Unfolding studies of the protease domain of urokinase-type plasminogen activator: the existence of partly folded states and stable subdomains.


Abstract

The domain structure and the stability against thermal and chemical denaturation of urokinase-type plasminogen activator (u-PA) have been investigated by NMR spectroscopy and differential scanning calorimetry (DSC). At least five structurally autonomous regions of this three-domain protein have been found to exist. Two of these are the EGF-like and the kringle domains; the others are all within the third domain, which is a serine protease. The latter undergoes three unfolding transitions in its enzymatically active form. Reaction with a specific affinity label (L-Glu-L-Gly-L-Arg-chloromethyl ketone) to produce an inactivated protein results in a stabilization of the structure involved in two of these transitions, and an increase in cooperativity to give a domain which unfolds in two, not three, distinct steps. These are attributed to the denaturation of the two major subdomains of the protease structure. One of the subdomains has exceptional stability, being unfolded only under extreme conditions such as 75 degrees C at pH 2.5 or 4 M GuDCl at pH 4.5 and 29 degrees C. This region has been identified by isolation and characterization of a fragment (residues Ile-159 to Thr-277) obtained by limited proteolysis with thermolysin under conditions where the protease domain was partly unfolded. The NMR data are consistent with this stable region being at the N-terminus of the protein and indicate that its structure and stability are similar to those of the corresponding region of the native protein. These results support the idea that the u-PA protease domain has structural resemblance to the digestive serine proteases, but that stabilizing interactions within the structure can differ significantly between a group of homologous proteins. Study holds ProTherm entries: 4486, 4487 Extra Details: transition I domain structure; unfolding transitions; specific affinity label

Submission Details

ID: LpzjFMbN

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:25 p.m.

Version: 1

Publication Details
Nowak UK;Cooper A;Saunders D;Smith RA;Dobson CM,Biochemistry (1994) Unfolding studies of the protease domain of urokinase-type plasminogen activator: the existence of partly folded states and stable subdomains. PMID:8130209
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1URK 1995-05-08 SOLUTION STRUCTURE OF THE AMINO TERMINAL FRAGMENT OF UROKINASE-TYPE PLASMINOGEN ACTIVATOR
1KDU 1993-10-31 SEQUENTIAL 1H NMR ASSIGNMENTS AND SECONDARY STRUCTURE OF THE KRINGLE DOMAIN FROM UROKINASE
5YC6 2018-10-03 1.18 The crystal structure of uPA in complex with 4-Bromobenzylamirne at pH4.6
4ZKO 2016-05-18 1.29 The crystal structure of upain-1-W3A in complex with uPA at pH7.4
4ZKR 2016-05-18 1.36 The crystal structure of upain-1-W3A in complex with uPA at pH9.0
4ZKN 2016-05-18 1.36 The crystal structure of upain-1-W3A in complex with uPA at pH5.5
2O8T 2007-12-25 1.45 Crystal Structure and Binding Epitopes of Urokinase-type Plasminogen Activator (C122A/N145Q) in complex with Inhibitors
3MHW 2010-04-21 1.45 The complex crystal Structure of Urokianse and 2-Aminobenzothiazole
