 2.0 A resolution.
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Protein Sci,
7,
875-885.
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G.De Simone,
A.Lombardi,
S.Galdiero,
F.Nastri,
R.Della Morte,
N.Staiano,
C.Pedone,
M.Bolognesi,
and
V.Pavone
(1998).
Hirunorms are true hirudin mimetics. The crystal structure of human alpha-thrombin-hirunorm V complex.
|
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Protein Sci,
7,
243-253.
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PDB code:
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M.M.Krem,
and
E.Di Cera
(1998).
Conserved water molecules in the specificity pocket of serine proteases and the molecular mechanism of Na+ binding.
|
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Proteins,
30,
34-42.
|
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P.C.Sanschagrin,
and
L.A.Kuhn
(1998).
Cluster analysis of consensus water sites in thrombin and trypsin shows conservation between serine proteases and contributions to ligand specificity.
|
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Protein Sci,
7,
2054-2064.
|
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R.Krishnan,
E.Zhang,
K.Hakansson,
R.K.Arni,
A.Tulinsky,
M.S.Lim-Wilby,
O.E.Levy,
J.E.Semple,
and
T.K.Brunck
(1998).
Highly selective mechanism-based thrombin inhibitors: structures of thrombin and trypsin inhibited with rigid peptidyl aldehydes.
|
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Biochemistry,
37,
12094-12103.
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PDB codes:
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T.V.Byzova,
and
E.F.Plow
(1998).
Activation of alphaVbeta3 on vascular cells controls recognition of prothrombin.
|
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J Cell Biol,
143,
2081-2092.
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A.Vindigni,
C.E.White,
E.A.Komives,
and
E.Di Cera
(1997).
Energetics of thrombin-thrombomodulin interaction.
|
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Biochemistry,
36,
6674-6681.
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M.G.Malkowski,
P.D.Martin,
J.C.Guzik,
and
B.F.Edwards
(1997).
The co-crystal structure of unliganded bovine alpha-thrombin and prethrombin-2: movement of the Tyr-Pro-Pro-Trp segment and active site residues upon ligand binding.
|
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Protein Sci,
6,
1438-1448.
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PDB codes:
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T.S.Morris,
S.Frormann,
S.Shechosky,
C.Lowe,
M.S.Lall,
V.Gauss-Müller,
R.H.Purcell,
S.U.Emerson,
J.C.Vederas,
and
B.A.Malcolm
(1997).
In vitro and ex vivo inhibition of hepatitis A virus 3C proteinase by a peptidyl monofluoromethyl ketone.
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Bioorg Med Chem,
5,
797-807.
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J.H.Matthews,
R.Krishnan,
M.J.Costanzo,
B.E.Maryanoff,
and
A.Tulinsky
(1996).
Crystal structures of thrombin with thiazole-containing inhibitors: probes of the S1' binding site.
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Biophys J,
71,
2830-2839.
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PDB codes:
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R.A.Engh,
H.Brandstetter,
G.Sucher,
A.Eichinger,
U.Baumann,
W.Bode,
R.Huber,
T.Poll,
R.Rudolph,
and
W.von der Saal
(1996).
Enzyme flexibility, solvent and 'weak' interactions characterize thrombin-ligand interactions: implications for drug design.
|
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Structure,
4,
1353-1362.
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PDB codes:
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T.Mather,
V.Oganessyan,
P.Hof,
R.Huber,
S.Foundling,
C.Esmon,
and
W.Bode
(1996).
The 2.8 A crystal structure of Gla-domainless activated protein C.
|
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EMBO J,
15,
6822-6831.
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PDB code:
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V.Ganesh,
A.Y.Lee,
J.Clardy,
and
A.Tulinsky
(1996).
Comparison of the structures of the cyclotheonamide A complexes of human alpha-thrombin and bovine beta-trypsin.
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Protein Sci,
5,
825-835.
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H.Brandstetter,
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R.Huber,
P.Lollar,
and
W.Bode
(1995).
X-ray structure of clotting factor IXa: active site and module structure related to Xase activity and hemophilia B.
|
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Proc Natl Acad Sci U S A,
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PDB code:
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K.Håkansson,
A.Tulinsky,
M.M.Abelman,
T.A.Miller,
G.P.Vlasuk,
P.W.Bergum,
M.S.Lim-Wilby,
and
T.K.Brunck
(1995).
Crystallographic structure of a peptidyl keto acid inhibitor and human alpha-thrombin.
|
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Bioorg Med Chem,
3,
1009-1017.
|
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P.Ascenzi,
G.Amiconi,
W.Bode,
M.Bolognesi,
M.Coletta,
and
E.Menegatti
(1995).
Proteinase inhibitors from the European medicinal leech Hirudo medicinalis: structural, functional and biomedical aspects.
|
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Mol Aspects Med,
16,
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|
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M.T.Stubbs,
and
W.Bode
(1994).
Coagulation factors and their inhibitors.
|
| |
Curr Opin Struct Biol,
4,
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The most recent references are shown first.
Citation data come partly from CiteXplore and partly
from an automated harvesting procedure. Note that this is likely to be
only a partial list as not all journals are covered by
either method. However, we are continually building up the citation data
so more and more references will be included with time.
Where a reference describes a PDB structure, the PDB
code is
shown on the right.
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Links
