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
1B2I 1999-09-24T00:00:00+0000 0 KRINGLE 2 DOMAIN OF HUMAN PLASMINOGEN: NMR SOLUTION STRUCTURE OF TRANS-4-AMINOMETHYLCYCLOHEXANE-1-CARBOXYLIC ACID (AMCHA) COMPLEX
1BML 1999-05-25T00:00:00+0000 2.9 COMPLEX OF THE CATALYTIC DOMAIN OF HUMAN PLASMIN AND STREPTOKINASE
1BUI 1998-09-04T00:00:00+0000 2.65 Structure of the ternary microplasmin-staphylokinase-microplasmin complex: a proteinase-cofactor-substrate complex in action
1CEA 1995-12-03T00:00:00+0000 2.06 THE STRUCTURE OF THE NON-COVALENT COMPLEX OF RECOMBINANT KRINGLE 1 DOMAIN OF HUMAN PLASMINOGEN WITH EACA (EPSILON-AMINOCAPROIC ACID)
1CEB 1995-12-03T00:00:00+0000 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)
1DDJ 1999-11-10T00:00:00+0000 2.0 CRYSTAL STRUCTURE OF HUMAN PLASMINOGEN CATALYTIC DOMAIN
1HPJ 1996-08-14T00:00:00+0000 0 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
1HPK 1996-08-14T00:00:00+0000 0 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
1I5K 2001-02-27T00:00:00+0000 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
1KI0 2001-12-02T00:00:00+0000 1.75 The X-ray Structure of Human Angiostatin

Relevant UniProtKB Entries

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