5Z1C 2018-12-26 1.45 The crystal structure of uPA in complex with 4-Iodobenzylamine at pH7.4
5WXF 2018-07-11 1.46 Crystal structure of uPA in complex with upain-2-2
4MNW 2014-02-05 1.49 Crystal structure of urokinase-type plasminogen activator (uPA) complexed with bicyclic peptide UK749
4XSK 2016-02-03 1.5 Structure of PAItrap, an uPA mutant
1GJ7 2002-04-27 1.5 ENGINEERING INHIBITORS HIGHLY SELECTIVE FOR THE S1 SITES OF SER190 TRYPSIN-LIKE SERINE PROTEASE DRUG TARGETS
4GLY 2013-05-15 1.52 Human urokinase-type plasminogen activator uPA in complex with the two-disulfide bridge peptide UK504
4JK5 2013-07-17 1.55 Human urokinase-type Plasminogen Activator (uPA) in complex with a bicyclic peptide inhibitor (UK18-D-Ser)
1GJA 2002-04-27 1.56 ENGINEERING INHIBITORS HIGHLY SELECTIVE FOR THE S1 SITES OF SER190 TRYPSIN-LIKE SERINE PROTEASE DRUG TARGETS
3OX7 2011-08-10 1.58 The crystal structure of uPA complex with peptide inhibitor MH027 at pH4.6
1OWE 2003-09-30 1.6 Substituted 2-Naphthamidine inhibitors of urokinase
4FUH 2012-08-22 1.6 Crystal Structure of the Urokinase
4X1P 2015-10-21 1.6 The crystal structure of mupain-1-17 in complex with murinised human uPA at pH4.6
4FU9 2012-08-22 1.6 Crystal Structure of the Urokinase
1OWH 2003-09-30 1.61 Substituted 2-Naphthamidine Inhibitors of Urokinase
5ZA9 2018-12-19 1.62 uPA-BB2-50F
1O5C 2004-09-21 1.63 Dissecting and Designing Inhibitor Selectivity Determinants at the S1 site Using an Artificial Ala190 Protease (Ala190 uPA)
1GJ8 2002-04-27 1.64 ENGINEERING INHIBITORS HIGHLY SELECTIVE FOR THE S1 SITES OF SER190 TRYPSIN-LIKE SERINE PROTEASE DRUG TARGETS
5WXO 2018-07-11 1.64 Crystal structure of uPA in complex with upain-2-2-W3A
5ZAF 2018-12-19 1.65 uPA-BB2-28F
1C5Y 2000-12-22 1.65 STRUCTURAL BASIS FOR SELECTIVITY OF A SMALL MOLECULE, S1-BINDING, SUB-MICROMOLAR INHIBITOR OF UROKINASE TYPE PLASMINOGEN ACTIVATOR
5ZAJ 2018-12-19 1.65 uPA-31F
1O5A 2004-09-21 1.68 Dissecting and Designing Inhibitor Selectivity Determinants at the S1 site Using an Artificial Ala190 Protease (Ala190 uPA)
3M61 2010-04-07 1.68 Crystal structure of complex of urokinase and a upain-1 variant(W3A) in pH4.6 condition
2O8U 2007-12-25 1.7 Crystal Structure and Binding Epitopes of Urokinase-type Plasminogen Activator (C122A/N145Q/S195A) in complex with Inhibitors
5ZA7 2018-12-19 1.7 uPA-HMA
4MNY 2014-02-05 1.7 Crystal structure of urokinase-type plasminogen activator (uPA) complexed with bicyclic peptide UK903
2VIV 2008-01-22 1.72 Fragment-Based Discovery of Mexiletine Derivatives as Orally Bioavailable Inhibitors of Urokinase-Type Plasminogen Activator
2VIP 2008-01-22 1.72 Fragment-Based Discovery of Mexiletine Derivatives as Orally Bioavailable Inhibitors of Urokinase-Type Plasminogen Activator
4FUC 2012-08-22 1.72 Crystal Structure of the Urokinase
1GJC 2002-04-27 1.73 ENGINEERING INHIBITORS HIGHLY SELECTIVE FOR THE S1 SITES OF SER190 TRYPSIN-LIKE SERINE PROTEASE DRUG TARGETS
5ZAE 2018-12-19 1.73 uPA-6F-HMA
5WXP 2018-07-11 1.75 Crystal structure of uPA in complex with upain-2-3-W3A
1GJD 2002-05-03 1.75 ENGINEERING INHIBITORS HIGHLY SELECTIVE FOR THE S1 SITES OF SER190 TRYPSIN-LIKE SERINE PROTEASE DRUG TARGETS
1C5X 2000-12-22 1.75 STRUCTURAL BASIS FOR SELECTIVITY OF A SMALL MOLECULE, S1-BINDING, SUB-MICROMOLAR INHIBITOR OF UROKINASE TYPE PLASMINOGEN ACTIVATOR
