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1 Human plasminogen kringle 2: Ligand binding properties and NMR solution structure
ORAL PRESENTATIONS: Plasminogen and plasminogen activators: molecular and structural aspects
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1 Human plasminogen kringle 2: Ligand
binding properties and NMR solution structure *MARTI DN, **SCHALLER J, and *LLIN,4S M *Department of Chemistry, Carnegie Mellon University, Pittsburgh, P.4 15213, USA, and **Department of Chemistry and Biochemistry, University of Bern, 3012 Bern, Switzerland We have established that human plasminogen kringle 2 (K2) has a higher affinity for 5-aminopentanoic acid (Ka ~ 3.4 mM'*; dipole length 1~ 6.2 ,~) than for 6aminohexanoic acid (Ka ~ 2.3 mM-J; l - 7.3/~), which makes the domain distinct among the plaminogen kringles [Marti et al. (1997) Biochemistry 36, 11591]. Lysine and arginine blocked at their 0~-aminoand carboxylate groups, mimicking intra-peptide chain residues, exhibit a significant interaction with K2, comparable to what is observed for the plasminogen K1, K4 and K5. Therefore, participation of K2 in intra- or intermolecular interactions on the same footing as K 1, K4 or K5 is likely. Among the 17 investigated ligands, the antifibrinolytic drug AMCHA reveals the strongest interaction (Ka ~ 7.3 mMl).
tein. 790 proton-proton distance and 85 dihedral angle constraints were employed for the structure calculations, which are based on distance-geometry, regularization/simulated annealing and energy minimization refinement protocols. The overall folding of K2 resembles that of K4. Dipolar (Overhauser) connectivities indicate close contact of the ligand AMCHA with aromatic/aliphatic residues Tyr 36,Pro61,TIp 62, Phe64,TIp 72, LeuTM(kringle residue numbering convention). Furthermore, proximity to the ionic side chain groups of Glu 57 and Arg71 is observed. The aromatic rings of Trp62 and Trp 72 are oriented perpendicular to each other and expose a lipophilic surface in van der Waals contact with the cyclohexane ring of AMCHA. By comparison to K4, Tip 72is more exposed to the solvent, which is also reflected in its aromatic proton chemical shifts. Furthermore, Phe64is positioned closer to the center of the kringle and is not in contact with Trp 72. Both Tyr36and Phe64stabilize the hydrophobic interaction with the ligand. The amino- and carboxylate groups of the ligand ion-pair the side chains of Asp53/Glu57 and Arg71, respectively.
The solution structure of the K2-AMCHA complex was obtained on the basis of homo and heteronuclear NMR data collected at 600 MHz. Resonance assignments were facilitated by resorting to the recombinant JSN and 13Clabeled prom
2 cDNA and gene structure of rat plasminogen BANGERT K and THORSEN S Department of Clinical Biochemistry, Rigshospitalet, Copenhagen University Hospital. Denmark Background. The rat is a commonly used experimental model for studying the plasminogen activator system and its role in tissue remodeling, thrombosis, wound healing and tumor growth. For this reason we have determined the cDNA and gene structure of rat plasminogen. Methods. The T - e n d o f rat plasminogen cDNA was isolated from a liver eDNA library by hybridization with a 475 bp rat plasminogen cDNA probe made by PCR with primers designed from the published sequence of a fragment of rat plasminogen cDNA (d. Biol. Chem. 1991; 266: 10825-9). The 5'-end of the eDNA was amplified by PCR using an upstream primer designed from the published human and murine plasminogen eDNA sequences. The cDNA was sequenced at least twice and more than 90% was sequenced in both directions. Introns were amplified with PCR using primers designed from the eDNA sequence. All exon/intron boundaries were sequenced twice and the sizes of the introns were determined by agarose gel electrophoresis.
Results. The rat plasminogen cDNA codes for 813 amino acids. Compared to human plasminogen, rat plasminogen has 3 amino acid insertions and 1 amino acid deletion. The overall identity of rat and human plasminogen is 79.1% and 78.4% at the of nucleotide and amino acid level, respectively. The catalytic triad, the Cys-residues, the key residues o f the lysine binding sites in kringles 1, 2, 4 and 5 and the region around the plasminogen activator cleavage site are all conserved. The 3'-UT region consists of 244 nucleotides including the stop codon. Four 3'-UT regions (6-16 nucleotides) are conserved between rat, human, murine, bovine and porcine plasminogen. One of these regions contains a typical polyadenylation signal (AAUAAA). The rat plasminogen gene comprises 44 kb from the start codon to the polyadenylation site and has like the human counterpart 19 exons and 18 introns. The positions of the introns are identical in the two species. The sizes of the introns in the rat plasminogen gene varies between 0.45 and 5 kb.
