Tetranectin-binding site on plasminogen kringle 4 involves the lysine-binding pocket and at least one additional amino acid residue

Kringle domains are found in a number of proteins where they govern protein -protein interactions. These interactions are often sensitive to lysine and lysine analogues, and the kringle-lysine interaction has been used as a mo del system for investigating kringle-protein interactions. In this study, w e analyze the interaction of wild-type and six single-residue mutants of re combinant plasminogen kringle 4 expressed in Escherichia coli with the reco mbinant C-type lectin domain of tetranectin and trans-aminomethyl-cyclohexa noic acid (t-AMCHA) using isothermal titration calorimetry. We find that al l amino acid residues of plasminogen kringle 4 found to be involved in r-AM CHA binding are also involved in binding tetranectin. Notably, one amino ac id residue of plasminogen kringle 4, Arg 32, not: involved in binding t-AMC HA, is critical for binding tetranectin. We also find that Asp 57 and Asp 5 5 of plasminogen kringle 4, which both were found to interact with the low molecular weight ligand with an almost: identical geometry in the crystal o f the complex, are not of equal functional importance in t-AMCHA binding. M utating Asp 57 to an Asn totally eliminates binding, whereas the Asp 55 to Asn, like the Arg 71 to Gin mutation, was found only to decrease affinity.