4OS6 2014-09-24 1.75 Crystal structure of urokinase-type plasminogen activator (uPA) complexed with bicyclic peptide UK604 (bicyclic 2)
5WXR 2018-07-11 1.75 Crystal structure of uPA in complex with upain-2-4-W3A
1GI8 2002-01-22 1.75 A NOVEL SERINE PROTEASE INHIBITION MOTIF INVOLVING A MULTI-CENTERED SHORT HYDROGEN BONDING NETWORK AT THE ACTIVE SITE
1GI7 2002-01-22 1.79 A NOVEL SERINE PROTEASE INHIBITION MOTIF INVOLVING A MULTI-CENTERED SHORT HYDROGEN BONDING NETWORK AT THE ACTIVE SITE
4OS2 2014-09-24 1.79 Crystal structure of urokinase-type plasminogen activator (uPA) complexed with bicyclic peptide UK602 (bicyclic 1)
5WXQ 2018-07-11 1.79 Crystal structure of uPA in complex with upain-2-4
2VIO 2008-01-22 1.8 Fragment-Based Discovery of Mexiletine Derivatives as Orally Bioavailable Inhibitors of Urokinase-Type Plasminogen Activator
4X1N 2015-03-25 1.8 The crystal structure of mupain-1-16 in complex with murinised human uPA at pH7.4
4MNV 2014-02-05 1.8 Crystal structure of bicyclic peptide UK729 bound as an acyl-enzyme intermediate to urokinase-type plasminogen activator (uPA)
4FUG 2012-08-22 1.8 Crystal Structure of the Urokinase
1GI9 2002-01-22 1.8 A NOVEL SERINE PROTEASE INHIBITION MOTIF INVOLVING A MULTI-CENTERED SHORT HYDROGEN BONDING NETWORK AT THE ACTIVE SITE
1GJ9 2002-04-27 1.8 ENGINEERING INHIBITORS HIGHLY SELECTIVE FOR THE S1 SITES OF SER190 TRYPSIN-LIKE SERINE PROTEASE DRUG TARGETS
1F5K 2001-06-15 1.8 UROKINASE PLASMINOGEN ACTIVATOR B-CHAIN-BENZAMIDINE COMPLEX
1EJN 2000-05-17 1.8 UROKINASE PLASMINOGEN ACTIVATOR B-CHAIN INHIBITOR COMPLEX
1O3P 2003-09-02 1.81 Elaborate Manifold of Short Hydrogen Bond Arrays Mediating Binding of Active Site-Directed Serine Protease Inhibitors
3IG6 2009-10-13 1.83 Low molecular weigth human Urokinase type Plasminogen activator 2-[6-(3'-Aminomethyl-biphenyl-3-yloxy)-4-(3-dimethylamino-pyrrolidin-1-yl)-3,5-difluoro-pyridin-2-yloxy]-4-dimethylamino-benzoic acid complex
1SQO 2004-04-27 1.84 Substituted 2-Naphthamidine Inhibitors of Urokinase
1O5B 2004-09-21 1.85 Dissecting and Designing Inhibitor Selectivity Determinants at the S1 site Using an Artificial Ala190 Protease (Ala190 uPA)
1C5Z 2000-12-22 1.85 STRUCTURAL BASIS FOR SELECTIVITY OF A SMALL MOLECULE, S1-BINDING, SUB-MICROMOLAR INHIBITOR OF UROKINASE TYPE PLASMINOGEN ACTIVATOR
4ZKS 2016-05-18 1.85 The crystal structure of upain-1-W3A in complex with inactive uPA (uPA-S195A) at pH7.4
4MNX 2014-02-05 1.85 Crystal structure of urokinase-type plasminogen activator (uPA) complexed with bicyclic peptide UK811
2O8W 2007-12-25 1.86 Crystal Structure and Binding Epitopes of Urokinase-type Plasminogen Activator (C122A/N145Q/S195A) in complex with Inhibitors
4X1S 2015-10-28 1.9 The crystal structure of mupain-1-16-D9A in complex with murinised human uPA at pH7.4
4FUB 2012-08-22 1.9 Crystal Structure of the Urokinase
4ZHM 2015-09-16 1.9 The crystal structure of mupain-1--16-IG in complex with murinised human uPA at pH7.4
1SQT 2004-04-27 1.9 Substituted 2-Naphthamidine Inhibitors of Urokinase
2FD6 2006-02-21 1.9 Structure of Human Urokinase Plasminogen Activator in Complex with Urokinase Receptor and an anti-upar antibody at 1.9 A
3QN7 2012-02-15 1.9 Potent and selective bicyclic peptide inhibitor (UK18) of human urokinase-type plasminogen activator(uPA)