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3 X-ray crystal structure of single chain human
tPA and vampire bat plasminogen activator help to explain the activation mechanism of tPA *RENATUS M, **KOHNERT U, ***SCHLEUNING W-D, and *BODE W *Max-Planck-lnstitute of Biochemistry, Department of Structural Research, D-82152 Martinsried, Germany, **Biochemical Research Center, Boehringer Mannheim GmbH, Nonnenwald 2, D-823 72 Penzberg, Germany, ***Research Laboratories, Schering A G, Miillerstr. 170-178, D- 13342 Berlin, Germany
lytic domains of single-chain human tPA and DSPA reveal Lys 156 to form an internal salt bridge with Asp 194, thereby generating the enzymatically active conformation, compatible with productive substrate binding. In other serine proteinases this active conformation is stabilized by the newly formed N-terminus o f the two-chain mature species liberated upon proteolytic activation.
Tissue type plasminogen activator (tPA) is the physiological initiator o f fibrinolysis, activating plasminogen via highly specific proteolysis; plasmin then proteinases, tPA is proteolytically active already in a single chain form, prior to activation cleavage. The related plasminogen activator from vampire bat (Desmodus rotundus) saliva (DSPA), which assists the ingestion of blood, has no activation cleavage site and functions only in a single chain form.
By comparison with the structures of other serine proteinases that also possess Lys156, such as trypsin, fXa and human uPA, we could identify a set o f secondary interactions which are required for Lys 156 to fulfill this activating role. Comparison of the tPA structure and that of DSPA provides explanations for the faint difference in the catalytic efficiency of both molecules. The availability o f the catalytic domain structure now allows the construction o f reliable models of the fulllength tPA molecule.
We have investigated the structural basis for this unusual single chain activity by X-ray crystallography. The crystal structures o f the cata-
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1 Human plasminogen kringle 2: Ligand
binding properties and NMR solution structure *MARTI DN, **SCHALLER J, and *LLIN,4S M *Department of Chemistry, Carnegie Mellon University, Pittsburgh, P.4 15213, USA, and **Department of Chemistry and Biochemistry, University of Bern, 3012 Bern, Switzerland We have established that human plasminogen kringle 2 (K2) has a higher affinity for 5-aminopentanoic acid (Ka ~ 3.4 mM'*; dipole length 1~ 6.2 ,~) than for 6aminohexanoic acid (Ka ~ 2.3 mM-J; l - 7.3/~), which makes the domain distinct among the plaminogen kringles [Marti et al. (1997) Biochemistry 36, 11591]. Lysine and arginine blocked at their 0~-aminoand carboxylate groups, mimicking intra-peptide chain residues, exhibit a significant interaction with K2, comparable to what is observed for the plasminogen K1, K4 and K5. Therefore, participation of K2 in intra- or intermolecular interactions on the same footing as K 1, K4 or K5 is likely. Among the 17 investigated ligands, the antifibrinolytic drug AMCHA reveals the strongest interaction (Ka ~ 7.3 mMl).
tein. 790 proton-proton distance and 85 dihedral angle constraints were employed for the structure calculations, which are based on distance-geometry, regularization/simulated annealing and energy minimization refinement protocols. The overall folding of K2 resembles that of K4. Dipolar (Overhauser) connectivities indicate close contact of the ligand AMCHA with aromatic/aliphatic residues Tyr 36,Pro61,TIp 62, Phe64,TIp 72, LeuTM(kringle residue numbering convention). Furthermore, proximity to the ionic side chain groups of Glu 57 and Arg71 is observed. The aromatic rings of Trp62 and Trp 72 are oriented perpendicular to each other and expose a lipophilic surface in van der Waals contact with the cyclohexane ring of AMCHA. By comparison to K4, Tip 72is more exposed to the solvent, which is also reflected in its aromatic proton chemical shifts. Furthermore, Phe64is positioned closer to the center of the kringle and is not in contact with Trp 72. Both Tyr36and Phe64stabilize the hydrophobic interaction with the ligand. The amino- and carboxylate groups of the ligand ion-pair the side chains of Asp53/Glu57 and Arg71, respectively.
