Expression, isolation and characterization of a mutated human plasminogen kringle 3 with a functional lysine binding site

Abstract.

Each kringle of human plasminogen (HPg) except kringle 3 (K3) exhibits affinity for ω-aminocarboxylic acids. Assuming that the K3 domain contains a preformed but nonfunctional lysine binding site (LBS), Lys³¹¹ was altered by site-directed mutagenesis into Asp³¹¹ in accordance with the consensus sequence of the LBS. Cys²⁹⁷ involved in the interkringle disulfide bridge was mutated into Ser²⁹⁷ to minimize dimerization and aggregation. The mutated K3 TYQ[K3_HPg/C297S/ K311D]DS (r-K3_mut) was expressed in Escherichia coli, isolated on an Ni²⁺-nitrilotriacetic acid-agarose column, refolded and purified on a lysine Bio-Gel column. Fluorescence titration indicates affinity of r-K3_mut for ω-aminocarboxylic acids with the following association constants (K_ass, mM⁻¹) 5-aminopentanoic acid 1.3; 6-aminohexanoic acid 4.2; 7-aminoheptanoic acid 0.5; trans-(aminomethyl)cyclohexanecarboxylic acid 12.7; p-benzylaminesulfonic acid 11.8. r-K3_mut exhibits an affinity similar to native and mutated (R220G, E221D) K2. The results indicate the presence of a preformed but nonfunctional LBS in native K3 of HPg. We were able to demonstrate for the first time that an appropriate mutation in the LBS of a kringle produced a weak but distinct affinity for ω-aminocarboxylic acids.