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159 Structure and ligand binding properties of the kringle 5 domain of human plasminogen
SESSION Xll: Structure and Structure Function
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THE "ACTIVATION DOMAIN": A HIGHLY COOPERATIVE EN1TI~ WITHIN THE CATALYTIC DOMAIN OF SERINE PROTEINASES.
CHICKEN PLASMINOGEN ACTIVATOR (chuPA): ALrrOACTIVATING AND SERPIN RESISTANT: A PROTEASE OUT OF CONTROL John D. Sipley, Daniela S. Alexander, Jacqueline E. Testa, Barbara Mucller°, and James P. Ouinley. Department of Pathology, S.U.N.Y. at Stony Brook, NY; "Scripps Research Institute, La Jolla, CA. As a structurally complex, multi-domain sefine protease a part of uPA's regulation and specificity is inherent in its complex structure. Chicken uPA (chuPA), although structurally and catalytically a member of the family of PA proteins, appears to be an unusual family member. When it is expressed at elevated levels, chuPA manifests its activity in an uncontrolled, unregulated manner: It is completely insensitive to the known uPA-speeifie serpins, it accumulates in plasminogen-freecultures as active enzyme, and most unusually it is activated by an autocatalytic mechanism. No other uPA or tPA is known to exhibit these characteristics. Comparative analysis of uPAs from various species reveals that ehuPA diverges from other uPAs and contains no positively charged amino acid residues in the surface loop, Variable Region 1 (VR.I). This motif is the binding site of the inhibitory serpin, PAI-1. The introduction of the positively charged residues from the VR1 of human uPA (huPA), 17SRRI-IRm, into chuPA, results in a chuPA mutant that is highly susceptible to inhibition by the serpins PAI-1, and PAI-2. Ceils transfected by this mutant progressively degrade the extracellular matrix but in contrast to cells expressing wild-type chuPA, are inhibited in their degradative capacity by nanomolar levels of PAIl. Conversely the 17~RRHRmotif in huPA has now been removed and replaced with the four residue motif, QNIM, present in the VR1 of chuPA. The mutant huPA~N~ is highly resistant to PAI-1 inhibition, and HeLa cells over expressing this mutant are highly invasive and metastatic. Comparison of a variety of mammalian cells with that of avian coils indicates that the uPA produced by the mammalian cells presents as the single chain, zymogen form while chicken uPA accumulates as the two chain, active enzyme. Mutational analysis, recombinant expression and use of anti catalytic monoclonal antibodies demonstrate that the activation of chicken uPA is through a unique autocatalytic mechanism. The impact of this spontaneous initiation of a serpin-resistant proteolytic cascade upon cell and tissue phenotype will be discussed.
Wolfram Bode. Doriano Lamba, Martin Renams, Richard Engh Max-Planek-lnstimt of Biochemistry D-82152 Martinsfied, Germany Chymotrypsin-like serine proteinases are normally secreted as single-chain zymogens with very weak reactivity towards unspecific active-site reagents and virtually no reactivity towards peptide inhibitor,s such as peptidyl chloromethyl ketonas. They are activated by pmteolytic liberation of a highly conserved Nterminus (typically with the sequence ne Val Gly Gly at residues 16 to 19 using ehymotrypsinogen numbering). The free a-amino group forms a solventinaccessible salt bridge with the Asp194 earboxylate group in the "llel6 pocket" specific for accommodation of the IleVal moiety. Formation ot the salt bridge creates a functional substrate recognition site by reorienting Asp194 compared to the prounzyme structure. This reorientation restructures the surrounding "activation domain", a seperam highly cooperative part of the catalytic domain, which includes the oxyaniun hole, the catalytic triad, and the S1 spacil'lcity pocket. The structuring of the activation domain can be triggered - besides through the endogenous IV le al N terminus - by interaction with exogenous substances such as the free ne Val-dipoptide, very tight binding protein inhibitors, or eofactors (TF-FVIIa, fibrin-tPA, streptokinase-plasminogen) without proteolytie cleavage. This design allows the robust regulation of critical biologieai processes in higher organisms. In the talk, structural details and biological examples will be presented and discussed.
159 STRUCTURE AND LIGAND BINDING PROPERTIES OF THE K_,RINGLE 5 DOMAIN OF HUMAN PLASMINOGEN. Castellino. F. j..1 McCance, S. G., I Chang, y.,l Mochalkin, L, 2 and Tulinsky, A.2, 1Department of Chemistry & Biochemistry, University of Notre Dame, Notre Dame, Indiana, and 2Department of Chemistry, Michigan State University, East Lansing, Michigan. The X-ray crystal structure of the kringle 5 domain of human plasminogen (K5HPg) has been determined. The unit cell was composed of a dimer of this kringle with the two molecules related by a noncrystailographic twofold rotation axis approximately parallel to the z-direction. The lysine binding site (LBS) is defined by His33-Thr37, Pro54-Va158,Pro61-Tyr64, and Leu71-Tyr74, which forms an elongated depression on the kringle surface approximately 9 A wide and 12 A long, and is occupied by in the apo-form by H20 molecules. A unique feature of the LBS of apo-K5HPg is the replacement by Leu71 of the cationic center that stabilizes ligand carboxylate group binding, a substitution which may also explain the affinity of alkylamines for this kringle domain. This consideration was examined by the construction of mutants at this position. Comparison of the steric relationships of apo-K5HPg with other w-amino acid binding kringle domains shows that, with the exception of the orientation of Asp 55, all other side chain interactions that stabilize ligand binding are present in K5HPg. The orientation of Asp55 and Asp57 of the anionic center differ by t 80°.