The solution structure of the K2-AMCHA complex was obtained on the basis of homo and heteronuclear NMR data collected at 600 MHz. Resonance assignments were facilitated by resorting to the recombinant JSN and 13Clabeled prom
2 cDNA and gene structure of rat plasminogen BANGERT K and THORSEN S Department of Clinical Biochemistry, Rigshospitalet, Copenhagen University Hospital. Denmark Background. The rat is a commonly used experimental model for studying the plasminogen activator system and its role in tissue remodeling, thrombosis, wound healing and tumor growth. For this reason we have determined the cDNA and gene structure of rat plasminogen. Methods. The T - e n d o f rat plasminogen cDNA was isolated from a liver eDNA library by hybridization with a 475 bp rat plasminogen cDNA probe made by PCR with primers designed from the published sequence of a fragment of rat plasminogen cDNA (d. Biol. Chem. 1991; 266: 10825-9). The 5'-end of the eDNA was amplified by PCR using an upstream primer designed from the published human and murine plasminogen eDNA sequences. The cDNA was sequenced at least twice and more than 90% was sequenced in both directions. Introns were amplified with PCR using primers designed from the eDNA sequence. All exon/intron boundaries were sequenced twice and the sizes of the introns were determined by agarose gel electrophoresis.
Results. The rat plasminogen cDNA codes for 813 amino acids. Compared to human plasminogen, rat plasminogen has 3 amino acid insertions and 1 amino acid deletion. The overall identity of rat and human plasminogen is 79.1% and 78.4% at the of nucleotide and amino acid level, respectively. The catalytic triad, the Cys-residues, the key residues o f the lysine binding sites in kringles 1, 2, 4 and 5 and the region around the plasminogen activator cleavage site are all conserved. The 3'-UT region consists of 244 nucleotides including the stop codon. Four 3'-UT regions (6-16 nucleotides) are conserved between rat, human, murine, bovine and porcine plasminogen. One of these regions contains a typical polyadenylation signal (AAUAAA). The rat plasminogen gene comprises 44 kb from the start codon to the polyadenylation site and has like the human counterpart 19 exons and 18 introns. The positions of the introns are identical in the two species. The sizes of the introns in the rat plasminogen gene varies between 0.45 and 5 kb.
m
3 X-ray crystal structure of single chain human
tPA and vampire bat plasminogen activator help to explain the activation mechanism of tPA *RENATUS M, **KOHNERT U, ***SCHLEUNING W-D, and *BODE W *Max-Planck-lnstitute of Biochemistry, Department of Structural Research, D-82152 Martinsried, Germany, **Biochemical Research Center, Boehringer Mannheim GmbH, Nonnenwald 2, D-823 72 Penzberg, Germany, ***Research Laboratories, Schering A G, Miillerstr. 170-178, D- 13342 Berlin, Germany
lytic domains of single-chain human tPA and DSPA reveal Lys 156 to form an internal salt bridge with Asp 194, thereby generating the enzymatically active conformation, compatible with productive substrate binding. In other serine proteinases this active conformation is stabilized by the newly formed N-terminus o f the two-chain mature species liberated upon proteolytic activation.
Tissue type plasminogen activator (tPA) is the physiological initiator o f fibrinolysis, activating plasminogen via highly specific proteolysis; plasmin then proteinases, tPA is proteolytically active already in a single chain form, prior to activation cleavage. The related plasminogen activator from vampire bat (Desmodus rotundus) saliva (DSPA), which assists the ingestion of blood, has no activation cleavage site and functions only in a single chain form.
By comparison with the structures of other serine proteinases that also possess Lys156, such as trypsin, fXa and human uPA, we could identify a set o f secondary interactions which are required for Lys 156 to fulfill this activating role. Comparison of the tPA structure and that of DSPA provides explanations for the faint difference in the catalytic efficiency of both molecules. The availability o f the catalytic domain structure now allows the construction o f reliable models of the fulllength tPA molecule.
We have investigated the structural basis for this unusual single chain activity by X-ray crystallography. The crystal structures o f the cata-