5ZA8 2018-12-19 1.9 uPA-BB2-27F
1GJB 2002-04-27 1.9 ENGINEERING INHIBITORS HIGHLY SELECTIVE FOR THE S1 SITES OF SER190 TRYPSIN-LIKE SERINE PROTEASE DRUG TARGETS
5ZC5 2018-12-19 1.9 uPA-NU-09F
2VIN 2008-01-22 1.9 Fragment-Based Discovery of Mexiletine Derivatives as Orally Bioavailable Inhibitors of Urokinase-Type Plasminogen Activator
6AG7 2019-08-14 1.9 The crystal structure of uPA in complex with HMA-55F
2I9A 2006-11-28 1.9 Crystal structure of the free aminoterminal fragment of urokinase type plasminogen activator (ATF)
1W0Z 2008-05-20 1.9 Urokinase type plasminogen activator
1C5W 2000-12-22 1.94 STRUCTURAL BASIS FOR SELECTIVITY OF A SMALL MOLECULE, S1-BINDING, SUB-MICROMOLAR INHIBITOR OF UROKINASE TYPE PLASMINOGEN ACTIVATOR
4DVA 2013-01-16 1.94 The crystal structure of human urokinase-type plasminogen activator catalytic domain
5ZAG 2018-12-19 1.95 uPA-BB2-94F
5HGG 2016-06-01 1.97 Crystal structure of uPA in complex with a camelid-derived antibody fragment
4FUD 2012-08-22 2.0 Crystal Structure of the Urokinase
4FU7 2012-08-22 2.0 Crystal Structure of the Urokinase
5YC7 2018-10-03 2.0 The crystal structure of uPA in complex with 4-Bromobenzylamirne at pH7.4
4OS4 2014-09-24 2.0 Crystal structure of urokinase-type plasminogen activator (uPA) complexed with bicyclic peptide UK603 (bicyclic 1)
1SQA 2004-04-27 2.0 Substituted 2-Naphthamidine Inhibitors of Urokinase
4FUF 2012-08-22 2.0 Crystal Structure of the Urokinase
4FUE 2012-08-22 2.0 Crystal Structure of the Urokinase
4OS7 2014-09-24 2.0 Crystal structure of urokinase-type plasminogen activator (uPA) complexed with bicyclic peptide UK607 (bicyclic)
1W11 2008-05-20 2.0 UROKINASE TYPE PLASMINOGEN ACTIVATOR
1VJA 2004-06-22 2.0 Urokinase Plasminogen Activator B-Chain-JT463 Complex
1W13 2008-05-20 2.0 UROKINASE TYPE PLASMINOGEN ACTIVATOR
1W10 2008-05-20 2.0 Urokinase type plasminogen activator
2VIQ 2008-01-22 2.0 Fragment-Based Discovery of Mexiletine Derivatives as Orally Bioavailable Inhibitors of Urokinase-Type Plasminogen Activator
4FUI 2012-08-22 2.0 Crystal Structure of the Urokinase
2R2W 2007-10-23 2.01 Urokinase plasminogen activator B-chain-GPPE complex
4H42 2013-10-30 2.01 Synthesis of a Weak Basic uPA Inhibitor and Crystal Structure of Complex with uPA
1U6Q 2004-10-19 2.02 Substituted 2-Naphthamadine inhibitors of Urokinase
3MWI 2011-06-01 2.03 The complex crystal Structure of Urokianse and 5-nitro-1H-indole-2-amidine
4FUJ 2012-08-22 2.05 Crystal Structure of the Urokinase
2VIW 2008-01-22 2.05 Fragment-Based Discovery of Mexiletine Derivatives as Orally Bioavailable Inhibitors of Urokinase-Type Plasminogen Activator
4ZHL 2015-09-16 2.06 The crystal structure of mupain-1-IG in complex with murinised human uPA at pH7.4
4X0W 2015-10-21 2.1 The crystal structure of mupain-1-17 in complex with murinised human uPA
4X1R 2015-03-25 2.1 The crystal structure of mupain-1-12 in complex with murinised human uPA at pH7.4
1F5L 2001-06-15 2.1 UROKINASE PLASMINOGEN ACTIVATOR B-CHAIN-AMILORIDE COMPLEX
5WXT 2018-07-11 2.1 Crystal structure of uPA-S195A in complex with S2444
2NWN 2007-10-16 2.15 New Pharmacophore for Serine Protease Inhibition Revealed by Crystal Structure of Human Urokinase-type Plasminogen Activator Complexed with a Cyclic Peptidyl Inhibitor, upain-1