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157
158
THE "ACTIVATION DOMAIN": A HIGHLY COOPERATIVE EN1TI~ WITHIN THE CATALYTIC DOMAIN OF SERINE PROTEINASES.
CHICKEN PLASMINOGEN ACTIVATOR (chuPA): ALrrOACTIVATING AND SERPIN RESISTANT: A PROTEASE OUT OF CONTROL John D. Sipley, Daniela S. Alexander, Jacqueline E. Testa, Barbara Mucller°, and James P. Ouinley. Department of Pathology, S.U.N.Y. at Stony Brook, NY; "Scripps Research Institute, La Jolla, CA. As a structurally complex, multi-domain sefine protease a part of uPA's regulation and specificity is inherent in its complex structure. Chicken uPA (chuPA), although structurally and catalytically a member of the family of PA proteins, appears to be an unusual family member. When it is expressed at elevated levels, chuPA manifests its activity in an uncontrolled, unregulated manner: It is completely insensitive to the known uPA-speeifie serpins, it accumulates in plasminogen-freecultures as active enzyme, and most unusually it is activated by an autocatalytic mechanism. No other uPA or tPA is known to exhibit these characteristics. Comparative analysis of uPAs from various species reveals that ehuPA diverges from other uPAs and contains no positively charged amino acid residues in the surface loop, Variable Region 1 (VR.I). This motif is the binding site of the inhibitory serpin, PAI-1. The introduction of the positively charged residues from the VR1 of human uPA (huPA), 17SRRI-IRm, into chuPA, results in a chuPA mutant that is highly susceptible to inhibition by the serpins PAI-1, and PAI-2. Ceils transfected by this mutant progressively degrade the extracellular matrix but in contrast to cells expressing wild-type chuPA, are inhibited in their degradative capacity by nanomolar levels of PAIl. Conversely the 17~RRHRmotif in huPA has now been removed and replaced with the four residue motif, QNIM, present in the VR1 of chuPA. The mutant huPA~N~ is highly resistant to PAI-1 inhibition, and HeLa cells over expressing this mutant are highly invasive and metastatic. Comparison of a variety of mammalian cells with that of avian coils indicates that the uPA produced by the mammalian cells presents as the single chain, zymogen form while chicken uPA accumulates as the two chain, active enzyme. Mutational analysis, recombinant expression and use of anti catalytic monoclonal antibodies demonstrate that the activation of chicken uPA is through a unique autocatalytic mechanism. The impact of this spontaneous initiation of a serpin-resistant proteolytic cascade upon cell and tissue phenotype will be discussed.
Wolfram Bode. Doriano Lamba, Martin Renams, Richard Engh Max-Planek-lnstimt of Biochemistry D-82152 Martinsfied, Germany Chymotrypsin-like serine proteinases are normally secreted as single-chain zymogens with very weak reactivity towards unspecific active-site reagents and virtually no reactivity towards peptide inhibitor,s such as peptidyl chloromethyl ketonas. They are activated by pmteolytic liberation of a highly conserved Nterminus (typically with the sequence ne Val Gly Gly at residues 16 to 19 using ehymotrypsinogen numbering). The free a-amino group forms a solventinaccessible salt bridge with the Asp194 earboxylate group in the "llel6 pocket" specific for accommodation of the IleVal moiety. Formation ot the salt bridge creates a functional substrate recognition site by reorienting Asp194 compared to the prounzyme structure. This reorientation restructures the surrounding "activation domain", a seperam highly cooperative part of the catalytic domain, which includes the oxyaniun hole, the catalytic triad, and the S1 spacil'lcity pocket. The structuring of the activation domain can be triggered - besides through the endogenous IV le al N terminus - by interaction with exogenous substances such as the free ne Val-dipoptide, very tight binding protein inhibitors, or eofactors (TF-FVIIa, fibrin-tPA, streptokinase-plasminogen) without proteolytie cleavage. This design allows the robust regulation of critical biologieai processes in higher organisms. In the talk, structural details and biological examples will be presented and discussed.
159 STRUCTURE AND LIGAND BINDING PROPERTIES OF THE K_,RINGLE 5 DOMAIN OF HUMAN PLASMINOGEN. Castellino. F. j..1 McCance, S. G., I Chang, y.,l Mochalkin, L, 2 and Tulinsky, A.2, 1Department of Chemistry & Biochemistry, University of Notre Dame, Notre Dame, Indiana, and 2Department of Chemistry, Michigan State University, East Lansing, Michigan. The X-ray crystal structure of the kringle 5 domain of human plasminogen (K5HPg) has been determined. The unit cell was composed of a dimer of this kringle with the two molecules related by a noncrystailographic twofold rotation axis approximately parallel to the z-direction. The lysine binding site (LBS) is defined by His33-Thr37, Pro54-Va158,Pro61-Tyr64, and Leu71-Tyr74, which forms an elongated depression on the kringle surface approximately 9 A wide and 12 A long, and is occupied by in the apo-form by H20 molecules. A unique feature of the LBS of apo-K5HPg is the replacement by Leu71 of the cationic center that stabilizes ligand carboxylate group binding, a substitution which may also explain the affinity of alkylamines for this kringle domain. This consideration was examined by the construction of mutants at this position. Comparison of the steric relationships of apo-K5HPg with other w-amino acid binding kringle domains shows that, with the exception of the orientation of Asp 55, all other side chain interactions that stabilize ligand binding are present in K5HPg. The orientation of Asp55 and Asp57 of the anionic center differ by t 80°.
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