Construction, expression, and purification of recombinant kringle 1 of human plasminogen and analysis of its interaction with omega-amino acids.


Abstract

An Escherichia coli expression vector, containing the alkaline phosphatase promoter and the stII heat-stable enterotoxin signal sequence, along with the cDNA of the kringle 1 (K1) region of human plasminogen (HPg), has been employed to express into the periplasmic space amino acid residues 82-163 (E163----D) of HPg. This region of the molecule contains the entire K1 domain (residues C84-C162) of HPg, as well as two non-kringle amino-terminal amino acids (S82-E83) that are present in their normal locations in HPg and a carboxyl-terminal amino acid, D163, that results from mutation of the E163, normally present at this location in the HPg amino acid sequence. After purification of r-K1 by chromatographic techniques, we have investigated its omega-amino acid binding properties by titration calorimetry, intrinsic fluorescence, and differential scanning microcalorimetry (DSC). The antifibrinolytic agent, epsilon-aminocaproic acid (EACA), possesses a single binding site for r-K1. The thermodynamic properties of this interaction, studied by calorimetric titrations of the heats of binding with this ligand, reveal a Kd of 12 +/- 2 microM at 25 degrees C and pH 7.4, a corresponding delta G of -6.7 +/- 0.1 kcal/mol, a delta H of -3.6 +/- 0.1 kcal/mol, and a delta S of 10.5 +/- 0.8 eu. The intrinsic fluorescence of r-K1 decreases by approximately 44% when its binding site is saturated with EACA, and titrations of this perturbation with EACA lead to calculation of a Kd of approximately 13 microM, a value in good agreement with that obtained from titration calorimetric analysis. EACA represents the strongest binding ligand of a variety of simple aliphatic omega-amino acids examined. A cyclic analogue of EACA, trans-4-(aminomethyl)cyclohexanecarboxylic acid, interacts with r-K1 with an approximate 12-fold tighter Kd (1.0 +/- 0.2 microM). Investigations by DSC, at pH 7.4, demonstrate that a significant stabilization of the r-K1 structure occurs when EACA binds to this domain. The temperature of maximum heat capacity change (Tm) in the thermal denaturation of r-K1 increases from approximately 340.8 to 359.1 K as a consequence of EACA binding. These studies demonstrate that a fully functional EACA-binding kringle from HPg can be expressed and secreted in E. coli, purified by techniques that do not require refolding, and investigated as an independent structural unit. Study holds ProTherm entries: 4246 Extra Details: omega-amino acid binding properties; thermodynamic properties;,binding ligand; independent structural unit

