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PDBsum entry 1a5h
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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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Structural mapping of the active site specificity determinants of human tissue-Type plasminogen activator. Implications for the design of low molecular weight substrates and inhibitors.
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Authors
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M.Renatus,
W.Bode,
R.Huber,
J.Stürzebecher,
D.Prasa,
S.Fischer,
U.Kohnert,
M.T.Stubbs.
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Ref.
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J Biol Chem, 1997,
272,
21713-21719.
[DOI no: ]
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PubMed id
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Abstract
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The recent structure determination of the catalytic domain of tissue-type
plasminogen activator (tPA) suggested residue Arg174 could play a role in P3/P4
substrate specificity. Six synthetic chromogenic tPA substrates of the type
R-Xaa-Gly-Arg-p-nitroanilide, in which R is an N-terminal protection group, were
synthesized to test this property. Although changing the residue Xaa (in its L
or D form) at position P3 from the hydrophobic Phe to an acidic residue, Asp or
Glu, gave no improvement in catalytic efficiency, comparative analysis of the
substrates indicated a preference for an acidic substituent occupying the S3
site when the S4 site contains a hydrophobic or basic moiety. The 2.9 A
structure determination of the catalytic domain of human tPA in complex with the
bis-benzamidine inhibitor 2, 7-bis-(4-amidinobenzylidene)-cycloheptan-1-one
reveals a three-site interaction, salt bridge formation of the proximal amidino
group of the inhibitor with Asp189 in the primary specificity pocket, extensive
hydrophobic surface burial, and a weak electrostatic interaction between the
distal amidino group of the inhibitor and two carbonyl oxygens of the protein.
The latter position was previously occupied by the guanidino group of Arg174,
which swings out to form the western edge of the S3 pocket. These data suggest
that the side chain of Arg174 is flexible, and does not play a major role in the
S4 specificity of tPA. On the other hand, this residue would modulate S3
specificity, and may be exploited to fine tune the specificity and selectivity
of tPA substrates and inhibitors.
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Figure 3.
Fig. 3. Experimental density (2F[o]  F[c]) for
the 186-loop of tPA, showing the cluster of Arg186A, Gln186C,
His188. Gly19 of the activation peptide is adjacent to this loop
(lower right), as is the strand His159-Arg161 (bottom).
Orientation as in Fig. 1.
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Figure 4.
Fig. 4. Active site region of human tPA in complex with the
bis-benzamidine inhibitor. The proximal benzamidine occupies
the^ specificity pocket, and the distal benzamidine reaches
toward^ an electrophilic pocket formed by the side chain
carbonyl atoms of Asp97, Thr98, and Arg174. The benzyl ring of
the inhibitor is perpendicular to the side^ chain of Tyr99; the
side chain of Arg174 is displaced by the amidino group. A,
experimental density (2F[o] F[c]) of
the active site region in complex with the bis-benzamidine^
inhibitor. B, active site region of human tPA in complex with
bis-benzamidine. Arg174 from b-tPA is superimposed (thin lines).
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(1997,
272,
21713-21719)
copyright 1997.
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Secondary reference #1
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Title
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Tissue-Type plasminogen activator: variants and crystal/solution structures demarcate structural determinants of function.
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Authors
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W.Bode,
M.Renatus.
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Ref.
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Curr Opin Struct Biol, 1997,
7,
865-872.
[DOI no: ]
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PubMed id
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Secondary reference #2
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Title
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The 2.3 a crystal structure of the catalytic domain of recombinant two-Chain human tissue-Type plasminogen activator.
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Authors
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D.Lamba,
M.Bauer,
R.Huber,
S.Fischer,
R.Rudolph,
U.Kohnert,
W.Bode.
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Ref.
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J Mol Biol, 1996,
258,
117-135.
[DOI no: ]
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PubMed id
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Figure 3.
Figure 3. Stereo section of the final electron density map (blue) around the bound benzamidine molecule (center),
superimposed on the t-PA model. Standard view as in Figures 1 and 2. Of the protein structure, only the entrance frame
Trpc215 to Cysc220 (around the benzamidine), the catalytic triad Serc195, Hisc57 and Aspc102 (to the east), and Lysc143
and Tyrc151 (to the south) are displayed; the spherical density east of the benzamidine molecule, which partially hides
the active Ser195, represents the bound phosphate ion. Contouring is at 1.0s. Figure made with O (Jones et al., 1991).
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Figure 5.
Figure 5. Stereo plot of the hypothetical docking complex of the catalytic domain (white connections) and kringle
2 (yellow connections; De Vos et al., 1992) in the covalent two-domain t-PA variant. The domains are superimposed
with a blue (catalytic domain) and a green Connolly surface (kringle 2 domain). This view is approximately rotated
135° from the standard orientation around a horizontal axis, so that the active site is now pointing to the east/back.
Charged side-chains of catalytic domain residues presumably involved in fibrin binding, and kringle 2 residues forming
the lysine binding site are labeled (chymotrypsinogen and kringle nomenclature). The plot was made with MAIN (Turk,
1992).
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The above figures are
reproduced from the cited reference
with permission from Elsevier
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