4OS1 2014-09-24 2.2 Crystal structure of urokinase-type plasminogen activator (uPA) complexed with bicyclic peptide UK601 (bicyclic 1)
1W14 2008-05-20 2.2 UROKINASE TYPE PLASMINOGEN ACTIVATOR
2VNT 2008-02-19 2.2 UROKINASE-TYPE PLASMINOGEN ACTIVATOR INHIBITOR COMPLEX WITH A 1-(7- SULPHOAMIDOISOQUINOLINYL)GUANIDINE
4JK6 2013-07-17 2.2 Human urokinase-type Plasminogen Activator (uPA) in complex with a bicyclic peptide inhibitor (UK18-D-Aba)
4FU8 2012-08-22 2.2 Crystal Structure of the Urokinase
4OS5 2014-09-24 2.26 Crystal structure of urokinase-type plasminogen activator (uPA) complexed with bicyclic peptide UK603 (bicyclic 2)
4X1Q 2015-03-25 2.28 The crystal structure of mupain-1 in complex with murinised human uPA at pH7.4
6NMB 2019-03-06 2.3 Tranexamic Acid is an Active Site Inhibitor of Urokinase Plasminogen Activator
3PB1 2010-12-29 2.3 Crystal Structure of a Michaelis Complex between Plasminogen Activator Inhibitor-1 and Urokinase-type Plasminogen Activator
5WXS 2018-07-11 2.3 Crystal structure of uPA in complex with S2444
3OY5 2011-08-10 2.31 The crystal structure of uPA complex with peptide inhibitor MH027 at pH7.4
3OY6 2011-08-10 2.31 The crystal structure of uPA complex with peptide inhibitor MH036 at pH4.6
1OWD 2003-09-30 2.32 Substituted 2-Naphthamidine inhibitors of urokinase
3KGP 2009-12-01 2.35 Crystal Structures of Urokinase-type Plasminogen Activator in Complex with 4-(Aminomethyl) Benzoic Acid and 4-(Aminomethyl-phenyl)-methanol
3KHV 2009-12-01 2.35 Crystal Structures of Urokinase-type Plasminogen Activator in Complex with 4-(Aminomethyl) Benzoic Acid and 4-(Aminomethyl-phenyl)-methanol
1W12 2008-05-20 2.4 UROKINASE TYPE PLASMINOGEN ACTIVATOR
1SC8 2004-06-22 2.4 Urokinase Plasminogen Activator B-Chain-J435 Complex
1VJ9 2004-06-22 2.4 Urokinase Plasminogen Activator B-Chain-JT464 Complex
1LMW 1996-01-29 2.5 LMW U-PA Structure complexed with EGRCMK (GLU-GLY-ARG Chloromethyl Ketone)
3BT2 2008-03-25 2.5 Structure of urokinase receptor, urokinase and vitronectin complex
1F92 2001-07-06 2.6 UROKINASE PLASMINOGEN ACTIVATOR B CHAIN-UKI-1D COMPLEX
3KID 2009-12-01 2.71 The Crystal Structures of 2-Aminobenzothiazole-based Inhibitors in Complexes with Urokinase-type Plasminogen Activator
1OWK 2003-09-30 2.8 Substituted 2-Naphthamidine Inhibitors of Urokinase
2I9B 2007-01-02 2.8 Crystal structure of ATF-urokinase receptor complex
3BT1 2008-03-25 2.8 Structure of urokinase receptor, urokinase and vitronectin complex
1OWI 2003-09-30 2.93 Substituted 2-Naphthamidine Inhibitors of Urokinase
4DW2 2013-01-16 2.97 The crystal structure of uPA in complex with the Fab fragment of mAb-112
5ZAH 2018-12-19 2.98 uPA-BB2-30F
5XG4 2017-07-26 3.0 Crystal structure of uPA in complex with quercetin
1FV9 2000-10-18 3.0 Crystal structure of human microurokinase in complex with 2-amino-5-hydroxy-benzimidazole
1OWJ 2003-09-30 3.1 Substituted 2-Naphthamidine Inhibitors of Urokinase
3U73 2012-04-18 3.19 Crystal structure of stabilized human uPAR mutant in complex with ATF
4K24 2014-02-26 4.5 Structure of anti-uPAR Fab ATN-658 in complex with uPAR

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
94.1 Urokinase-type plasminogen activator P16227 UROK_PAPCY
98.1 Urokinase-type plasminogen activator Q5RF29 UROK_PONAB
100.0 Urokinase-type plasminogen activator P00749 UROK_HUMAN