Submission Details

ID: 2ZmXge3z3

Submitter: Connie Wang

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

Version: 1

Publication Details
Menhart N;Sehl LC;Kelley RF;Castellino FJ,Biochemistry (1991) Construction, expression, and purification of recombinant kringle 1 of human plasminogen and analysis of its interaction with omega-amino acids. PMID:1993205
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
1HPK 1997-03-12 SOLUTION NMR STRUCTURE OF THE HUMAN PLASMINOGEN KRINGLE 1 DOMAIN COMPLEXED WITH 6-AMINOHEXANOIC ACID AT PH 5.3, 310K, DERIVED FROM RANDOMLY GENERATED STRUCTURES USING SIMULATED ANNEALING, MINIMIZED AVERAGE STRUCTURE
2L0S 2010-08-04 Solution Structure of Human Plasminogen Kringle 3
2KNF 2009-10-27 Solution structure and functional characterization of human plasminogen kringle 5
1B2I 1999-11-19 KRINGLE 2 DOMAIN OF HUMAN PLASMINOGEN: NMR SOLUTION STRUCTURE OF TRANS-4-AMINOMETHYLCYCLOHEXANE-1-CARBOXYLIC ACID (AMCHA) COMPLEX
1HPJ 1997-03-12 SOLUTION NMR STRUCTURE OF THE HUMAN PLASMINOGEN KRINGLE 1 DOMAIN COMPLEXED WITH 6-AMINOHEXANOIC ACID AT PH 5.3, 310K, DERIVED FROM RANDOMLY GENERATED STRUCTURES USING SIMULATED ANNEALING, 12 STRUCTURES
5UGG 2017-05-31 1.2 Protease Inhibitor
6D3Y 2019-01-23 1.32 Highly Potent and Selective Plasmin Inhibitors Based on the Sunflower Trypsin Inhibitor-1 Scaffold Attenuate Fibrinolysis in Plasma
5UGD 2017-05-31 1.38 Protease Inhibitor
6D40 2019-01-23 1.43 Highly Potent and Selective Plasmin Inhibitors Based on the Sunflower Trypsin Inhibitor-1 Scaffold Attenuate Fibrinolysis in Plasma
5HPG 1998-03-25 1.66 STRUCTURE AND LIGAND DETERMINANTS OF THE RECOMBINANT KRINGLE 5 DOMAIN OF HUMAN PLASMINOGEN
1KRN 1997-01-11 1.67 STRUCTURE OF KRINGLE 4 AT 4C TEMPERATURE AND 1.67 ANGSTROMS RESOLUTION
6OG4 2019-07-24 1.7 plasminogen binding group A streptococcal M protein
1KI0 2002-05-29 1.75 The X-ray Structure of Human Angiostatin
4CIK 2014-06-18 1.78 plasminogen kringle 1 in complex with inhibitor
6D3X 2019-01-23 1.8 Highly Potent and Selective Plasmin Inhibitors Based on the Sunflower Trypsin Inhibitor-1 Scaffold Attenuate Fibrinolysis in Plasma
1PK4 1993-10-31 1.9 CRYSTAL AND MOLECULAR STRUCTURE OF HUMAN PLASMINOGEN KRINGLE 4 REFINED AT 1.9-ANGSTROMS RESOLUTION
1QRZ 1999-10-14 2.0 CATALYTIC DOMAIN OF PLASMINOGEN
1DDJ 2000-02-18 2.0 CRYSTAL STRUCTURE OF HUMAN PLASMINOGEN CATALYTIC DOMAIN
6D3Z 2019-01-23 2.0 Protease SFTI complex
4DCB 2012-06-06 2.03 Y. pestis Plasminogen Activator Pla in Complex with Human Plasminogen Activation Loop Peptide ALP11
1CEA 1996-04-03 2.06 THE STRUCTURE OF THE NON-COVALENT COMPLEX OF RECOMBINANT KRINGLE 1 DOMAIN OF HUMAN PLASMINOGEN WITH EACA (EPSILON-AMINOCAPROIC ACID)
1CEB 1996-04-03 2.07 THE STRUCTURE OF THE NON-COVALENT COMPLEX OF RECOMBINANT KRINGLE 1 DOMAIN OF HUMAN PLASMINOGEN WITH AMCHA (TRANS-4-AMINOMETHYLCYCLOHEXANE-1-CARBOXYLIC ACID)
1PMK 1994-06-22 2.25 KRINGLE-KRINGLE INTERACTIONS IN MULTIMER KRINGLE STRUCTURES
2PK4 1993-10-31 2.25 THE REFINED STRUCTURE OF THE EPSILON-AMINOCAPROIC ACID COMPLEX OF HUMAN PLASMINOGEN KRINGLE
2DOH 2006-12-05 2.3 The X-ray crystallographic structure of the angiogenesis inhibitor, angiostatin, bound a to a peptide from the group A streptococcal surface protein PAM
1L4D 2002-12-11 2.3 CRYSTAL STRUCTURE OF MICROPLASMINOGEN-STREPTOKINASE ALPHA DOMAIN COMPLEX
1RJX 2003-12-02 2.3 Human PLASMINOGEN CATALYTIC DOMAIN, K698M MUTANT
4DUR 2012-03-28 2.45 The X-ray Crystal Structure of Full-Length type II Human Plasminogen
1PKR 1994-01-31 2.48 THE STRUCTURE OF RECOMBINANT PLASMINOGEN KRINGLE 1 AND THE FIBRIN BINDING SITE
1BUI 1999-09-02 2.65 Structure of the ternary microplasmin-staphylokinase-microplasmin complex: a proteinase-cofactor-substrate complex in action
1I5K 2001-08-01 2.7 STRUCTURE AND BINDING DETERMINANTS OF THE RECOMBINANT KRINGLE-2 DOMAIN OF HUMAN PLASMINOGEN TO AN INTERNAL PEPTIDE FROM A GROUP A STREPTOCOCCAL SURFACE PROTEIN
3UIR 2012-12-26 2.78 Crystal structure of the plasmin-textilinin-1 complex
1L4Z 2002-12-11 2.8 X-RAY CRYSTAL STRUCTURE OF THE COMPLEX OF MICROPLASMINOGEN WITH ALPHA DOMAIN OF STREPTOKINASE IN THE PRESENCE CADMIUM IONS
1BML 1999-08-02 2.9 COMPLEX OF THE CATALYTIC DOMAIN OF HUMAN PLASMIN AND STREPTOKINASE
2DOI 2006-12-05 3.1 The X-ray crystallographic structure of the angiogenesis inhibitor, angiostatin, bound to a peptide from the group A streptococcus protein PAM
4A5T 2012-05-23 3.49 STRUCTURAL BASIS FOR THE CONFORMATIONAL MODULATION
4DUU 2012-03-28 5.2 The X-ray Crystal Structure of Full-Length type I Human Plasminogen

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
92.8 Plasminogen P12545 PLMN_MACMU
95.9 Plasminogen Q5R8X6 PLMN_PONAB
100.0 Plasminogen P00747 PLMN_HUMAN
95.8 Plasminogen Q02325 PLGB_HUMAN
95.8 Plasminogen Q15195 PLGA_HUMAN