Comparative Analyses of the Lysine Binding Site Properties of Apolipoprotein(a) Kringle IV Types 7 and 10

Apolipoprotein(a) [apo(a)] shares extensive sequence similarity with plasminogen and consists of multiple tandem repeats of domains similar to plasminogen kringle IV (KIV), followed by domains homologous to the plasminogen KV and protease domains. The apo(a) KIV domains can be classified into 10 types on the basis of amino acid sequence (KIV1−KIV10) of which KIV10 contains a canonical lysine binding site (LBS); KIV10 mediates the lysine-dependent interaction of Lp(a) with certain biological substrates. Molecular modeling studies indicated the presence of weak LBS in each of KIV5−KIV8, and subsequent biochemical studies have revealed contributions of these kringles to lysine-mediated interactions involving apo(a). The present study describes the direct demonstration of a weak LBS within KIV7, as well as the first characterization of the ligand specificity of an LBS outside that of KIV10. We have expressed both KIV7 and KIV10 from bacterial cells and purified them to homogeneity from cell lysates. Equilibrium binding analyses of the KIV7 LBS using intrinsic fluorescence revealed an affinity for l-lysine and its analogues ∼10-fold weaker (KD = 230 ± 42 μM for ε-aminocaproic acid) than that of KIV10 (KD = 33 ± 4 μM for ε-aminocaproic acid). Moreover, we demonstrated differences in specificity of the LBS of KIV7 in comparison with KIV10 in that KIV7 preferentially bound l-proline. Both kringles bind 4-aminobutyric acid with similar affinities albeit with apparently different mechanisms. Key Phe62 → Tyr and Asp56 → Glu substitutions in the KIV7 LBS result in alterations in the size of the LBS and in the spatial relationship between the cationic and anionic centers in the LBS and thus account for the differences in the binding properties of KIV7 and KIV10.